(12) Patent Application Publication (10) Pub. No.: US 2016/0184387 A1 Charmot Et Al

US 2016O184387A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0184387 A1 Charmot et al. (43) Pub. Date: Jun. 30, 2016

(54) COMPOUNDS AND METHODS FOR A 6LX3 L/7072 (2006.01) INHIBITING PHOSPHATE TRANSPORT A613 L/4525 (2006.01) A63L/382 (2006.01) (71) Applicants: Dominique CHARMOT, Campbell, CA A63L/27 (2006.01) (US); Jason G. LEWIS, Castro Valley, A613 L/42 (2006.01) CA (US); Jeffrey W. JACOBS, San A613 L/353 (2006.01) Mateo, CA (US); Ingrid A613 L/4439 (2006.01) LANGSETMO, Fremont, CA (US); A613 L/4045 (2006.01) Christopher RERAS, Belmont, CA A633/26 (2006.01) (US) A613 L/475 (2006.01) A613 L/352 (2006.01) (72) Inventors: Dominique Charmot, Campbell, CA A 6LX3L/95 (2006.01) (US); Jason G. Lewis, Castro Valley, CA A6II 45/06 (2006.01) (US); Jeffrey W. Jacobs, San Mateo, CA GOIN33/50 (2006.01) (US); Ingrid Langsetmo, Fremont, CA GOIN33/84 (2006.01) (US); Christopher Carreras, Belmont, A613 L/506 (2006.01) CA (US) (52) U.S. Cl. CPC ...... A61K 38/10 (2013.01); A61 K3I/506 (21) Appl. No.: 14/911,225 (2013.01); A61 K3I/4418 (2013.01); A61 K (22) PCT Filed: Aug. 8, 2014 31/7072 (2013.01);ER 2. A61 (2013.01); K31/4525 (2013.01);R E; (2013.01); A61 K3I/421 (2013.01); A61 K (86). PCT No.: PCT/US2O14/OSO290 3 1/353 (2013.01); A61 K3I/4439 (2013.01): S371 (c)(1), A6 IK3I/4045 (2013.01); A61K33/26 (2) Date: Feb. 9, 2016 (2013.01); A61 K3I/475 (2013.01); A61 K 31/352 (2013.01); A61K 31/195 (2013.01); Related U.S. Application Data A61K 45/06 (2013.01); G0IN33/5044 (60) Provisional application No. 61/936,715, filed on Feb. (2013.01); G0IN33/5038 (2013.01); G0IN 6, 2014, provisional application No. 61/864,215, filed 33/84 (2013.01) on Aug. 9, 2013. (57) ABSTRACT Publication Classification Provided are non-NHE3-binding agents having activity as phosphate transport/uptake inhibitors in the gastrointestinal (51) Int. Cl. tract, including in the Small intestine, methods for their use as A6 IK38/10 (2006.01) therapeutic or prophylactic agents, and related methods of A6 IK3I/448 (2006.01) drug discovery. Patent Application Publication Jun. 30, 2016 Sheet 1 of 55 US 2016/O184387 A1

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COMPOUNDS AND METHODS FOR levels is required during therapy with calcium-based phos INHIBITING PHOSPHATE TRANSPORT phate binders. Other calcium and aluminum-free phosphate binders. Such as sevelamer, a crosslinked polyamine polymer, CROSS-REFERENCE TO RELATED have drawbacks that include the amount and frequency of APPLICATIONS dosing required to be therapeutically active. The relatively 0001. This application claims priority under 35 U.S.C. modest phosphate binding capacity of those drugs in vivo S119(e) to U.S. Application No. 61/864,215, filed Aug. 9, obliges patients to escalate the dose (up to 7 grams per day or 2013; and U.S. Application No. 61/936,715, filed on Feb. 6, more). Such quantities have been shown to produce gas 2014: each of which is incorporated by reference in its trointestinal discomfort, such as dyspepsia, abdominal pain entirety. and, in some extreme cases, bowel perforation. 0008. An alternative approach to the prevention of phos STATEMENT REGARDING SEQUENCE phate absorption from the intestine in patients with elevated LISTING phosphate serum levels is through inhibition of the intestinal 0002 The Sequence Listing associated with this applica transport system which mediates phosphate uptake in the tion is provided in text format in lieu of a paper copy, and is intestine. It is understood that phosphate absorption in the hereby incorporated by reference into the specification. The upper intestine is mediated at least in part by a carrier-medi name of the text file containing the Sequence Listing is ated mechanism which couples the absorption of phosphate ARDE 017 01WO ST25.txt. The text file is 193 KB, was to that of sodium Inhibition of intestinal phosphate transport created on Aug. 8, 2014, and is being Submitted electronically will reduce body phosphorus overload. In patients with Via EFS-Web. advanced kidney disease (e.g. stage 4 and 5), the body phos phorus overload manifests itself by serum phosphorus con BACKGROUND centration above normal levels, i.e., hyperphosphatemia. Hyperphosphatemia is directly related to mortality and mor 0003 1. Technical Field bidity Inhibition of intestinal phosphate transport will reduce 0004. The present invention relates to non-NHE3-binding serum phosphorus concentration and therefore improve out agents having activity as phosphate transport inhibitors in the come in those patients. In chronic kidney disease patients at gastrointestinal tract, including in the Small intestine, meth stage 2 or 3, the body phosphorus overload does not neces ods for their use as therapeutic or prophylactic agents, and sarily lead to hyperphosphatemia, i.e., Some patients remain related methods of drug discovery. normophosphatemic, but there is a need to reduce or prevent 0005 2. Description of the Related Art body phosphorus overload even at those early stages to avoid 0006 Patients with inadequate renal function, hypopar associated bone and vascular disorders, and ultimately athyroidism, or certain other medical conditions (such as improve mortality rate. Similarly, inhibition of intestinal hereditary hyperphosphatemia, Albrighthereditary osteodys phosphate transport would be particularly advantageous in trophy, amyloidosis, etc.) often have hyperphosphatemia, or patients that have a disease that is treatable by inhibiting the elevated serum phosphorus levels (where the level, for uptake of phosphate from the intestines. Furthermore, inhi example, is more than about 6 mg/dL). Hyperphosphatemia, bition of phosphate transport may slow the progression of especially if present over extended periods of time, leads to renal failure and reduce risk of cardiovascular events. severe abnormalities in calcium and phosphorus metabolism, often manifested by secondary hyperparathyroidism, bone 0009. The luminal pole of the intestinal epithelia com disease and ectopic calcification in the cardiovascular system, prises a so-called unstirred water layer (UWL) where trans joints, lungs, eyes and other soft tissues. Higher serum phos port is essentially of diffusive nature because of the viscosity phorus levels are strongly associated with the progression of of the mucus layer. This unstirred layer is defined as a stag renal failure, cardiovascular calcification and mortality in nant layer adjacent to the membrane on the apical side acting end-stage renal disease (ESRD) patients. High-normal serum as a diffusion barrier so that rapidly permeating Substances phosphorus levels have been associated with cardiovascular could actually be rate-limited by diffusion. This limited dif events and mortality among individuals who have chronic fusion applies to H' and therefore the UWL contributes to kidney disease (CKD) and among those who have normal establishing a pH microclimate due to the outward flux of kidney function (see, e.g., Joy et al., J. Manag. Care Pharm., proton and the diffusion limit imposed by the mucus layer. 13:397-411, 2007) The progression of kidney disease can be The acidic environment at the vicinity of the cell surface slowed by reducing phosphate retention. Thus, for renal fail maintains a relatively large electrochemical gradient across ure patients who are hyperphosphatemic and for chronic kid the epithelial membrane—a cross epithelial pH gradient, or ney disease patients who have serum phosphorus levels CEPG. within the normal range or only slightly elevated, therapy to 0010 Strong evidence exists for the involvement of this reduce phosphate retention is beneficial. CEPG in the transport of nutrients via proton co-transporters 0007 For patients who experience hyperphosphatemia, and —OH antiporters, such as PEPT1, folate/OH anti calcium salts have been widely used to bind intestinal phos porter, and B-alanine/H+ cotransporter. See, e.g., Ikuma, J phate and prevent its absorption. Different types of calcium Med Chem. 50: 1166-1176, 1996. The disturbance of the pH salts, including calcium carbonate, acetate, citrate, alginate, microclimate, for example, a decrease of the CEPG, can alter and ketoacid salts have been utilized for phosphate binding. the absorption of nutrients. This has been shown in the case of However, these therapies often cause hypercalcemia, a con proton-mediated absorption of peptide via PEPT1. See, e.g., dition which results from absorption of high amounts of Thwaites et al., Gastroenterology. 122:1322-1333, 2002; and ingested calcium. Hypercalcemia causes serious side effects Thwaites and Anderson, Exp. Physiol. 92:603-619, 2007. Such as cardiac arrhythmias, renal failure, and skin and vas However, no role for the CEPG has been established in the cular calcification. Frequent monitoring of serum calcium absorption of phosphate ions across the intestinal membrane. US 2016/O 184387 A1 Jun. 30, 2016

