Patent Application
20220280514
The present invention is directed to methods of treating bile acid diarrhea by administering a CFTR chloride channel inhibitor.
Bile acid diarrhea (BAD) is commonly seen following 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 enterohepatic circulation, which results in the delivery of excess bile acids to the colon where they cause fluid secretion and increase motility.
Current therapy for primary BAD, or BAD caused by ileal resection or Crohn's disease, includes 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, in which BAD is likely involved in pathogenesis in 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 significant side effects such as ischemic colitis with alosetron and pancreatitis with eluxadoline.
An unmet need for improved and alternative therapeutics for BAD persists.
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 has been associated with secretory diarrhea.
CFTR inhibitors have clinical application in the therapy of secretory diarrheas. Secretory diarrheas caused by enterotoxins, such as cholera and Travelers' diarrhea (enteropathogenic E. coli), require functional CFTR for primary chloride secretion into the intestinal lumen, which secondarily 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 indicated that intestinal chloride secretion in enterotoxin-mediated secretory diarrheas occurs mainly 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)).
U.S. Pat. No. 9,062,073 describes a series of benzopyrimido-pyrrolo-oxazine-dione (BPO) compounds and certain pyrimido-pyrrolo-quinoxalinedione (PPQ) compounds which are useful as CFTR chloride channel inhibitors. The BPO compounds have the generic formula:
The PPQ compounds have the generic formula:
Aspects of the present invention are directed to methods of treating a subject having bile acid diarrhea, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to treat the bile acid diarrhea.
Other aspects of the invention are directed to methods 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 the bile acid diarrhea.
In addition, aspects of the present invention are also directed to methods 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 said intestinal fluid secretion resulting from bile acid-induced activation of apical CFTR chloride channels.
Other aspects of the invention are directed to methods 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 said intestinal fluid secretion resulting from bile acid-induced activation of apical CFTR chloride channels.
Further aspects of the present invention are directed to methods of reducing bile acid-induced apical CFTR chloride channel current in the intestinal epithelium of a subject in need thereof, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce said bile acid-induced apical CFTR chloride channel current.
Other aspects of the invention are directed to methods of reducing bile acid-induced apical CFTR chloride channel current in the intestinal epithelium of 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 said bile acid-induced apical CFTR chloride channel current.
In some aspects, the invention is directed to methods 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 said diarrhea or to alleviate the symptoms of said diarrhea.
Other aspects of the invention are directed to methods for treating diarrhea, or alleviating symptoms associated with diarrhea, in a subject who has undergone ileal resection, comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel effective to treat said diarrhea or to alleviate the symptoms of said diarrhea.
The present invention may be understood by reference to the following detailed description which forms a part of this disclosure. The invention is not limited to the specific methods, conditions or parameters described and/or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.
Scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art, unless otherwise defined herein.
As used herein, the terms “composition,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” or “medicament” are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or mammal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action.
As used herein, the terms “treatment” or “therapy” (as well as different forms thereof) 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 of a condition, disease or disorder.
The term “administering” means either directly administering a compound or composition of the present invention.
The term “subject,” is used hereinto refer to a human being, or a domesticated mammal (e.g., dog, cat) to whom treatment, including prophylactic treatment, with the compounds according to the present invention, is provided.
Within the present invention, the disclosed compounds may be prepared in the form of pharmaceutically acceptable salts. “Pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. These pharmaceutically acceptable salts are prepared by methods known in the art, e.g., by dissolving the free amine bases with an excess of the acid in aqueous alcohol, or neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with an amine.
The present invention is directed to methods of treating a subject having bile acid diarrhea, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to treat the bile acid diarrhea.
In some aspects, the present invention is directed to methods of treating a subject having bile acid diarrhea. Bile acid diarrhea (BAD) is a clinical diagnosed condition in which diarrhea is thought to result from bile acid malabsorption or disregulation. See, e.g., M. Camilleri, Advances in understanding of bile acid diarrhea, Expert Rev Gastroenterol Hepatol. 2014 January; 8(1): 49-61. Methods of diagnosing BAD are known in the art. See, e.g., id.
In some aspects of the methods disclosed herein, the subject is administered an amount of a CFTR chloride channel inhibitor (CFTR-CCI). As used herein, a CFTR chloride channel refers to the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), 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 medicinal 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. 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 A S. 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 by reference herein in its entirety.
In some embodiments, the CFTR chloride channel inhibitor is a BPO CFTR chloride channel inhibitor (BPO-CFTR-CCI). BPO-CFTR-CCIs are compounds encompassed by the generic structure
In some embodiments, the BPO-CFTR-CCI is a compound of formula
BPO-CFTR-CCIs are described in U.S. Pat. No. 9,062,073, which is incorporated herein by reference in its entirety.
In some embodiments, the BPO-CFTR-CCI is (R)-BPO-27, a compound having the structure:
In other embodiments, the CFTR chloride channel inhibitor is a PPQ CFTR chloride channel inhibitor (PPQ-CFTR-CCI). PPQ-CFTR-CCIs are compounds encompassed by the generic structure
PPQ-CFTR-CCIs are described in described in U.S. Pat. No. 9,062,073, which is incorporated herein by reference in its entirety.
In some embodiments, the CFTR chloride channel inhibitor is a thiazolidinone CFTR chloride channel inhibitor (TD-CFTR-CCI). TD-CFTR-CCIs are compounds encompassed by the generic structure:
TD-CFTR-CCIs are also compounds encompassed by the generic structure:
TD-CFTR-CCIs are also the compounds 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 A S. Thiazolidinone CFTR inhibitors with improved water solubility identified by structure-activity analysis. Bioorg. Med. Chem. 2008; 16:8187-8195; U.S. Pat. Nos. 7,235,573, 7,638,543, and International Patent Application Publication No. WO 09/120803. The entirety of each of these references is incorporated by reference herein.
In some embodiments, the TD-CFTR-CCI is CFTRinh-172, which has the structure:
In some embodiments, the CFTR chloride channel inhibitor is a glycine hydrazide CFTR chloride channel inhibitor (GH-CFTR-CCI). GH-CFTR-CCIs are compounds encompassed by the structure:
GH-CFTR-CCIs are also compounds encompassed by the generic formula:
GH-CFTR-CCIs are also compounds encompassed by the generic formula:
GH-CFTR-CCIs are also the compounds 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. Pat. Nos. 7,414,037 and 7,888,332; U.S. Application Publication No. 2009/0253799; International Patent Application Publication No. WO 09/146144. The entirety of each of these references is incorporated by reference herein.
In some embodiments, the GH-CFTR-CCI is GlyH-101, which has the structure
Other CFTR chloride channel inhibitors are glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino) benzoate, and niflumic acid.
In some embodiments of the methods disclosed herein, the subject is administered a CFTR chloride channel inhibitor that is a BPO-CFTR-CCI, a PPQ-CFTR-CCI, a TD-CFTR-CCI, or a GH-CFTR-CCI.
In some embodiments of the methods disclosed herein, the subject is administered (R)-BPO-27, CFTRinh-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 a BPO-CFTR-CCI.
In some embodiments, the CFTR chloride channel inhibitor is a PPQ-CFTR-CCI.
In some embodiments, the CFTR chloride channel inhibitor is a TD-CFTR-CCI. In some embodiments, the TD-CFTR-CCI is CFTRinh-172.
In some embodiments, the CFTR chloride channel inhibitor is a GH-CFTR-CCI. In some embodiments, the GH-CFTR-CCI 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.
In some embodiments of the methods of the disclosure, the subject is administered a pharmaceutical composition comprising a pharmaceutical excipient and an amount of a CFTR chloride channel inhibitor.
In some aspects, the disclosure is directed to methods 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 the bile acid diarrhea. In some embodiments, the CFTR chloride channel inhibitor is a BPO-CFTR-CCI, a PPQ-CFTR-CCI, a TD-CFTR-CCI, or a GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a 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 a PPQ-CFTR-CCI. In other embodiments, the CFTR chloride channel inhibitor is a TD-CFTR-CCI. In some embodiments, the TD-CFTR-CCI is CFTRinh-172. In other embodiments, the CFTR chloride channel inhibitor is a GH-CFTR-CCI. In some embodiments, the GH-CFTR-CCI is GlyH-101. In yet 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.
In those embodiments wherein the 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 these embodiments, the amount of (R)-BPO-27 in the pharmaceutical composition will be substantially equal to, or more 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, the (R)-BPO-27 will be present in an enantiomeric excess (ee), as compared to the (S)-BPO-27. For example, the (R)-BPO-27 can be present in about a 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 include (R)-BPO-27 wherein the % ee is greater than 99%.