0.011 Evidence also exists for the involvement of water 0018. In some embodiments, the method results in a absorption in the transport of ions across the epithelia of the method selected from one or more of: Small intestine particularly the jejenum. Juan et al., J. Clin 0019 (a) a method for treating hyperphosphatemia, Endocrinol Metab. 43:517-22, 1976. But such mechanisms optionally postprandial hyperphosphatemia; have been little-explored in the area of phosphate-lowering 0020 (b) a method for treating a renal disease, optionally therapeutics. chronic kidney disease (CKD) or end-stage renal disease (ESRD); 0021 (c) a method for reducing serum creatinine levels; BRIEF SUMMARY 0022 (d) a method for treating proteinuria; 0012. The present invention relates generally to non 0023 (e) a method for delaying time to renal replacement NHE3-binding compounds having activity as phosphate therapy (RRT), optionally dialysis: transport inhibitors in the gastrointestinal tract, especially in (0024 (f) a method for reducing FGF23 levels: the Small intestine, including stereoisomers, pharmaceuti 0025 (g) a method for reducing the hyperphosphatemic cally acceptable salts and prodrugs thereof, and the use of effect of active vitamin D; Such compounds to inhibit phosphate uptake and to thereby 0026 (h) a method for attenuating hyperparathyroidism, treat any of a variety of conditions or diseases in which optionally secondary hyperparathyroidism; modulation of phosphate uptake provides a therapeutic ben 0027 (i) a method for reducing serum parathyroid hor mone (PTH) efit. 0028 (i) a method for improving endothelial dysfunction, 0013 Embodiments of the present invention thus include optionally induced by postprandial serum phosphorus; methods for inhibiting phosphate uptake in the gastrointesti 0029 (k) a method for reducing vascular calcification, nal tract of a patient in need of phosphate lowering, compris optionally intima-localized vascular calcification; ing administering to the patient a compound that does not 0030 (1) a method for reducing urinary phosphorous; bind NHE3, where the compound is substantially active in the 0031 (m) a method for normalizing serum phosphorus gastrointestinal tract to inhibit transport of phosphate ions levels; (Pi) therein upon administration to the patient in need thereof. 0032 (n) a method for reducing phosphate burden in an 0014. In specific embodiments, the compound is a guany elderly patient; late cyclase C receptor (GC-C) agonist compound. 0033 (o) a method for decreasing dietary phosphate 0015. In certain embodiments, the compounds are pH uptake; modulatory agents. These and related embodiments include 0034 (p) a method for reducing renal hypertrophy; and methods for inhibiting phosphate uptake in the gastrointesti 0035 (q) a method for reducing heart hypertrophy. nal tract of a patient in need of phosphate lowering, compris 0036. In certain embodiments, the compound decreases ing administering to the patient a compound that decreases the intracellular pH of the epithelial cells of the surface of the the cross-epithelial pH gradient (CEPG) in the small intes Small intestine, optionally at the Subapical Surface of the tine, where the CEPG is defined as the difference in pH epithelial cell. In certain embodiments, the compound between (i) the cytoplasm of the epithelial cells of the surface increases the pH of the unstirred layer at the apical surface of of the Small intestine, optionally at the Subapical Surface of the Small intestine. In some embodiments, the compound (a) the epithelial cell, and (ii) the unstirred layer at the apical stimulates bicarbonate secretion in the small intestine, or (b) Surface of the Small intestine, where the compound is Sub inhibits acid secretion in the Small intestine, or (c) stimulates stantially active in the gastrointestinal tract to inhibit trans bicarbonate secretion and inhibits acid secretion in the small port of phosphate ions (Pi) therein upon administration to the intestine. patient in need thereof, and where the compound does not 0037. In certain embodiments, the compound increases bind NHE3. one or more intracellular secondary messengers of epithelial cells of the surface of the small intestine. In some embodi 0016. In some embodiments, the compounds reduce water ments, the one or more intracellular secondary messengers absorption in the Small intestine, optionally the jejunum. are selected from Ca", cyclic adenosine monophosphate These and related embodiments include methods for inhibit (cAMP), and cyclic guanosine monophosphate (cGMP). ing phosphate uptake in the gastrointestinal tract of a patient 0038. In certain embodiments, the compound is substan in need of phosphate lowering, comprising administering to tially systemically non-bioavailable upon enteral administra the patient a compound that decreases water absorption in the tion to the patient. In particular embodiments, the compound Small intestine, optionally the jejunum, where the compound is substantially impermeable to the epithelium of the gas does not bind NHE3, and where the compound is substan trointestinal tract. In some embodiments, the compound is tially active in the gastrointestinal tract to inhibit transport of substantially permeable to the epithelium of the gastrointes phosphate ions (Pi) therein upon administration to the patient tinal tract. in need thereof. 0039. In certain embodiments, administration to the 0017. In some embodiments, the compound decreases the patient in need thereof (a) reduces serum phosphorus concen CEPG in the small intestine and also decreases water absorp trations or levels to about 150% or less of normal serum tion in the Small intestine. In some embodiments, the com phosphorus levels, and/or (b) reduces uptake of dietary phos pound decreases the CEPG in the small intestine without phorous by at least about 10% relative to an untreated state. In significantly decreasing water absorption in the Small intes Some embodiments, administration to the patient in need tine. In other embodiments, the compound decreases water thereof increases phosphate levels in fecal excretion by at absorption in the Small intestine without significantly least about 10% relative to an untreated state. In some decreasing the CEPG in the small intestine (e.g., without embodiments, administration to the patient in need thereof significantly stimulating bicarbonate secretion and/or inhib reduces urinary phosphate concentrations or levels by at least iting acid secretion). about 10% relative to an untreated state. US 2016/O 184387 A1 Jun. 30, 2016

0040. In some embodiments, the patient in need thereof CyS Xaas Xaa, Xaa-7 Xaas Xaao Xaao Xaa11 CyS12 Xaas has ESRD, and administration to the patient reduces serum Xaa, Xaas Xaa, (SEQID NO:5), where Xaa is: Ser, Asn. phosphorus concentrations or levels by at least about 10% Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is missing: Xaa is His, relative to an untreated State. Asp, Glu, Ala, Ser, Asn., Gly, or is missing: Xaa is Thr, Asp, 0041. In some embodiments, the patient in need thereof Ser, Glu, Pro, Val or Leu; Xaas is Asp, Ile or Glu; Xaa is Ile, has CKD, and administration to the patient reduces FGF23 Trp or Leu; Xaa, is Cys, Ser, or Tyr; Xaas is Ala, Val, Thr, Ile, levels and serum intact parathyroid hormone (iPTH) levels by Metor is missing: Xaa, is Phe, Tyr, ASn, or Trp. Xaao is Ala, at least about 10% relative to an untreated state. Val, Met, Thr or Ile: Xaa is Ala or Val: Xaa is Thr or Ala; 0042. In certain embodiments, the compound is selected Xaa is Gly, Ala or Ser; Xaas is Cys, Tyr or is missing; and from one or more of a guanylate cyclase C receptor (GC-C) Xaa is His, Leu or Ser. agonist, a P2Yagonist, an adenosine A2b receptoragonist, a 0061. In some embodiments, the peptide comprises the soluble guanylate cyclase agonist, an adenylate cyclase amino acid sequence: ASn Asp Glu CyS Glu Leu CyS Val ASn receptoragonist, an imidazoline-1 receptoragonist, a cholin Val Ala Cys Thr Gly Cys Leu (SEQ ID NO:6), or a variant ergicagonist, a prostaglandin EP4 receptoragonist, a dopam thereof having 1, 2, 3, 4, or 5 deletions, insertions, and/or ine D1 agonist, a melatonin receptoragonist, a 5HT4agonist, Substitutions. an atrial natriuretic peptide receptoragonist, a carbonic anhy 0062. In certain embodiments, the P2Yagonist is selected drase inhibitor, a phosphodiesterase inhibitor, and a Down from a compound in FIG. 4 or FIGS. 5A-5C. In certain Regulated in Adenoma (DRA or SLC26A3) agonist. embodiments, the adenosine A2b receptoragonist is selected 0043. In some embodiments, the GC-C agonist is a pep from a compound in FIGS. 6A-6C. In some embodiments, the tide, optionally a bacterial heat stable enterotoxin, guanylin, soluble guanylate cyclase agonist is selected from a com proguanylin, uroguanylin, prouroguanylin, lymphoguanylin, pound in FIGS. 9A-9L. In certain embodiments, the adeny or a variant or analog of any of the foregoing. late cyclase receptor agonist is selected from a compound in 0044. In some embodiments, the GC-C agonist peptide FIG. 10. In some embodiments, the imidazoline-1 receptor comprises the amino acid sequence (I): Xaa, Xaa, Xaa, Xaa, agonist is selected from moXonidine and a compound in FIG. Xaas CyS CyS7 Xaas Xaao CySo CyS Xaa12 Xaa, Xaa1a. 11. In certain embodiments, the cholinergic agonist is Cysis Xaa, Xaa, Cysis Xaao Xaao Xaa-1 (SEQID NO:1) selected from a compound in FIG. 12. In particular embodi where: Xaa, Xaa, Xaa, Xaa, Xaas is ASn Ser Ser ASn Tyr ments, the prostaglandin EP4 receptor agonist is selected (SEQ ID NO:2) or is missing or Xaa, Xaa, Xaa, Xaa, is from PGE or its analogs/derivatives and a compound in FIG. missing. 7 or FIG. 13. In certain embodiments, the dopamine D1 0045. In certain embodiments, Xaas is ASn, Trp, Tyr, Asp, agonist is selected from a compound in FIG. 14. In some or Phe. embodiments, the melatonin receptoragonist is selected from 0046) In certain embodiments, Xaas is Thr or Ile. melatonin and a compound in FIG. 15. In some embodiments, 0047. In certain embodiments, Xaas is Tyr, Asp, or Trp. the 5HT4 agonist is selected from serotonin and its analogs, 0048. In certain embodiments, Xaas is Glu, Asp, Gln, Gly, prucalopride, metoclopramide, cleobopride, mosapride, pru or Pro. calopride, renzapride, tegaserod, Zacopride, norcisapride, 0049. In certain embodiments, Xaa, is Leu, Ile, Val, Ala, naronopride, and Velusetrag. Lys, Arg, Trp, Tyr, or Phe. 0063. In some embodiments, the atrial natriuretic peptide 0050. In certain embodiments, Xaa, is Leu, Ile, Val, Lys, receptor agonist comprises or consists of an amino acid Arg, Trp, Tyr, or Phe. sequence selected from: Ser Leu Arg Arg Ser Ser Cys Phe Gly 0051. In certain embodiments, Xaa, is ASn, Tyr, Asp, or Gly Arg Ile Asp Arg Ile-Gly Ala Glin Ser Gly Leu Gly Cys Asn Ala. Ser Phe Arg Tyr (SEQID NO:7), Cys Phe Gly Gly Arg Ile Asp 0052. In certain embodiments, Xaa, is Ala, Pro, or Gly. Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys (SEQID NO:8) and 0053. In certain embodiments, Xaa, is Ala, Leu, Ser, Gly, Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp. Gly Leu Gly Cys Asn Ser Phe Arg (SEQID NO:9), including 0054) In certain embodiments, Xaa is Thr, Ala, Asn. Lys, variants thereof having 1, 2, 3, 4, or 5 deletions, insertions, Arg, or Trp. and/or Substitutions. 0055. In certain embodiments, Xaa, is Gly, Pro, or Ala. 0064. In certain embodiments, the carbonic anhydrase 0056. In certain embodiments, Xaa, is Trp, Tyr, Phe, ASn, inhibitor is selected from a compound in FIG. 17. In certain or Leu. embodiments, the phosphodiesterase inhibitor is selected 0057. In certain embodiments, Xaa, is Lys or Arg. from a compound in FIG. 18. In some embodiments, the DRA 0058. In certain embodiments, Xaao Xaa, is Asplphe or agonist is selected from FIGS. 21A-B. Xaao is ASnor Glu and Xaa is missing. In certain embodi 0065. In some embodiments, the compound is substan ments, Xaao Xaao Xaa- is missing. tially systemically non-bioavailable upon enteral administra 0059. In specific embodiments, the GC-C agonist peptide tion to the patient and has (i) atPSA of at least about 200 A. comprises the amino acid sequence: ASn Ser Ser ASn Tyr Cys In certain embodiments, the compound has a tRSA of at least Cys Glu Tyr Cys Cys ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ about 250A, a tBSA of at least about 270 A, a tRSA of at ID NO:3), or a variant thereofhaving 1, 2, 3, 4, or 5 deletions, least about 300 A, a tFSA of at least about 350 A, a tFSA of insertions, and/or Substitutions. In particular embodiments, at least about 400 A, or a tBSA of at least about 500 A. In the peptide comprises the amino acid sequence: CyS CyS Glu particular embodiments, the compound has a molecular Tyr Cys Cys ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID weight of at least about 500 Da, at least about 1000 Da, at least NO:4), or a variant thereof having 1, 2, 3, 4, or 5 deletions, about 2500 Da, or at least about 5000 Da or more. In some insertions, and/or substitutions. embodiments, the compound has (i) a total number of NH 0060. In certain embodiments, the GC-C agonist peptide and/or OH and/or other potential hydrogen bond donor moi comprises the amino acid sequence (III): Xaa, Xaa, Xaa eties greater than about 5; (ii) a total number of O atoms US 2016/O 184387 A1 Jun. 30, 2016