The amount of CFTR chloride channel inhibitor administered is an amount that is effective to treat the bile acid diarrhea. The amount that is effective in this regard will vary depending on the subject's characteristics and condition.
In some aspects of the disclosed methods, the amount of CFTR chloride channel inhibitor is effective to reduce intestinal fluid secretion resulting from the bile acid diarrhea.
In other aspects of the disclosed methods, the amount of CFTR chloride channel inhibitor is effective to reduce bile acid-induced activation of apical CFTR chloride channels.
In some aspects of the disclosed methods, subject is also administered an amount of a second agent effective to treat the 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 p 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 p opioid receptor agonist, preferably eluxadoline. In other embodiments, the second agent is a broad-spectrum gut-specific antibiotic, preferably rifaximin.
In some aspects, the present invention is directed to methods 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 the intestinal fluid secretion resulting from bile acid-induced activation of CFTR chloride channels.
As used herein, intestinal fluid secretion refers to secretion of fluid from the intestinal epithelia into the intestinal lumen.
In some embodiments, the intestinal fluid secretion results from bile acid-induced activation of CFTR chloride channels in the intestinal epithelium. In other embodiments, the CFTR chloride channels are apical CFTR chloride channels in the intestinal epithelium. Apical CFTR chloride channels are CFTR chloride channels located in the apical cell membrane (i.e., the cell membrane facing the intestinal lumen). Thus, in some embodiments, the present invention is directed to methods 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 the intestinal fluid secretion resulting from bile acid-induced activation of apical CFTR chloride channels.
In some aspects, the intestinal fluid secretion is reduced by administering an amount of a CFTR chloride channel inhibitor effective to reduce said intestinal fluid secretion. As used herein, reduction of intestinal fluid secretion refers to a decrease in the amount of fluid that is secreted into the intestinal lumen relative to the amount of fluid that is secreted into the intestinal lumen in the absence of administration of a CFTR chloride channel inhibitor. Methods of measuring decrease in fluid secretion are known to those skilled in the art, and include measuring the water content of the intestinal luminal contents, and diarrhea output.
In some embodiments, the intestinal fluid secretion is reduced by administering a CFTR chloride channel inhibitor that is a BPO-CFTR-CCI, a PPQ-CFTR-CCI, a TD-CFTR-CCI, or a GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a BPO-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a PPQ-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a TD-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a GH-CFTR-CCI. In some embodiments, the intestinal fluid secretion is reduced by administering a CFTR chloride channel inhibitor that is (R)-BPO-27, CFTRinh-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 CFTRinh-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.
In this aspect of the invention, the amount of CFTR chloride channel inhibitor administered is an amount that is effective to reduce said intestinal fluid secretion resulting from bile acid-induced activation of CFTR chloride channels (e.g., the apical CFTR chloride channels). The amount that is effective in this regard will vary depending on the subject's characteristics and condition.
In some aspects, the present invention is directed to methods 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 the intestinal fluid secretion resulting from bile acid-induced activation of CFTR chloride channels. In some embodiments, the CFTR chloride channel inhibitor is a BPO-CFTR-CCI, a PPQ-CFTR-CCI, a TD-CFTR-CCI, or a GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a 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 a PPQ-CFTR-CCI. In other embodiments, the CFTR chloride channel inhibitor is a TD-CFTR-CCI. In some embodiments, the TD-CFTR-CCI is CFTRinh-172. In other embodiments, the CFTR chloride channel inhibitor is a GH-CFTR-CCI. In some embodiments, the GH-CFTR-CCI is GlyH-101. In yet 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.
In other aspects, the disclosure is directed to methods of reducing bile acid-induced CFTR chloride channel current in the intestinal epithelium of a subject in need thereof, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce said bile acid-induced CFTR chloride channel current.
As used here, “current” refers to the passage of ions through the channel. Thus, CFTR chloride channel current refers to the passage of chloride ions through the CFTR chloride channel.
In some embodiments, the CFTR chloride channel current results from bile acid-induced activation of apical CFTR chloride channels in the intestinal epithelium. Thus, in some embodiments, the present invention is directed to methods of reducing bile acid-induced apical CFTR chloride channel current in the intestinal epithelium of a subject in need thereof, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce said bile acid-induced apical CFTR chloride channel current.
In some embodiments, the bile acid-induced CFTR chloride channel current in the intestinal epithelium is reduced by administering a CFTR chloride channel inhibitor that is a BPO-CFTR-CCI, a PPQ-CFTR-CCI, a TD-CFTR-CCI, or a GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a BPO-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a PPQ-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a TD-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a GH-CFTR-CCI. In some embodiments, the bile acid-induced CFTR chloride channel current in the intestinal epithelium is reduced by administering a CFTR chloride channel inhibitor that is (R)-BPO-27, CFTRinh-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 CFTRinh-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.
In this aspect of the invention, the amount of CFTR chloride channel inhibitor administered is an amount that is effective to reduce the bile acid-induced CFTR chloride channel (e.g., the apical CFTR chloride channels) current. The amount that is effective in this regard will vary depending on the subject's characteristics and condition.
In some aspects, the present invention is directed to methods of reducing bile acid-induced CFTR chloride channel current in the intestinal epithelium of 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 said bile acid-induced CFTR chloride channel current. In some embodiments, the CFTR chloride channel inhibitor is a BPO-CFTR-CCI, a PPQ-CFTR-CCI, a TD-CFTR-CCI, or a GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a 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 a PPQ-CFTR-CCI. In other embodiments, the CFTR chloride channel inhibitor is a TD-CFTR-CCI. In some embodiments, the TD-CFTR-CCI is CFTRinh-172. In other embodiments, the CFTR chloride channel inhibitor is a GH-CFTR-CCI. In some embodiments, the GH-CFTR-CCI is GlyH-101. In yet 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.
In some aspects, the invention is directed to methods 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 said diarrhea or to alleviate the symptoms of said diarrhea. As used here, the symptoms of diarrhea include one or more of (1) increased stool water content, (2) increased defecation frequency; (3) increased stool output; (4) abdominal pain; (5) abdominal bloating; and (6) nausea.
In some embodiments, the methods for treating diarrhea, or alleviating symptoms associated with diarrhea, in a subject who has undergone ileal resection, comprises administering to the subject a CFTR chloride channel inhibitor that is a BPO-CFTR-CCI, a PPQ-CFTR-CCI, a TD-CFTR-CCI, or a GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a BPO-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a PPQ-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a TD-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a GH-CFTR-CCI.
In some embodiments, the methods for treating diarrhea, or alleviating symptoms associated with diarrhea, in a subject who has undergone ileal resection, comprises administering to the subject a CFTR chloride channel inhibitor that is (R)-BPO-27, CFTRinh-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 CFTRinh-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.
In this aspect of the invention, the amount of CFTR chloride channel inhibitor administered is an amount that is effective to treat the subject's diarrhea, or alleviating the subject's symptoms associated with diarrhea. The amount that is effective in this regard will vary depending on the subject's characteristics and condition.
In some aspects, the invention is directed to methods for treating diarrhea, or alleviating symptoms associated with diarrhea, in a subject who has undergone ileal resection, comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to treat said diarrhea or to alleviate the symptoms of said diarrhea. In some embodiments, the CFTR chloride channel inhibitor is a BPO-CFTR-CCI, a PPQ-CFTR-CCI, a TD-CFTR-CCI, or a GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is a 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 a PPQ-CFTR-CCI. In other embodiments, the CFTR chloride channel inhibitor is a TD-CFTR-CCI. In some embodiments, the TD-CFTR-CCI is CFTRinh-172. In other embodiments, the CFTR chloride channel inhibitor is a GH-CFTR-CCI. In some embodiments, the GH-CFTR-CCI is GlyH-101. In yet 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.
In all of the methods of the disclosure, the 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.
In some embodiments, the subject has been diagnosed with Crohn's disease.
In other embodiments, the subject has been diagnosed with IBS-D.
In yet other embodiments, the subject has been diagnosed with functional diarrhea.
In some aspects, the methods of the present invention result in a reduction in the water content of the subject's stool. The extent of water reduction can be determined by measuring the amount of water in the subject's stool before administering the CFTR chloride channel inhibitor, and comparing that amount to the amount of water in the subject's stool after administering the CFTR chloride channel inhibitor.