and/or Natoms and/or other potential hydrogen bond accep cells under conditions Sufficient to form organoids. In some tors greater than about 10; and/or (iii) a Moriguchi partition embodiments, step (a) comprises culturing intestinal section coefficient greater than about 10 or less than about 10. In (s) in a Ussing chamber. Some embodiments, the compound has a permeability coef 0074. In certain embodiments, step (c)(i) comprises con ficient, P., of less than about 100x10 cm/s, or less than tacting the cells with a pH-sensitive fluorescent dye and mea about 10x10 cm/s, or less than about 1x10 cm/s, or less Suring fluorescence of the dye. In some embodiments, step than about 0.1x10 cm/s. (c)(ii) comprises contacting the cells with P-labeled phos 0066 Certain methods further comprise administering phate ions and measuring uptake of the labeled phosphate one or more additional biologically active agents. In some 1O.S. embodiments, the compound and the one or more additional 0075. In some embodiments, the increase and/or decrease biologically active agents are administered as part of a single of (d) is statistically significant. pharmaceutical composition. In certain embodiments, the 0076. In certain embodiments, the test compound is a compound and the one or more additional biologically active Small molecule or peptide that is known or Suspected to agents are administered as individual pharmaceutical compo stimulate bicarbonate secretion and/or inhibit acid secretion sitions. In some embodiments, the individual pharmaceutical in the Small intestine. compositions are administered sequentially. In some embodi 0077. In certain embodiments, the test compound is ments, the individual pharmaceutical compositions are selected from one or more of a P2Yagonist, an adenosine A2b administered simultaneously. receptor agonist, a guanylate cyclase C receptor agonist, a soluble guanylate cyclase agonist, an adenylate cyclase 0067. In certain embodiments, the additional biologically receptoragonist, an imidazoline-1 receptoragonist, a cholin active agent is selected from vitamin D (ergocalciferol), ergic agonist, a prostaglandin EP4 receptoragonist, a dopam vitamin D (cholecalciferol), active vitamin D (calcitriol) and ine D1 agonist, a melatonin receptoragonist, a 5HT4agonist, active vitamin Danalogs (e.g. doxercalciferol, paricalcitol). an atrial natriuretic peptide receptoragonist, a carbonic anhy 0068. In certain embodiments, the additional biologically drase inhibitor, a phosphodiesterase inhibitor, and a Down active agent is a phosphate binder. In some embodiments, the Regulated in Adenoma (DRA or SLC26A3) agonist, as phosphate binder is selected from the group consisting of described herein and/or known in the art. sevelamer (e.g., Renvela R. (sevelamer carbonate), Renagel(R) 0078. These and other aspects of the invention will be (sevelamer hydrochloride)), lanthanum carbonate (e.g., Fos apparent upon reference to the following detailed description. renolR), calcium carbonate (e.g., Calcichew(R), TitralacR), calcium acetate (e.g. PhosLoR), PhoseXR), calcium acetate/ BRIEF DESCRIPTION OF THE DRAWINGS magnesium carbonate (e.g., Renepho(R), OsvaRenR), MCI 196, ferric citrate (e.g., ZerenexTM) magnesium iron hydroxy (0079 FIGS. 1A-1B shows that linaclotide (a GC-C recep carbonate (e.g., FermagateTM), aluminum hydroxide (e.g., tor agonist) reduces the uptake of phosphate uptake in the Alucaps(R), Basaliel R), APS1585, SBR-759, and PA-21. gastrointestinal tract of rats. 0080 FIGS. 2A-2B show that moxonidine (an imidazo 0069. In certain embodiments, the additional biologically line Subtype 1 (I) receptor agonist) and the water Soluble active agent is a NaPi2b inhibitor. In some embodiments, the forskolin analog colforsin (an adenylate cyclase agonist) additional biologically active agent is niacin or nicotinamide reduce the uptake of phosphate uptake in the gastrointestinal 0070. In certain embodiments, the subject has CKD and tract of rats. the additionally active biological agentis selected from one or I0081 FIG. 3 shows that the P2Y2 receptoragonist Up.U more of ACE inhibitors, antiogensin II receptor blockers, reduces the uptake of phosphate uptake in the gastrointestinal beta-blockers, calcium channel blockers, direct renin inhibi tract of rats. tors, diuretics, vasodilators, erythropoietin therapy, iron I0082 FIG. 4 shows exemplary small molecule P2Y recep replacement therapy, inhibitors of advanced glycation end toragonists. products, vitamin D, and statins. I0083 FIGS.5A-5C show exemplary small molecule P2Y 0071. In certain embodiments, the compound or compo receptor agonists. sition is administered orally, optionally where the compound I0084 FIGS. 6A-6C show exemplary small molecule or composition is administered orally once-a-day. adenosine A2b receptor agonist, including representative 0072 Also included are methods of screening for an adenosine-like A2b agonists (6B) and representative dicyan inhibitor of phosphate uptake, comprising (a) culturing intes opyridine A2b agonists (6C). tinal cells, (b) contacting the cultured intestinal cells with a I0085 FIG. 7 shows a list of exemplary prostaglandin EP4 test compound, and (c) measuring (i) the pH at the apical receptor agonists. surface of the intestinal cells, (ii) the intracellular pH of the I0086 FIGS. 8A-8B show the photophysical properties of intestinal cells, and/or (iii) phosphate uptake by the intestinal exemplary near-neutral pH indicators (8A) and acidic pH cells, and (d) identifying the test compound as an inhibitor of indicators (8B). phosphate uptake where the pH from (c)(i) increases relative I0087 FIGS. 9A-9L show exemplary soluble guanylate to a control, the intracellular pH from (c)(ii) decreases relative cyclase (sGC) agonists, including heme-dependent and to a control, and/or phosphate uptake from (c)(iii) decreases heme-independentagonists (9A). relative to a control. I0088 FIG. 10 shows exemplary adenylate cyclase recep 0073. In some embodiments, step (a) comprise culturing toragonists. intestinal cells to monolayers. In certain embodiments, step I0089 FIG. 11 shows exemplary imidazoline receptorago (a) comprises isolating the cells from intestinal crypts and nists. culturing under conditions sufficient to form enteroids. In 0090 FIG. 12 shows exemplary cholinergic agonists and certain embodiments, step (a) comprises culturing isolated the antagonists atropine and (-)-hyosine. embryonic stem cells, endoderm cells, or pluripotent stem 0091 FIG. 13 shows exemplary EP4 receptor agonists. US 2016/O 184387 A1 Jun. 30, 2016

0092 FIG. 14 shows exemplary dopamine D1 receptor Piuptake is a general phenomenon not necessarily linked to a agonists. specific phosphate transporter. Pit-1 and Pit-2 transport the 0093 FIG. 15 shows exemplary melatonin (MT2) recep monobasic form of phosphate NaH2PO whereas NaPi2b toragonists. transports the dibasic form NaHPO.'. The observation that 0094 FIG. 16 shows the structures of exemplary peptide the cell acidification affects phosphate uptake with both trans agonists (SEQID Nos. 7, 8 and 9) of the NP receptor(s). porters is inconsistent with a mechanism based on a change in 0095 FIG. 17 shows exemplary carbonic anhydrase the H electrochemical gradient alone. inhibitors. 0.107 These observations are counterintuitive in the least 0096 FIG. 18 shows exemplary phosphodiesterase inhibi because an increase in Pi uptake could have been expected. tOrS. For example, a decrease in intracellular pH (e.g., without any 0097 FIG. 19 illustrates the pH gradients found in the corresponding change in the extracellular pH) could have intestine, including the pH gradient across the cell membrane, been expected to create a driving force for the uptake of basic and the pH gradient at the immediate vicinity of the epithelial anions such as the dibasic form of phosphate (NaPO). membrane and the gut lumen. 0108) Nonetheless, a reduction in phosphate uptake was 0098 FIG. 20 shows a phase diagram of the solubility observed, presenting the potential of using direct or indirect calcium and phosphate ions in an aqueous environment (at pH-modulatory agents, particularly those having activity as RT) over a range of pH values. pH-modulatory agents in the gastrointestinal tract (e.g., Small 0099 FIGS. 21A-21B depict representative examples of intestine), to reduce phosphate uptake in a patient in need of subtype selective PKC inhibitors. phosphate lowering. This potential is Supported by the obser 0100 FIGS. 22A-22C show that acidification of the inte Vation that a variety of pH-modulatory agents are capable of rior of HEK-293 cells led to a significant reduction in phos reducing phosphate uptake in the mammalian gastrointestinal phate uptake, as measured by uptake of Plabeled Pi. tract (see the accompanying Examples). The term "pH-modu latory agents, as used herein, includes agents or compounds DETAILED DESCRIPTION that are capable of directly or indirectly increasing bicarbon 0101. In the following description, certain specific details ate (HCO) secretion and/or decreasing acid/proton (e.g., are set forth in order to provide a thorough understanding of H') secretion into the lumen of the gastrointestinal tract, for various embodiments of the invention. However, one skilled example, the Small intestine or duodenum. Some pH-modu in the art will understand that the invention may be practiced latory compounds may act, for example, by modulating (e.g., without these details. increasing) certain intracellular secondary messengers of epi 0102 Unless the context requires otherwise, throughout thelial cells of the gastrointestinal tract, such as Ca", cAMP. the present specification and claims, the word “comprise' and cGMP, and others. Some exemplary compounds thus either variations thereof. Such as, "comprises' and "comprising are directly or indirectly stimulate bicarbonate secretion into the to be construed in an open, inclusive sense, that is, as “includ lumen of the small intestine, inhibit acid secretion into the ing, but not limited to”. lumen of the Small intestine, or stimulate bicarbonate secre 0103 Reference throughout this specification to “one tion and inhibit acid secretion into the lumen of the small embodiment' or “an embodiment’ means that a particular intestine. In some aspects, the compound decreases the cyto feature, structure or characteristic described in connection plasmic or intracellular pH of the epithelial cells of the sur with the embodiment is included in at least one embodiment face of the Small intestine, optionally at the Subapical Surface of the present invention. Thus, the appearances of the phrases of the epithelial cell, without or without modulating the pH of “in one embodiment' or “in an embodiment” in various the adjacent extracellular environment. In certain embodi places throughout this specification are not necessarily all ments, the compound does not bind to and inhibit the sodium referring to the same embodiment. Furthermore, the particu hydrogen antiporter 3 (NHE3). lar features, structures, or characteristics may be combined in 0109. In some aspects, the compound decreases the pH of any Suitable manner in one or more embodiments. the “unstirred layer at the apical surface of the small intes 0104 Embodiments of the present invention relate gener tine. The “unstirred layer” refers to a stagnant layer adjacent ally to the discovery that non-NHE3-binding compounds, to the membrane on the apical side (e.g., about 600 um deep) Such as guanylate cyclase agonist compounds, are able to which acts as a diffusion barrier so that rapidly permeating inhibit phosphate uptake in the gastrointestinal tract, for substances (e.g., "H") can be rate-limited by diffusion. With example, in the Small intestine. out wishing to be bound to theory, such an approach would 0105. According to one non-limiting theory, the cellular elicit a flux of bicarbonate across the epithelial cells of the uptake of phosphate ions (Pi) can be influenced by changes to gastrointestinal tract, increase the pH in immediate vicinity of intracellular pH and/or the pH of the adjacent extracellular the cell exterior (UWL), and therefore decrease the pH gra environment. For instance, as shown in the accompanying dient at the mucosal Surface. Because of the continuous Examples, acidification of the cell interior of Human Embry exchange of proton and bicarbonate ions at the apical Surface onic Kidney (HEK-293) cells (while maintaining the extra of the intestinal cells via co-transporters, antiporters and cellular pH at about 7.4) led to a significant reduction in channels, a pH gradient is maintained across the cell mem phosphate uptake, as measured by uptake of Plabeled Pi. brane. As a result of the unstirred layer, another pH gradient 0106. In related experiments, where the phosphate trans is established between the immediate vicinity of the epithelial porter NaPi2b (SLC34A2) was transiently expressed in membrane and the gut lumen. The two pH gradients are HEK-293 cells, the same phenomenon was observed. represented schematically in FIG. 19. Because the endogenous Pi transporters, Pit-1 and/or Pit-2 0110. Accordingly, in some aspects, a compound (SLC20A2) are responsible for Piuptake in non-transformed decreases the cross-epithelial pH gradient (CEPG) in the HEK-293 cells (to satisfy cell metabolic demands), it was gastrointestinal tract. The term “CEPG’ includes the differ concluded that the effect of a decrease in intracellular pH on ence in pH between (i) the cytoplasm of the epithelial cells of US 2016/O 184387 A1 Jun. 30, 2016