In some embodiments, the reduction in the water content of the subject's stool is demonstrated by measurement using a clinical instrument such as, for example, the Bristol Stool Form Scale (BSFS). The BSFS, which is known in the art, assigns the subject's stool a consistency score ranging from 1 (hard) to 7 (watery). Thus, in some embodiments, the reduction in the water content of the subject's stool using the present methods is demonstrated by a decrease in score using the Bristol Stool Form Scale. In some embodiments, the reduction in the water content of the subject's stool using the present methods is demonstrated by a decrease of 1 point (e.g., 7 to 6, 6 to 5, 5 to 4, 4 to 3, 3 to 2, 2 to 1) using the Bristol Stool Form Scale. In other embodiments, the reduction in the water content of the subject's stool using the present methods is demonstrated by a decrease of 2 points (e.g., 7 to 5, 6 to 4, 5 to 3, 4 to 2, 3 to 1) using the Bristol Stool Form Scale. In other embodiments, the reduction in the water content of the subject's stool using the present methods is demonstrated by a decrease of 3 points (e.g., 7 to 4, 6 to 3, 5 to 2, 4 to 1) using the Bristol Stool Form Scale. In other embodiments, the reduction in the water content of the subject's stool using the present methods is demonstrated by a decrease of more than 3 points using the Bristol Stool Form Scale.
It will be understood by those skilled in the art that the reduction in the water content of the subject's stool can be demonstrated using other clinical scales known the art.
In some aspects, the methods of the present invention result in a reduction in the subject's frequency of defecation. The extent of defecation frequency reduction can be determined by comparing the subject's defecation frequency before administering the CFTR chloride channel inhibitor to the subject's defection frequency after administering the CFTR chloride channel inhibitor. Here, defection frequency may be measured by, for example, interviewing the subject, or administering to the subject an instrument designed to elicit this information. Methods of determining defecation frequency are known to those in the art.
In other aspects, the methods of the present invention result in a reduction in the subject's stool output. The extent of stool output reduction can be determined by measuring the quantity of subject's stool before administering the CFTR chloride channel inhibitor, and comparing that amount to the quantity of the subject's stool after administering the CFTR chloride channel inhibitor. Here, the quantity may be measured by weight or by volume. Methods for measuring stool output are known to those in the art.
In other aspects, the methods of the present invention result in a reduction in the subject's abdominal pain. The extent of abdominal pain reduction can be determined by comparing the subject's abdominal pain before administering the CFTR chloride channel inhibitor with the subject's abdominal pain after administering the CFTR chloride channel inhibitor. Here, abdominal pain may be measured by, for example, interviewing the subject, or administering to the subject an instrument designed to elicit this information. Methods of determining abdominal pain are known to those in the art.
In other aspects, the methods of the present invention result in a reduction in the subject's abdominal bloating. The extent of abdominal bloating reduction can be determined by comparing the subject's abdominal bloating before administering the CFTR chloride channel inhibitor with the subject's abdominal bloating after administering the CFTR chloride channel inhibitor. Here, abdominal bloating may be measured by, for example, interviewing the subject, or administering to the subject an instrument designed to elicit this information. Methods of determining abdominal bloating are known to those in the art.
In other aspects, the methods of the present invention result in a reduction in the subject's nausea. The extent of nausea reduction can be determined by comparing the subject's nausea before administering the CFTR chloride channel inhibitor with the subject's nausea after administering the CFTR chloride channel inhibitor. Here, nausea may be measured by, for example, interviewing the subject, or administering to the subject an instrument designed to elicit this information, such as the Nausea Questionnaire. Methods of measuring nausea are known to those in the art.
The following examples are provided to provide a better understanding of the subject matter described herein. These examples should not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the scope of the present invention.
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
CFTRinh-172: 4-[[4-Oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid
DCA: deoxycholic acid
DRA: down-regulated in adenoma
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]glycinehydrazide
IBS-D: diarrhea-predominant irritable bowel syndrome
IC50: half-maximal inhibitory concentration
Unless specified otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.). Sodium salts of tauro-chenodeoxycholic acid and tauro-deoxycholic acid were purchased from Spectrum Chemicals (Gardena, Calif.). 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, Mass.). Forskolin was purchased from Lc Laboratories (Woburn, Mass.). CFTRinh-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.
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, N.Y.) at 37° C. in 5% CO2/95% air and used 7-14 days after plating.