the surface of the small intestine (i.e., the intracellular pH), (iii) specific numbers of NH and/or OH and/or potentially optionally at the subapical surface of the epithelial cell, and hydrogen bond donor moieties (e.g., greater than about five); (ii) the unstirred layer at the apical surface of the small intes (iv) specific numbers of rotatable bonds (e.g., greater than tine. Certain embodiments exclude compounds (e.g., antac about five); (iv) specific permeability features (e.g., P. less ids) that merely increase the luminal pH of the gastrointesti than about 100x10 cm/s); and/or any of a number of other nal tract without modulating bicarbonate and/or acid features and characteristics as described herein. secretion or without altering the pH in the unstirred layer or 0.115. In patients with advanced kidney disease (e.g. stage UWL 4 and 5), the body phosphorus overload manifests itself by 0111. In some embodiments, and without wishing to be serum phosphorus concentration above normal levels, i.e., bound by any one theory, intraluminal free calcium ions may hyperphosphatemia. Hyperphosphatemia is directly related contribute to the inhibition of Pi uptake induced by a to mortality and morbidity Inhibition of intestinal phosphate decrease, in the CEPG. A phase diagram of calcium and transport will reduce serum phosphorus concentration and phosphate ions in an aqueous environment at room tempera therefore improve outcome in those patients. In stage 2 and 3 ture shows that the solubility of calcium (and therefore phos chronic kidney disease patients, the body phosphorus over phate) is pH dependent, that is, phosphate solubility load does not necessarily lead to hyperphosphatemia, i.e., decreases as pH increases. See FIG. 20. This phenomenon patients remain normophosphatemic, but it does trigger an would suggest that, all things being equal, a drug-induced pH increase in FGF-23, a risk factor in mortality and morbidity in increase in the microenvironment of the mucosal Surface those patients. Therefore, there is a need to reduce body would minimize free Piavailability, thus reducing its cellular phosphorus overload even at those early stages to avoid asso uptake in the gastrointestinal tract. ciated bone and vascular disorders, and ultimately improve 0112 According to another non-limiting theory, the mortality rate. uptake of phosphate ions can be influenced by the absorption 0116. Inhibition of intestinal phosphate transport will be of water in the Small intestine, primarily in the jejunum. particularly advantageous in patients that have a disease that Specifically, increased water absorption in the Small intestine is treatable by inhibiting the uptake of phosphate from the associates with increased phosphate uptake, and vice versa. In intestines. Furthermore, inhibition of phosphate transport Such instances, non-NHE3-binding compounds that reduce may slow the progression of renal failure and reduce the risk water absorption in the small intestine can be used to inhibit of cardiovascular events, among other diseases or conditions phosphate uptake. Certain embodiments thus relate to meth associated with the need for phosphate lowering. ods for inhibiting phosphate uptake in the gastrointestinal I. Compounds that Inhibit Phosphate Transport tract of a patient in need of phosphate lowering, comprising 0117 Embodiments of the present invention relate to com administering to the patient a compound that decreases water pounds that are able to inhibit or reduce phosphate transport/ absorption in the Small intestine, where the compound does uptake in the gastrointestinal tract, for instance, by modulat not bind NHE3, and where the compound is substantially ing the pH within or adjacent to the epithelial membrane of active in the gastrointestinal tract to inhibit transport of phos the gastrointestinal lumen, by decreasing water absorption in phate ions (Pi) therein upon administration to the patient in the small intestine, or both. Examples of pH-modulatory need thereof. In certain embodiments, the compound compounds include those that stimulate bicarbonate secre decreases “net' water absorption, for instance, by modulating tion in the Small intestine (i.e., duodenal bicarbonate secre the balance between secretion and absorption, e.g., by tion or DBS), inhibit acid/proton secretion in the small intes decreasing absorption, increasing secretion, or both. In some tine, or both. embodiments, the compound decreases water absorption in 0118. The compounds provided herein can include small the jejunum. molecules of synthetic or biologic origin and peptides or 0113. In some aspects, inhibition of phosphate uptake in polypeptides. The terms “peptide' and “polypeptide' are the gastrointestinal tract may be achieved by the administra used interchangeably herein; however, in certain instances, tion of certain compounds, and/or pharmaceutical composi the term "peptide' can refer to shorter polypeptides, for tions comprising them, which may advantageously be example, polypeptides that consist of about 2, 3, 4, 5, 6, 7, 8, designed Such that little, or Substantially none, of the com 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, pound is absorbed into the blood stream (that is, it is designed or 50 amino acids, including all integers and ranges (e.g., to be non-systemic or Substantially non-systemic). In this 5-10, 8-12, 10-15) in between. Polypeptides and peptides can regard, the compounds have features that give rise to little or be composed of naturally-occurring amino acids and/or non Substantially no systemic availability upon enteral adminis naturally occurring amino acids. Antibodies are also included tration, including oral administration. In other words, the as polypeptides. compounds are not absorbed into the bloodstream at mean 0119. In some embodiments, the compound is selected ingful levels and therefore have no activity there, but instead from one or more of aP2Y receptoragonist, an adenosine A2b have their activity localized substantially within the GI tract. receptor agonist, a guanylate cyclase C receptor agonist, a 0114. Therefore, in certain illustrative embodiments as soluble guanylate cyclase agonist, an adenylate cyclase further described herein, the compounds of the invention receptoragonist, an imidazoline-1 receptoragonist, a cholin generally require a combination of structural and/or func ergic agonist, a prostaglandin EP4 receptoragonist, a dopam tional features relating or contributing to their activity in the ine D1 agonist, a melatonin receptoragonist, a 5HT4agonist, GI tract and/or their substantial non-systemic bioavailability. an atrial natriuretic peptide receptoragonist, a carbonic anhy Such features may include, for example, one or more of (i) drase inhibitor, a phosphodiesterase inhibitor, or a Down specific tRSA and/or MW values (e.g., at least about 190 A Regulated in Adenoma (DRA or SLC26A3) agonist. In some and/or at least about 736 Daltons, respectively), (ii) specific aspects, as noted above, Such agonist compounds induce levels of fecal recovery of the compound and/or its metabo bicarbonate secretion and/or inhibit acid secretion in the lites after administration (e.g., greater than 50% at 72 hours); upper gastrointestinal tract, including the duodenum and the US 2016/O 184387 A1 Jun. 30, 2016 proximal jejunum. In some aspects, the mechanism of action (0.126 P2Y receptors have been shown to mediate Ca" directly or indirectly modulates apical proton and bicarbonate signaling in duodenocytes and contribute to duodenal transporters to produce a decrease in CEPG or a relatively mucosal bicarbonate secretion. See, e.g., Dong et al., Am J basic microenvironment at the mucosal Surface, which Physiol Gastrointest Liver Physiol 296:G424-G432, 2009. thereby reduces phosphate uptakefabsorption. Without being bound by any one mechanism, in certain 0120 In specific aspects, the compound directly or indi aspects a P2Y receptor agonist inhibits or reduces phosphate rectly stimulates duodenal bicarbonate secretion (DBS). DBS uptake in the gastrointestinal tract by stimulating bicarbonate is a natural defense of the mucosa which operates in the secretion into the small intestine (also referred to as duodenal duodenal and proximal jejunum segments of the gut to neu bicarbonate secretion; DBS). tralize acidic gastric fluid. DBS can be stimulated by a num I0127. In some embodiments, and without being bound by ber of biological pathways, including those which regulate any one mechanism, a P2Y receptor agonist inhibits or the activity of chloride/bicarbonate antiporters such as reduces phosphate uptake in the gastrointestinal tract by SLC26A3 (DRA) and SLC26A3 (PAT-1), chloride and bicar decreasing water absorption in the Small intestine. bonate channels via CFTR, and calcium-activated chloride I0128. Some P2Y receptors are selectively activated, for channels, among others. In some aspects, these pathways are example, by adenine nucleotides such as ATP and ADP, and stimulated by an increase in one or more secondary messen others are by uracil nucleotides or UDP-glucose. The P2Y gers, such as intracellular Ca", cAMP, and/or cGMP. receptor accounts for the functionality of the defined P2Y 0121. In some aspects, the compound directly or indirectly purinoreceptor. It operates in a variety of tissues including decreases water absorption in the Small intestine. In particular Smooth muscle, endothelium and neuronal tissues as well as aspects, the compound decreases water absorption in the in blood platelets. The P2Y receptor is selective for adenine jejunum. The specific aspects, the compound decreases water nucleotides. ADP is the most potent physiological agonist. In absorption in the small intestine by about or at least about Some embodiments, the compound is a P2Y receptor ago nist, optionally a selective P2Y receptor agonist relative to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% other P2Y receptors. One example of a P2Y receptoragonist relative to a control compound or no compound. is 2-methylthio-ADP. 0122) The term “agonist includes a compound that binds to a target molecule Such as a receptor and triggers or stimu I0129. In particular embodiments, the compound is a P2Y lates a cellular response by that target molecule. Included are and/or P2Y receptor agonist, optionally a selective P2Y Super agonists, full agonists, partial agonists, and selective receptor agonist relative to other P2Y receptors. These two agonists. Super agonists produce a greater maximal response receptors display the highest identity in the sequences of their than the endogenous agonist(s) for the target molecule, full TM domains (66.8%) of all the P2Y receptor subtypes. The agonists produce a comparable response relative to the P2Y receptor can be activated, for instance, by uracil nucle otides, UDP-sugar derivatives, and adenine nucleotides such endogenous agonist(s) for the target molecule, and partial as ATP P2Y receptors are expressed in many tissues includ agonists produce a significantly lesser (e.g., 10%. 20%, 30%, ing lung, heart, skeletal muscle, spleen, kidney, liver and 40%, 50%, 60%, 70%, 80%) maximal response than the epithelia. These receptors play an important role in regulating endogenous agonist(s) for the target molecule. ion transport in epithelial cells. Triphosphate nucleotides 0123. Further to its activity as an agonist, in certain including UTP ATP, UTPYS and ATPYS act as full agonists of embodiments a compound can also be characterized by its the P2Y receptor. In addition to the above-mentioned ago “specific binding to a target. For instance, in Some embodi nists, the P2Y receptor also responds to diadenosine-tetra ments a compound (e.g., a direct-acting compound) can spe phosphate (AP4A) and Up4U (diquafosol, INS365, used for cifically bind to a target described herein with a binding the treatment for dry eye disease). The analogue P-(uridine affinity (K) of at least about 0.01, 0.05, 0.1, 0.2,0.3, 0.4,0.5, 5')-P4-(2'-deoxycytidine 5') tetraphosphate (INS37217 is a 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, potentagonist at the P2Y receptor with some agonist effects 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or on the P2Y receptor. Denufosol ((3S,5R)-5-(4-amino-2-ox 50 nM. In particular embodiments, the target is selected from opyrimidin-1-yl)-3-hydroxyoxolan-2-yl)methoxy-hydroxy one or more of a P2Y receptor, an adenosine A2b receptor, a phosphoryl III(2R.3S.4R,5R)-5-(2,4-dioxopyrimidin-1-yl)- guanylate cyclase C receptor, an adenylate cyclase receptor, 3,4-dihydroxyoxolan-2-yl)methoxy-hydroxyphosphoryl an imidazoline-1 receptor, an acetylcholine receptor, a pros oxy-hydroxyphosphoryl hydrogen phosphate; including its taglandin EP4 receptor, a dopamine D1 receptor, a melatonin tetrasodium salt) is also an exemplary P2Y receptoragonist. receptor, 5HT4, an atrial natriuretic peptide receptor, a car Also included is PSB1114. bonic anhydrase, a phosphodiesterase, and Down-Regulated 0.130 For ribose and uracil modifications, both 2'-deoxy in Adenoma (DRA or SLC26A3), as described herein. 2'-amino-UTP and 2-thio-UTP preserve the agonist potency (0.124 A. P2Y Agonists of UTP at the P2Y receptor. The combination of these two 0.125. In certain embodiments, the compound is a P2Y modifications yields 2'-amino-2-thio-UTP, which synergizes agonist (or P2Y receptor agonist). P2Y receptors refer to a to enhance both potency (8 nM ECs) and selectivity (300 family of purinergic G protein-coupled receptors. Examples fold P2Y-selective versus P2Y). Modifications at position of human P2Y receptors include P2Y, P2Y, P2Y P2Ys, 5, such as 5-bromo-UTP (EC-0.75 uM) and 5-iodo-UTP P2Y P2Y, P2YP2YP2Y, P2Y, P2Y, and P2Y (ECso 0.83 uM), Suggest that introducing a small hydropho The main native or endogenous ligands of the P2Y receptors bic group might be beneficial at the P2Y receptor. are adenosine 5'-triphosphate (ATP), adenosine 5'-diphos I0131 The P2Y receptor agonists provided herein include phate (ADP), uridine 5'-triphosphate (UTP), uridine mononucleotides, dinucleotides, and nucleotide-sugars, 5'-diphosphate (UDP), and UDP-glucose (or other UDP sug among other agonists known in the art. See, e.g., U.S. Pat. No. ars). Dinucleotides such as ApU are also naturally-occurring 6,624,150; EP 1196396; WO 2008/060632: Cosyn et al., P2Y agonists. Bioorg Med Chem Lett. 19:3002-5, 2009 (describing uridine US 2016/O 184387 A1 Jun. 30, 2016

5'-(phospho)phosphonate and a 5'-methylenephosphonate nal. 5:3-19, 2009. Activation at either site results in Cl equivalent of UMP); Ko et al., Bioorg Med Chem. 16:6319 secretion via direct activation of the cAMP-activated Cl— 32, 2008 (describing, for example, alpha.beta-methylene channel cystic fibrosis transmembrane conductance regulator UDP, a P2Y receptoragonist; Up(4)-phenyl ester and Up(4)- (CFTR). CFTR modulates the secretion of both chloride and Illglucose, Selective P2Y receptor agonists; bicarbonate. For example, in rats the A2B receptor has been dihalomethylene phosphonate analogues, selective P2Y immuno-localized to the brush border membrane of duodenal receptor agonists; a 2-thio analogue of INS37217 (P(1)-(uri villi, where luminal adenosine has been shown to stimulate dine-5')-P(4)-(2'-deoxycytidine-5') tetraphosphate), a potent bicarbonate secretion via A2B receptors and CFTR. See, e.g., and selective P2Y receptor agonist; Ivanov et al., J Med Ham et al., J Pharmacol Exp Ther. 335:607-13, 2010. With Chem. 50: 1166-76, 2007: Brookings et al., Bioorg Med Chem out being bound by any one mechanism, in certain aspects an Lett. 17:562-5, 2007 (describing the synthesis and P2Yago adenosine A2b receptoragonist inhibits or reduces phosphate nist activities of a series of nucleoside triphosphates); and uptake in the gastrointestinal tract by stimulating bicarbonate Jacobson et al., Purinergic Signal. 5:75-89, 2009; each of secretion into the Small intestine, e.g., by decreasing the which is incorporated by reference in its entirety. CEPG. 0132) Additional examples of P2Y receptor agonists 0.138. In some embodiments, and without being bound by include those described in WO 1999/09998 and U.S. Appli any one mechanism, an adenosine A2b receptor agonist cation Nos. 2002/0052336 and 2003/0027785, including inhibits or reduces phosphate uptake in the gastrointestinal PP-diadenosinetetraphosphate (AP); uridine-5'-diphos tract by decreasing water absorption in the Small intestine. phate (UDP); uridine-5'-O-(2-thiodiphosphate) (UDPBS); 0.139 General examples of adenosine A2b receptor ago 5-bromouridine-5'-triphosphate (5-BrUTP): 5-(1-phenyl nists include adenosine, adenosine-like compounds, and non ethynyl)-uridine-5'-triphosphate (5-(1-phenylethynyl)UTP); adenosine compounds. In some embodiments, nucleoside 5-methyluridine-5'-diphosphate (5-methylUDP): 4-hexylth based adenosine A2b receptor agonists include modified iouridine-5'-triphosphate (4-hexylthioUTP): 4-thiouridine adenosine compounds, such as adenosine compounds Substi 5'-triphosphate (4-thioUTP); 2-methoxyuridine-5'-triphos tuted at the N (6)-position of the purine heterocycle, the phate (2-methoxyl JTP); 4-(1-morpholino)uridine-5'- C(2)-position of the purine heterocycle, the 5'-position of the tetraphosphate (4-(1-morpholino))UP 4-hexyloxyuridine ribose moiety, and any combination of the foregoing. Also 5'-diphosphate (4-hexyloxyl JDP): 4-(N, N-dimethyl) included are non-ribose ligands such as Substituted dicarbo cytidine-5'-triphosphate (N, N-dimethylCTP): 4-(N-hexyl) nitrilepyridines, among which 2-6-amino-3,5-dicyano-4-4- cytidine-5'-triphosphate (N-hexylCTP); P'-(cytidine-5')-P'- (cyclopropylmethoxy)phenylpyridin-2-ylsulfanyl)aceta (uridine-5'-)tetraphosphate (CPU); P'-O-(methyl)-P'- mide is an example. See, e.g., Baraldi et al., Purinergic (uridine-5'-)tetraphosphate (MePU) and 4-(N-cyclopentyl) Signal. 4:287-303, 2008; and Baraldi et al., Purinergic Sig thymidine-5'-triphosphate (N-cyclopentylCTP). nal. 5:3-19, 2009; each of which is incorporated by reference 0.133 Also included are 5'-adenosine-triphosphate (ATP), in its entirety. 5'-uridine-triphosphate (UTP), uridine-5'-O-(3-thiotriphos 0140. Additional non-limiting examples of adenosine A2b phate) (UTPYS), P'-(uridine-5')-Psup.4-(uridine-5'-)tetra receptoragonists include BAY 60-6583, CV 1808, AMP579, phosphate (UP), 5'-4-(thiouridine)-triphosphate (4-thio NECA (N-ethylcarboxamidoadenosine), (S)-PHPNECA, UTP), and P-(cytidine-5')-P-(uridine-5'-) tetraphosphate LUF-5835, 6-guanyl NECA, and LUF-584. See also Beukers (CPU). The identification and preparation of certain thio et al., J. Med. Chem, 47:3707-3709, 2004 (describing, for phosphate analogues of nucleoside diphosphates (such as example, non-adenosine agonists such as LUF5834 UDP-3-S) are described in U.S. Pat. No. 3,846,402 and (2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethyl Goody and Eckstein (J. Am. Chem. Soc. 93: 6252-6257. sulfanyl)pyridine-3,5-dicarbonitrile) and LUF5835 (a 3-hy 1971). Alternatively, UTP and other analogs thereofare also droxyphenyl analogue)); Beukers et al., Med Res Rev. 26:667 commercially available from Vendors such as Sigma (St. 98, 2006 (describing, for example, (S)PHPNECA and certain Louis, Mo.) and Pharmacia (Uppsala, Sweden). Exemplary non-ribose ligands as adenosine A2b receptor agonists); and methods of identifying P2Y receptoragonists are described, Liu et al., Basic Res Cardiol. 105:129-37, 2010. Also for example, in U.S. Application No. 2003/0175810. included are the A2b receptor agonists described in U.S. 0134. In some embodiments, the P2Y receptoragonist is a Application No. 2002/0156076. These references are incor non-endogenous Small molecule agonist. Additional porated by reference in their entireties. examples of P2Y receptoragonists are shown in FIGS. 4 and 01.41 Examples of adenosine A2b receptor agonists are SA-5C. shown in FIGS. 6A-6C, and further disclosed, together with 0135 B. Adenosine A2b Receptor Agonists methods for their synthesis, in U.S. Application No. 2009/ 0136. In certain embodiments, the compound is an 0221649 and PCT Publication Nos. WO 2006/027142, WO adenosine A2b receptor agonist, optionally a selective ago 2007/101531, and WO 2003/008384, each of which is incor nist. Adenosine exerts most of its physiological functions by porated by reference in its entirety. acting as a local modulator at four receptor Subtypes named 0142. C. Guanylate Cyclase C Receptor Agonists A1, A2A, A2B and A3 adenosine receptors (ARs). The 0143. In certain embodiments, the compound is a guanylyl adenosine A2b receptor (or ADORA2B) is a G-protein cyclase C (GC-C) agonist, optionally a selective agonist. coupled adenosine receptor integral membrane protein that GC-C is an isoform of the guanylate cyclase family that is stimulates adenylate cyclase activity in the presence of highly concentrated at the apical membrane of intestinal epi adenosine. thelial cells. It is also the target receptor for bacterially-se 0.137 The A2b receptor is expressed in a variety of tissues, creted heat stable-enterotoxins, which are responsible for and high concentrations have been Suggested in the caecum acute secretory diarrhea. GC-C is also known as guanylate and large intestine on both the mucosal and basolateral aspect cyclase 2C, intestinal guanylate cyclase, guanylate cyclase C of colonic epithelial cells. See Baraldi et al., Purinergic Sig receptor, and heat-stable enterotoxin receptor (hSTAR). US 2016/O 184387 A1 Jun. 30, 2016