Human colonoid cultures are generated from de-identified tissue samples (other than age and sex) obtained from endoscopic or surgical procedures from three different subjects. Human cultures are generated from isolated intestinal crypts embedded in Matrigel (Corning, Tewksbury, Mass.) in 24-well plates and cultured in the presence of Wnt3A, R-spondin-1 and Noggin containing undifferentiated media (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 media are used for the propagation of colonoids. To generate monolayer cultures, colonoids are triturated in Cultrex Organoid Harvesting Solution (Trevigen, Gaithersburg, Md.), and the 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, Mass.) pre-coated with human collagen IV (34 μg/mL; Millipore Sigma). Monolayers are cultured in UDM at 37° C., 5% CO2. Under these conditions, cultures reach confluency in 7-14 days as monitored by transepithelial resistance.
T84 cells are mounted in Ussing chambers and bathed in symmetrical HCO3−-buffered solution containing (in mM): 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2), 10 D-glucose, 5 HEPES and 25 NaHCO3 (pH 7.4). The solutions are aerated with 95% O2/5% CO2 and maintained at 37° C. In one study, for measurement of apical Cl− conductance, a basolateral to apical Cl− gradient is applied in which the basolateral hemichamber 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 g/ml amphotericin B. Short-circuit current is measured using an EVC4000 multichannel voltage clamp (World Precision Instruments, Sarasota, Fla.).
For intestinal short-circuit current measurement, CD1 mice are anesthetized with isoflurane. The colon is removed, washed with ice-cold Krebs buffer, opened along the mesenteric border, and a full-thickness fragment is mounted in a micro-Ussing chamber (area 0.7 cm2, World Precision Instruments). Hemichambers are filled with oxygenated Krebs-bicarbonate solution.
Measurements in human colonoid cultures are done as described (18). The apical and basolateral hemichambers are filled with Krebs-Ringer bicarbonate (KBR) buffer that is gassed with 95% O2/5% CO2 at 37° C. The basolateral hemichamber is supplemented with 10 mM glucose and the apical hemichamber is supplemented with 10 mM mannitol to maintain osmotic balance.
T84 cells are plated in 96-well black-walled microplates. Confluent cells are loaded with Fluo-4 NW (Invitrogen, Carlsbad, Calif.) at 72 h after plating. For Ca2+ measurement Fluo-4 fluorescence is measured with a FluoStar fluorescence plate reader (BMG Lab Technologies, Durham, N.C.) at excitation/emission wavelengths of 485/538 nm. In some studies cells are pretreated for 30 min with BAPTA-AM. Ca2+ measurement in human colonoid cultures is done in monolayers that are transduced with the fluorescent Ca2+ sensor Adeno-GCsMP6s. For cAMP assay, T84 cells are grown in 24-well plates, treated for 30 min with CDCA and/or forskolin, lysed by repeating freeze/thaw, centrifuged to remove cell debris, and the supernatant is assayed for cAMP using the Parameter cAMP immunoassay kit according to the manufacturer's instructions (R&D Systems, Minneapolis, N. Mex.).
CD1 mice (age 8-10 weeks) are given access to 5% dextrose water but not solid food for 24 h before experiments. Mice are administered (R)-BPO-27 (5 mg/kg) or vehicle (5% DMSO, 10% Kolliphor HS in saline) intraperitoneally 60 min before creation of closed intestinal (mid-jejunal or distal colonic) loops and injection of CDCA or PBS vehicle. This dose of (R)-BPO-27 was previously shown to give therapeutic serum levels for several hours in mice. 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 creation of loops, mice are anesthetized with isoflurane, and body temperature is maintained during surgery at 36-38° C. using a heating pad. A small abdominal incision is made to expose the intestine. For the mid-jejunal closed-loop model, 2-3 cm loops are created by sutures. Loops are injected with 100 μl PBS containing CDCA or PBS vehicle. The abdominal incision is closed with sutures, and mice are allowed to recover from anesthesia. In the colonic closed-loop model, mice are given an enema (500 μl of mineral oil) 12 hours before surgery to cleanse 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 1-2 cm closed loop is created by sutures. Intestinal loops are surgically removed at specified times, and loop length and weight is measured to quantify fluid secretion. In some experiments, CF mice (ΔF508 homozygous, age 8-10 weeks) are used.