0144 GC-C has an extracellular ligand-binding domain, a 1989) having the mature amino acid sequence Asn Thr Phe single transmembrane region, a region similar to protein Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys kinases, and a C-terminal guanylate cyclase domain. Tyrosine Tyr (SEQ ID NO:13): Y enterocolitica ST peptides, Y-ST(Y- kinase activity mediates the GC-C signaling pathway within STa), Y-STh, and Y-STc (reviewed in Huang et al., Microb, the cell. Guanylin and uroguanylin are endogenous peptide Pathog. 22:89, 1997) having the following pro-form amino ligands for GC-C. Activation of GC-C leads, for example, to acid sequences: Gln Ala Cys Asp Pro Pro Ser Pro Pro Ala Glu intracellular cGMP elevation, PKGII-dependent phosphory Val Ser Ser Asp Trp-Asp Cys Cys-Asp Val Cys Cys Asn Pro Ala lation of the cystic fibrosis transmembrane regulator (CFTR), Cys Ala Gly Cys (SEQID NO:14) (as well as a Ser-7 to Leu-7 and other downstream signals which trigger increased chlo variant of Y-STa (SEQ ID NO:15), (Takao et al., Eur: J. ride and bicarbonate intraluminal secretion (via CFTR, and Biochem. 152:199, 1985); Lys Ala Cys Asp Thr Gln Thr Pro possibly DRA or PAT-1). Ser Pro Ser Glu Glu Asn Asp Asp Trp Cys Cys Glu Val Cys 0145 GC-C agonists such as linaclotide, guanylin, and E. Cys ASn Pro Ala Cys Ala Gly Cys (SEQID NO:16); Gln Glu coli heat stable enterotoxins (STa) have been shown to stimu Thr Ala Ser Gly Gln Val Gly Asp Val Ser Ser Ser Thr Ile Ala late duodenal bicarbonate secretion. See, e.g., Rao et al., Am Thr GluVal Ser Glu Ala Glu Cys GlyThr Gln Ser Ala Thr Thr J Physiol Gastrointest Liver Physiol 286:G95-G101, 2004; Gln Gly Glu Asn Asp Trp Asp Tip Cys CysGlu Leu Cys Cys Busby et al., EurJ Pharmacol. 649:328-35, 2010; Bryant et ASn Pro Ala Cys Phe Gly Cys (SEQID NO:17), respectively: al., Life Sci. 86:760-5, 2010. Without being bound by any one Y kristensenii ST peptide having the mature amino acid mechanism, in certain aspects a GC-C agonist inhibits or sequence Ser Asp Trp Cys CysGlu Val Cys Cys Asn Pro Ala reduces phosphate uptake in the gastrointestinal tract by Cys Ala Gly Cys (SEQ ID NO:18); V cholerae non-01 ST stimulating bicarbonate secretion into the Small intestine. peptide (Takao et al., FEBS Lett. 193:250, 1985) having the 0146 In some embodiments, and without being bound by mature amino acid sequence Ile Asp CyS CyS Glu Ile CyS Cys any one mechanism, a GC-Cagonist inhibits or reduces phos ASn Pro Ala Cys Phe Gly Cys Leu Asn (SEQID NO:19); and phate uptake in the gastrointestinal tract by decreasing water V mimicus ST peptide (Arita et al., FEMS Microbiol. Lett. absorption in the Small intestine. 79:105, 1991) having the mature amino acid sequence Ile Asp 0147 General examples of GC-Cagonists include peptide Cys CysGlu Ile Cys Cys Asn Pro Ala Cys Phe Gly Cys Leu agonists and analogs thereof, including synthetic analogs of Asn (SEQID NO:20). Table A1 below shows the sequences endogenous GC-C peptide agonists. Particular examples of of exemplary mature ST peptides. GC-Cagonists include, without limitation, heat stable entero toxins (ST or STa peptides) including those from E. coli, TABLE A1 guanylin, proguanylin, uroguanylin, prouroguanylin, lym phoguanylin, linaclotide (Linzess), SP-333, and plecanatide. SEQ See, e.g., Drug Des Devel Ther: 7:351-60, 2013. Linaclotide Mature ST Peptides ID NO : is a STa synthetic analog marketed for the treatment of irri NSSNYCCELCCNPACTGCY 10 table bowel syndrome—constipation dominant (IBS-C). See, e.g., Bryant et al., Life Sci. 86:760-5, 2010. Plecanatide is a NTFYCCELCCNPACAGCY 11 synthetic analog of uroguanylin developed for the treatment NTFYCCELCCNPACAPCY 21 of IBS-C. See, e.g., Pitari, supra; and Shailubhai et al., Dig Dis Sci. 2013 Apr. 27. Epub ahead of print. Additional NTFYCCELCCYPACAGCN 12 examples of GC-C agonists are described in U.S. Application IDCCEICCNPACFGCLN 19 Nos. 2012/0064039, 2004/0258687, 2005/0287067, 2006/ 0281682, 2006/0258593, 2006/0094658, 2008/0025966, IDCCEICCNPACFGCLN 19 2003/0073628, 2004/012 1961 and 2004/0152868 and in U.S. Pat. Nos. 5,140,102, 7,041,786, and 7,304,036. These refer IDCCEICCNPACE 22 ences are incorporated by reference in their entireties. IDCCEICCNPACFG 23 0148. In some embodiments, the GC-C agonist is a bacte rial ST (or STa) peptide, or a variant or analog or derivative IDCCEICCNPACFGCLN 19 thereof. In bacteria, ST or STapeptides are derived from a IDRCEICCNPACFGCLN 24 preproprotein that generally has at least 70 amino acids. The pre and pro regions are cleaved as part of the secretion pro DWDCCDWCCNPACAGC 25 cess, and the resulting mature protein, which generally DWDCCDWCCNPACAGC 26 includes fewer than about 20 amino acids, is biologically active. NDDWCCEWCCNPACAGC 27 0149 Exemplary bacterial ST peptides include: E. coli ST Ib (Moseley et al., Infect. Immun. 39:1167, 1983) having the WDWCCELCCNPACFGC 28 mature amino acid sequence Asn Ser Ser Asn Tyr Cys CysGlu SDWCCEWCCNPACAGC 18 Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:10); E. coli ST Ia (So and McCarthy, PNAS USA. OACDPPSPPAEWSSDWDCCDVCCDPACAGC 29 77:4011, 1980) having the mature amino acid sequence Asn OACDPPSPPAEWSSDWDCCDVCCNPACAGC 14 Thr Phe Tyr Cys Cys Glu Leu Cys Cys ASn Pro Ala Cys Ala Gly Cys Tyr (SEQID NO:11); E. coli STI (Chan and Gian KACDTOTPSPSEENDDTCCEVCCNPACAGC 16 nella, J. Biol. Chem. 256: 7744, 1981) having the mature amino acid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys OETASGOVGDVSSSTIATEVSEAECGTOSAT 3 O Cys Tyr Pro Ala Cys Ala Gly Cys Asn (SEQ ID NO:12); C. TOGENDWDWCCELCCNPACFGC 31 freundii ST peptide (Guarino et al., Infect. Immun. 57:649, US 2016/O 184387 A1 Jun. 30, 2016 10

TABLE A1 - continued receptor. In the human body an inactive form of chymot rypsin, chymotrypsinogen is produced in the pancreas. When SEQ this inactive enzyme reaches the Small intestine it is converted Mature ST Peptides ID NO : to active chymotrypsin by the excision of two di-peptides. Active chymotrypsin can cleave peptides at the peptide bond MKKLMLAIFISWLSFPSFSOSTESLDS 32 on the carboxy-terminal side of Trp, Tyr, or Phe. The presence ofactive chymotrypsin in the intestinal tract can lead to cleav SKEKITLETKKCDWWKNNSEKKSEN 33 age of certain of the GC-C peptide agonists having an appro MNNTFYCCELCCNPACAGCY 34 priately positioned functional chymotrypsin cleavage site. In Some instances, it is expected that chymotrypsin cleavage will MKKSILFIFLSVLSFSPFAODAKPVES 35 moderate the action of a GC-C peptide agonist having an appropriately positioned chymotrypsin cleavage site as the SKEKITLESKKCNIAKKSNKSGPESM 36 peptide passes through the intestinal tract. NSSNYCCELCCNPACTGCY 37 0153. Certain of the GC-C agonist peptides include a potentially functional trypsin cleavage site, e.g., Lys or Arg. MKKIVFWLVLMLSSFGAFGOETVSG 38 Trypsinogen, like chymotrypsin, is a serine protease that is OFSDALSTPITAEWYKOACDPPLPPA 39 produced in the pancreas and is present in the digestive tract. The active form, trypsin, will cleave peptides having a Lys or EWSSDWDCCDWCCNPACAGC 4 O Arg. The presence of active trypsin in the intestinal tract can lead to cleavage of certain of the GC-C agonist peptides 0150. The immature (including pre and pro regions) form having an appropriately positioned functional trypsin cleav of E. coli ST-IA (STP) protein has the sequence: mkklm age site. In certain instances, it is expected that trypsin cleav laifisvlsfpsfsqstesldsskekit age will moderate the action of a GC-C peptide agonist hav letkkcdvvknnsekksenmnntfy.ccelccnpacagcy (SEQ ID ing an appropriately positioned trypsin cleavage site as the NO:41); see GenBank R. Accession No. PO1559 (gii: 123711). peptide passes through the intestinal tract. The pre sequence extends from residues 1-19. The pro 0154) In certain embodiments, the peptide comprises at sequence extends from residues 20-54. The mature protein least six cysteines that can form three disulfide bonds. In extends from residues 55-72. The immature (including pre certain embodiments, the disulfide bonds are replaced by and pro regions) form of E. coli ST-1B (ST-H) protein has the other covalent cross-links and in some cases the cysteines are Sequence: mkksilfiflsvlsfspfaqdak substituted by other residues to provide for alternative cova pVesskekitleskkcniakkSnksgpesmns.Snyccelccnpactgcy lent cross-links (described elsewhere herein). Certain pep (SEQ ID NO:42); see GenBank R. Accession No. P07965 tides include a functional chymotrypsin or trypsin cleavage (gi:3915589)). The immature (including pre and pro regions) site located so as to allow inactivation of the peptide upon form of Y. enterocolitica ST protein has the sequence: cleavage. Certain peptides having a functional cleavage site mkkivfvlylmlssfgafgqetVSgqfs undergo cleavage and gradual inactivation in the digestive dalstpitaevykdacdpplppaevssawdccdvccnpacagc (SEQ ID tract, and this is desirable in some circumstances. In certain NO:43); see GenBank R. Accession No. 525659 (gi:282047)). peptides, a functional chymotrypsin site is altered, increasing Accordingly, a GC-C agonist peptide may comprise or con the stability of the peptide in vivo. sist of any one or more of the bacterial ST peptide sequences 0.155. In certain embodiments, the peptides include either described herein, including variants thereof. one or two or more contiguous negatively charged amino 0151. The bacterial ST peptides typically have six Cys acids (e.g., Asp or Glu) or one or two or more contiguous residues. These six Cys residues form three disulfide bonds in positively charged residues (e.g., Lys or Arg) or one or two or the mature and active form of the peptide. If the six Cys more contiguous positively or negatively charged amino residues are identified, from the amino to carboxy terminus of acids at the carboxy terminus. In these and related embodi the peptide, as A, B, C, D, E, and F, then the disulfide bonds ments, all of the flanking amino acids at the carboxy terminus usually form as follows: A-D, B-E, and C-F. The formation of are either positively or negatively charged. In some embodi these bonds is believed to contribute GC-C receptor binding. ments, the carboxy terminal charged amino acids are pre Hence, in certain embodiments, a GC-C agonist peptide has ceded by a Leu. For example, the following amino acid at least one, two, or three disulfide bonds selected from any sequences can be added to the carboxy terminus of the pep combination of A-D, B-E, and C-F, as shown above. In some tide: Asp; Asp Lys: Lys Lys Lys Lys Lys Lys (SEQIDNO:44); embodiments, however, one or more cysteines of the GC-C Asp Lys Lys Lys Lys Lys Lys (SEQID NO:45); Leu Lys Lys: peptide agonists described herein are deleted or replaced with and Leu Asp. In particular embodiments, a Leu is added to the a different amino acid. In some embodiments, 1, 2, 3, 4, 5, or carboxy terminus. 6 cysteines are deleted or replaced with a different amino 0156. In some aspects, the (bacterial ST analog) GC-C acid. In particular aspects, the most N-terminal cysteine resi agonist peptide comprises, consists, or consists essentially of dues (e.g., A, B, or A and B) and/or the most C-terminal cysteine residue or residues (e.g., E, F, or E and F) are deleted the amino acid sequence shown below (I): or replaced with a different amino acid. In certain embodi ments, the different amino acid is alanine or serine. (SEQ ID NO: 46) 0152 Certain of the GC-C agonist peptides include a Xaa1 Xaa2 Xaa3 Xaa, Xaas Cys, CyS7 Xa as Xaag Cyso potentially functional chymotrypsin cleavage site, e.g., a Trp, Tyr or Phe located between either Cys B/Cys D or between CyS Xaa12 Xaa13 Xaa14 Cys 15 Xaa16 Xaa17 Cys 18 Xaa1g CyS E/CyS F. Cleavage at either chymotrypsin cleavage site may reduce the ability of the peptide to bind to the GC-C US 2016/O 184387 A1 Jun. 30, 2016

O157. In some embodiments, Xaa, Xaa-Xaa-Xaa-Xaasis Some embodiments, Xaa is Thr, Ala or Trp. In some Asn Ser Ser Asn Tyr (SEQ ID NO:2) or is missing or Xaa, embodiments, Xaa is Thr. In some embodiments, Xaa, is Xaa- Xaa, Xaa is missing. In certain embodiments, Xaas. Trp, Tyr or Phe. In some embodiments, Xaa is Thror Ala. In Xaao, Xaa12, Xaa14, Xaa16. Xaa17, and Xaa1s are any amino specific embodiments, Xaa, it is Val. In particular embodi acid. In certain embodiments, Xaas, Xaao, Xaa12, Xaa1a. ments, Xaa is Gly. In some embodiments, Xaa is Thr, Ser, Xaa, Xaa17, and Xaao are any natural or non-natural amino Metor Val. In some embodiments, Xaa, is Val, Ala, or Thr. In acid or amino acid analog. Some embodiments, Xaa is Ile, Val, LyS, ASn, Glu, Asp, or 0158. In certain embodiments, Xaas is Asn., Trp, Tyr, Asp, Thr. or Phe. In other embodiments, Xaas is Thr or Ile. In some (0165. In certain embodiments, Xaa, is any natural or non embodiments, Xaas is Tyr, Asp or Trp. In certain embodi natural amino acid or amino acid analog. In some embodi ments, Xaas is ASn, Trp, Tyr, Asp, Ile, Thr or Phe. In specific ments, Xaa, is Gly, Pro or Ala. In specific embodiments, embodiments Xaas is ASn. Xaa, is Gly. In particular embodiments, Xaa, is Ala. In 0159. In certain embodiments, Xaas is any natural or non some embodiments, Xaa, is Gly or Ala. In some embodi natural amino acid or amino acid analog. In some embodi ments, Xaa, is Gly, ASn, Ser or Ala. In some embodiments, ments, Xaas is Glu, Asp, Gln, Gly or Pro. In other embodi Xaa, is ASn, Glu, Asp, Thr, Ala, Ser, or Gly. In some embodi ments, Xaas is Glu. In some embodiments, Xaa, is Glu or ments, Xaa, is Asp, Ala, Ser, or Gly. Asp. In some embodiments, Xaas is ASn, Glu, or Asp. In some 0166 Incertain embodiments, Xaac is any natural or non embodiments, Xaas is Glu, His, LyS, Gln, ASn, or Asp. In natural amino acid or amino acid analog. In some embodi Some embodiments, Xaas is Glu, His, Gln, ASn, or Asp. In ments, Xaao is Trp, Tyr, Phe, ASn, Ile, Val. His, Leu, or Arg. Some embodiments, Xaas is Glu, ASn, His, Gln, Lys, Asp or In some embodiments, Xaao is Trp, Tyr, ASnor Leu. In some Ser. In specific embodiments, Xaas is Pro. embodiments, Xaa, is Trp, Tyr or Phe. In some embodi 0160. In certain embodiments, Xaa, is any natural or non ments, Xaao is Tyr, Phe or His. In some embodiments, Xaao natural amino acid or amino acid analog. In some embodi is Tyr or Trp. In specific embodiments, Xaa, is Tyr. In some ments, Xaa, is any natural or non-natural aromatic amino acid embodiments, Xaao is Leu, Ile or Val. In particular embodi oramino acid analog. In some embodiments, Xaao is Leu, Ile, ments, Xaa, is His. In some embodiments, Xaa, is Trp, Tyr. Val, Ala, Lys, Arg, Trp, Tyr or Phe. In some embodiments, Phe, ASn, Ile, Val, His or Leu. In some embodiments, Xaac is Xaa is Leu, Ile, Val, LyS, Arg, Trp, Tyr or Phe. In some Trp, Tyr, Phe or Leu. In some embodiments, Xaa, is Tyr or embodiments, Xaa, is Leu, Ile, Val, Trp, Tyr or Phe. In some Leu. In some embodiments, Xaac is Lys or Arg. In some embodiments, Xaa, is Leu, Ile or Val. In some embodiments, embodiments, Xaao is any amino acid other than Pro, Arg, Xaa is Trp, Tyr or Phe. In some embodiments, Xaag is Leu, LyS, Asp or Glu. In some embodiments, Xaa, is any amino Ile, Lys, Arg, Trp, Tyr, or Phe. In some embodiments, Xaa, is acid other than Pro. In some embodiments, Xaac is missing. Leu, Val, Ile, or Met. In some embodiments, Xaa, is Leu or (0167. In certain embodiments Xaa, is Asp or Asn. In Phe. In some embodiments, Xaa, is Leu, Phe, or Tyr. In some certain embodiments Xaa-Xaa is AspPhe or is missing. In embodiments, Xaa, is Tyr, Phe or His. In some embodiments, Some embodiments, Xaao is ASnor Glu and Xaa is missing. Xaa is Phe, His, Trp, or Tyr. In certain embodiments, Xaa, is In Some embodiments, Xaao Xaao Xaa-1 is missing. not Leu. In specific embodiments, Xaa, is Tyr. 0.168. In some aspects, the GC-C agonist peptide com 0161 In certain embodiments, Xaa is any natural or non prises, consists, or consists essentially of the amino acid natural amino acid or amino acid analog. In certain embodi sequence shown below (II): ments, Xaa is ASn, Tyr, Asp or Ala. In specific embodi ments, Xaa, Asn. In certain embodiments, Xaa, is ASn, (SEO ID NO: 47) Met, Arg, Lys, His, or Gln. In certain embodiments, Xaa is Xaa1a.a. Xaa2Xaa- Xaa,Xala Xaa,Xala XaasXala Cys.S CyS7St. Xa as XaagXala CysoS ASn, Lys, His, or Gln. In certain embodiments, Xaa is ASn, Asp, Glu or Gln. In certain embodiments, Xaa, is ASn, Thr, CyS11 Asn12 Pro 13 Ala Cysis Xaa16. Gly17 Cy S18 Xaa1g Ser, Arg, Lys, Gln, or His. In some embodiments, Xaa is ASn, Ser, or His. Xaa2o Xaa-21 0162. In certain embodiments, Xaa is Ala, Pro or Gly. In 0169 where Xaa, Xaa-Xaa-Xaa-Xaas is Asn Ser Ser Asn certain embodiments, Xaa is Pro or Gly. In specific embodi Tyr (SEQID NO:2) or is missing or Xaa, Xaa, Xaa, Xaa, is ments, Xaa is Pro. In particular embodiments, Xaa is Gly. missing and Xaas is ASn; 0163. In certain embodiments, Xaa, is any natural or non 0170 Xaas is Glu or Asp; natural amino acid or amino acid analog. In certain embodi (0171 Xaa, is Leu, Ile, Val, Trp, Tyr or Phe: ments, Xaa, is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Thr, 0172 Xaa is Thr, Ala, Trp; Lys, Arg, or Asp. In certain embodiments, Xaa, is Ala or Gly. 0173 Xaa, is Trp, Tyr, Phe or Leu or is missing; and In some embodiments, Xaa, is Val or Ala. In certain embodi Xaa-oXaa is AspPhe. ments, Xaa, is Ala or Thr. In specific embodiments, Xaa, is 0.174. In some aspects, the GC-C agonist peptide com Ala. In certain embodiments, Xaa, is Val, Gln, Asn., Glu, prises, consists, or consists essentially of the amino acid Asp, Thr, or Ala. In certain embodiments, Xaa is Gly, Cys or sequence (II): Xaa, Xaa, Xaa-Xaa, Xaas CyS CyS7 Xaas Ser. Xaa-CySoCyS ASn 12 Pros Alala Cysis Xaa16 Gly17 Cysis 0164. In certain embodiments, Xaa is any natural or non Xaa, Xaa, Xaa, (SEQID NO:48) where, Xaa, is Leu, Ile natural amino acid or amino acid analog. In some embodi or Val and Xaa is Trp, Tyr or Phe; Xaa, is Trp, Tyr or Phe, ments, Xaa is any natural or non-natural non-aromatic and Xaa is Thr or Ala; Xaa, is Trp, Tyr, Phe and amino acid or amino acid analog. In certain embodiments, Xaa-oxaa is AspPhe, and Xaa, Xaa-Xaa-Xaa is missing Xaac Thr, Ala, ASn, Lys, Arg, Trp, Gly or Val. In certain and Xaas is ASn; the peptide comprises fewer than 50, 40, 30 embodiments, Xaa is Thr, Ala, ASn, Lys, Arg or Trp. In or 25 amino acids; or fewer than five amino acids precede certain embodiments, Xaa is Thr, Ala, Lys, Arg or Trp. In CyS. US 2016/O 184387 A1 Jun. 30, 2016