Female Sprague-Dawley rats (age 8-10 weeks) are administered 500 μl CDCA (10 mM in PBS) or PBS vehicle by a mid-colonic infusion using a flexible plastic tube (15 gauge, 78 mm length, Instech Laboratories, Plymouth Meeting, Pa.). Rats are intraperitoneally administered (R)-BPO-27 (10 mg/kg) (or vehicle control) 30 min before infusion. (R)-BPO-27 is dissolved in saline containing 5% DMSO and 10% Kolliphor HS. Following the infusion, rats are placed individually in metabolic cages and given free access to water and food. Stool samples are collected prior to infusion (for baseline stool water content) and over 4 hours after the infusion. Stools are weighed immediately when visualized to determine wet weight. To determine stool water content, the stool samples are dried at 70° C. for 24 hours and water content is calculated as (wet weight−dry weight)/wet weight.
Data are presented as mean±S.E.M. Statistical analysis is performed using Prism 5 GraphPad Software package (San Diego, Calif.). Statistical comparisons are made using the-Student's t-test when there are two groups or one-way analysis of variance when there are three groups or more. A p value of <0.05 is taken as statistically significantly.
In initial studies multiple bile acids are screened by short-circuit current measurements for their ability to induce a prosecretory 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, produce a robust secretory current that is blocked by (R)-BPO-27 (
The increase in short-circuit current following CDCA application is fully blocked by the selective and chemically unrelated inhibitors (R)-BPO-27 and CFTRinh-172 (
To investigate the sidedness of CDCA action in T84 cells, CDCA is added to either the apical or basolateral bathing solutions.
Possible CDCA actions on cAMP and Ca2+ signaling pathways in T84 cells are investigated. See Example 4. CDCA up to 0.75 mM does not significantly affect the short-circuit response to addition of the cAMP agonist forskolin, but a small reduction in forskolin-induced current is seen with 1 mM CDCA (
The CDCA-induced increase in short-circuit current in T84 cells is largely blocked by pretreatment with the Ca2+ chelator BAPTA-AM (
The major findings obtained using T84 cells, including the CFTR Cl− secretory response to apical CDCA, and the involvement Ca2+ signaling, are investigated 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 but not basolateral bathing solution, which is reversed by (R)-BPO-27 (
A closed-intestinal loop model is established in mice to investigate the prosecretory action 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 a single animal and their extensive prior use in studying intestinal fluid secretion in models of cholera and Traveler's diarrhea.
To study effects of CFTR inhibition, (R)-BPO-27 is administered 60 min prior to creation of closed intestinal loops and injection of CDCA (or control PBS vehicle) (
Since CFTR inhibition largely but not completely blocks loop fluid accumulation, whether CFTR-independent mechanisms might in part be responsible from CDCA effect is investigated. For these experiments closed-loop studies are done in cystic fibrosis mice lacking functional CFTR. Mid-jejunal and distal colonic closed loops injected with 10 mM CDCA show a small but significantly greater loop fluid content compared to control loops injected with PBS vehicle, suggesting mechanisms other than CFTR also playing a role in CDCA-induced fluid accumulation, which based on prior studies might include inhibition of Na+/H+ and Cl−/HCO3− exchangers (pro-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 Ca2+ 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
A model is established to test (R)-BPO-27 effect in which CDCA (or vehicle control) is administered to rats by a mid-colonic infusion, with stool collected over 4 hours for measurement of stool weight and water content (by wet-to-dry weight ratio) (
Without being bound by theory, the data herein support of the utility of CFTR inhibition by CFTR inhibitors, particularly (R)-BPO-27, in the treatment of bile acid-associated diarrheas. Short-circuit current measurements in T84 cells and human colonoid cultures show a prosecretory current in response to exposure of the cell apical surface to CDCA, which is fully reversed by CFTR inhibition. Complete inhibition of secretory current by (R)-BPO-27 is also seen in CDCA-exposed mouse colonic tissue ex vivo. Systemically administered (R)-BPO-27 largely prevents fluid accumulation in closed colonic loops in mice in vivo, and prevents the increase in stool water following intracolonic infusion of a CDCA-containing solution. These results implicate CFTR as a major prosecretory ion channel in CDCA-induced diarrhea in human colonic cell cultures and rodents.
This application claims the benefit of priority to U.S. Provisional Application No. 62/860,539 filed Jun. 12, 2019, the entirety of which is incorporated by reference herein.
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 this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/036683 | 6/8/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62860539 | Jun 2019 | US |