0.175. In some aspects, the GC-C agonist peptide com ridyl)-Ala; Amino-Phe: Fluoro-Phe: Cyclohexyl-Gly; thBu prises, consists, or consists essentially of the amino acid Gly; beta-(3-benzothienyl)-Ala; beta-2-thienyl)-Ala; 5-Me sequence Xaa, Xaa, Xaa, Xaa, Xaas CyS CyS Glu Xaa, Cys thyl-Trp.; and 4-Methyl-Trp. Cys ASn Pro Ala Cys Thr Gly Cys Tyr Xaa, Xaa, (II) (SEQ (0179. In some embodiments, Xaa, can be an N(alpha)-C ID NO:49) where Xaa, is any amino acid: where Xaa, is any (alpha) cyclized amino acid analogues with the structure: amino acid other than Leu; where Xaa, is selected from Phe, Trp and Tyr; where Xaa, is selected from any other natural or non-natural aromatic amino acid; where Xaa, is Tyr, where Xaa is Phe; where Xaa, is Trp; where Xaa, Xaa, Xaa, Xaa, Xaas is ASn Ser Ser ASn Tyr, where Xaa, Xaa, Xaa, Xaa, and Xaas are missing; where Xaa, Xaa, Xaa, and Xaa, are missing; where Xaa, Xaa, and Xaa- are missing; where Xaa, and Xaa, are missing; where Xaa is missing; where Xaa-Xaa is AspPhe or is missing or Xaao is ASn or Glu and Xaa1 is missing or Xaa1-Xaa-oXaa-1 is missing: where Xaa, Xaa-Xaa-Xaa, Xaas and Tyr Xaao Xaa, are missing. In some aspects, the GC-C agonist peptide comprises, con 0180 Xaa, can also be homopro (L-pipecolic acid); sists, or consists essentially of the amino acid sequence Xaa, hydroxy-Pro; 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-me Xaa, Xaa, Xaa, Xaas CyS CyS7 Xaas Xaao CySo Cys thyl-Pro. Xaa12 Xaa1s Xaa14 Cysis Xaa16 Xaa17 CyS is Xaa19 Xaao 0181 In aspects where Xaa is Gly, Ala, Leu or Val, Xaa, Xaa, (I) (SEQID NO:50) where: Xaa, Xaa-Xaa-Xaa-Xaas can be: is missing and/or the sequence Xaao Xaao Xaa-1 is missing. where the peptide optionally comprises additional carboxy terminal and/or amino-terminal amino acids. In instances where the peptide is missing one or more terminal amino acids such as Xaa, or Xaa, the peptide can optionally com prise additional carboxy-terminal and/or amino-terminal amino acids. (0176). In certain embodiments, the peptide includes disul fide bonds between Cys and Cys, between Cys, and Cysis and between CySo and Cys. In some embodiments, the peptide is a reduced peptide having no disulfide bonds. In still 0182 In certain aspects, Xaa, can be an alpha-Substituted other embodiments, the peptide has one or two disulfide or N-methylated amino acid such as alpha-amino isobutyric bonds selected from: a disulfide bond between Cys and acid (aib), L/D-alpha-ethylalanine (L/D-isovaline), L/D-me Cys, a disulfide bond between Cys, and Cyss and a disul thylvaline, or L/D-alpha-methyleucine or a non-natural fide bond between CySo and Cys. amino acid Such as beta-fluoro-Ala. 0177. In certain embodiments, one or more amino acids 0183 In some aspects, Xaa, can be alpha-amino isobu are replaced by a non-naturally occurring amino acid, or a tyric acid (aib) or L/D-alpha-ethylalanine (LID-isovaline). naturally or non-naturally occurring amino acid analog. 0184. Additional examples of non-natural amino acids There are many amino acids beyond the standard 20 amino and amino acid analogs are known in the art and described acids. Some are naturally-occurring others non-naturally-oc elsewhere herein. curring (see, e.g., Hunt, The Non-Protein Amino Acids: In 0185. In some instances, for example, where Xaa, is Trp, Chemistry and Biochemistry of the Amino Acids, Barrett, Tyr, or Phe or where Xaa is Trp, the peptide has a potentially Chapman and Hall, 1985). For example, an aromatic amino functional chymotrypsin cleavage site that is located at a acid can be replaced by 3,4-dihydroxy-L-phenylalanine, position where cleavage may alter GC-C receptor binding by 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylgly the peptide. When Xaao is Lys or Arg or when Xaa is Lys or cine (Phg) or nor-tyrosine (norTyr). Phg and norTyrand other Arg, the peptide has a potentially functional trypsin cleavage amino acids including Phe and Tyr can be substituted by, for site that is located at a position where cleavage may alter example, a halogen, —CH, -OH, -CH2NH, -C(O)H, GC-C receptor binding by the peptide. —CHCH. —CN, —CHCHCH, -SH, or another group. 0186. In certain instances, for example, where Xaac is Any amino acid can be substituted by the D-form of the amino Trp, Tyr, or Phe, the peptide has a chymotrypsin cleavage site acid. that is located at a position where cleavage will liberate the 0.178 With regard to non-naturally occurring amino acids portion of the peptide carboxy-terminal to Xaas. When or naturally and non-naturally occurring amino acid analogs, Xaao is Leu, Ile or Val, the peptide can have a chymotrypsin a number of substitutions in the peptide of formula I or the cleavage site that is located at a position where cleavage will peptide of formula II are possible. For example, in some liberate the portion of the peptide amino-terminal to Xaao. At aspects Xaas can be replaced by gamma-Hydroxy-Glu or relatively high pH the same effect can be seen if Xaa, is His. gamma-Carboxy-Glu. In some aspects, Xaa, can be replaced Where Xaao is Lys or Arg, the peptide has a trypsin cleavage by an alpha Substituted amino acid Such as L-alpha-meth site that is located at a position where cleavage will liberate ylphenylalanine or by analogues such as: 3-Amino-Tyr, Tyr portion of the peptide carboxy-terminal to Xaa. (CH); Tyr(PO(CH)); Tyr(SOH); beta-Cyclohexyl-Ala; 0187. In some instances, for example, where Xaa, or the beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; beta amino-terminal amino acid of the peptide (e.g., Xaa, or Xaa-) Cyclopropyl-Ala; beta-Quinolyl-Ala; beta-2-Thiazolyl)-Ala; is Trp, Tyr, or Phe, the peptide has a chymotrypsin cleavage beta-(Triazole-1-yl)-Ala; beta-(2-Pyridyl)-Ala; beta-(3-Py site that is located at a position where cleavage will liberate US 2016/O 184387 A1 Jun. 30, 2016

the portion of the peptide amino-terminal to Xaa (or Xaa, or Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa-Xaa (II) (SEQ Xaas) along with Xaa, Xaa, or Xaas. If Xaa, or the amino ID NO:49) are shown in Table A2 below. terminal amino acid of the peptide of the invention (e.g., Xaa or Xaa) is Lys or Arg, the peptide has a trypsin cleavage site TABLE A2 that is located at a position where cleavage will liberate por tion of the peptide amino-terminal to Xaa, along with Xaa, Glin Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Xaa- or Xaa). If Xaa, or the amino-terminal amino acid of Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 51) the peptide of the invention is Leu, Ile or Val, the peptide can Asn Lieu. Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn have a chymotrypsin cleavage site that is located at a position Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 52) where cleavage will liberate the portion of the peptide amino Asn Ser Ser Glin Tyr Cys Cys Glu Tyr Cys Cys Asn terminal to Xaa. At relatively high pH the same effect is seen Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 53) when Xaa is His. 0188 If fully-folded, disulfide bonds may be present Glin Ser Ser Glin Tyr Cys Cys Glu Tyr Cys Cys Asn between: Cys and Cys: Cys, and Cysis; and Cys and Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO. 54) Cyss. In some aspects, the GC-C agonist peptides are iden Asn Ser Ser Asn Tyr Cys Cys Glu Ala Cys Cys Asn tical to or have sequence similarity to ST peptides. However, Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 55) in Some aspects the GC-C agonist peptides comprise amino Asn Ser Ser Asn Tyr Cys Cys Glu Asn. Cys Cys Asn acid changes and/or additions that improve functionality. Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 56 These changes can, for example, increase or decrease activity (e.g., increase or decrease the ability of the peptide to reduce Asn Ser Ser Asn Tyr Cys Cys Glu. Cys Cys Cys Asn phosphate uptake), alter the ability of the peptide to fold Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 57) correctly, alter the stability of the peptide, alter the ability of Asn Ser Ser Asn Tyr Cys Cys Glu Glu. Cys Cys Asn the peptide to bind the GC-C receptor, and/or decrease tox Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 58) icity. In some instances, the peptides may function more desirably thana wild-type ST peptide. For example, in certain Asn Ser Ser Asn Tyr Cys Cys Glu. His Cys Cys Asn instances, undesirable side effects such as diarrhea and dehy Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO. 59) dration are reduced. Asn Ser Ser Asn Tyr Cys Cys Glu Lys Cys Cys Asn 0189 In the case of a peptide comprising or consisting of Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 6O) the sequence (I) Xaa, Xaa, Xaa, Xaa, Xaas Cys. CyS7 Xaas Asn Ser Ser Asn Tyr Cys Cys Glu Phe Cys Cys Asn Xaao CySo CyS Xaa12 Xaa1s Xaa14 CyS is Xaa16 Xaa17 Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 61) Cyss Xaa, Xaa-Xaa (SEQIDNO:50) or Xaa, Xaa-Xaa Xaa-Xaas Cys Cys Glu Xaa, Cys Cys Asn Pro Ala Cys Thr Asn Ser Ser Asn Tyr Cys Cys Glu Ser Cys Cys Asn Gly Cys Tyr Xaa, Xaa, (II) (SEQID NO:49) where: Xaa, Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 62) Xaa-Xaas Xaa, Xaas is missing and/or the sequence Xaao Asn Ser Ser Asn Tyr Cys Cys Glu Trp Cys Cys Asn Xaao Xaa-1 is missing, the peptide can optionally comprise Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 63) additional carboxy-terminal and/or amino-terminal amino acids. For example, the peptide can include an amino terminal Cys Cys Glu Ala Cys Cys Asn Pro Ala Cys Thr Gly sequence that facilitates recombinant production of the pep Cys Tyr (SEQ ID NO: 64) tide and is cleaved prior to administration of the peptide to a patient. The peptide can also include other amino-terminal or Cys Cys Glu Asn Cys Cys Asn Pro Ala Cys Thr Gly carboxy-terminal amino acids. In some instances, the addi Cys Tyr (SEQ ID NO: 65) tional amino acids protect the peptide, Stabilize the peptide, Cys Cys Glu. Cys Cys Cys Asn Pro Ala Cys Thr Gly and/or alter the activity of the peptide. In instances. Some or Cys Tyr (SEQ ID NO: 66) all of the additional amino acids are removed prior to admin istration of the peptide to a patient. The peptide can include 1. Cys Cys Glu Glu. Cys Cys Asn Pro Ala Cys Thr Gly 2,3,4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 7080,90, 100 or more Cys Tyr (SEQ ID NO: 67) amino acids at its amino-terminus and/or carboxy-terminus. Cys Cys Glu. His Cys Cys Asn Pro Ala Cys Thr Gly The number offlanking amino acids need not be the same. For Cys Tyr (SEQ ID NO: 68) example, there can be 10 additional amino acids at the amino terminus of the peptide and none at the carboxy-terminus. Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys Thr Gly 0190. In some embodiments, the peptide comprises the Cys Tyr (SEQ ID NO: 69) amino acid sequence (I): Xaa, Xaa, Xaa- Xaa, Xaas Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly CySCyS7Xaas Xaao CySo CyS Xaa12 Xaa, Xaa1a Cysis Cys Tyr (SEQ ID NO: 7 O) XaaXaa,Cysis Xaao Xaao Xaa, (SEQ ID NO:50) where: Xaa, Xaa, Xaa, Xaa, Xaas is missing: Xaas is Glu; Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Xaa is Leu, Ile, Lys, Arg, Trp, Tyror Phe, Xaa is ASn; Xaa, Cys Tyr (SEQ ID NO: 71) is Pro; Xaa, is Ala; Xaa is Thr, Ala, Lys, Arg, Trp; Xaa, is Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Gly, Xaac is Tyr or Leu; and Xaao Xaa is Asp Phe or is Cys Tyr (SEQ ID NO: 72) missing. In instances where Xaao Xaa-1 and/or Xaa, Xaa Xaa-Xaa-Xaas are missing, the peptide may optionally com Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly prise additional flanking amino acids. Cys (SEQ ID NO: 73) 0191 Examples of GC-C agonist peptides which com Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys Thr Gly prise, consist, or consist essentially of the amino acid Cys (SEQ ID NO: 74) sequence Xaa, Xaa, Xaa, Xaa, Xaas CyS CyS Glu Xaa, Cys

US 2016/O 184387 A1 Jun. 30, 2016

TABLE A3 - continued amino acid is inserted between Cys and Cys; e) one amino acid is inserted between Cys and Cys and one amino acid is Cys Cys Llys Trp Cys Cys Asn Pro Ala Cys Thr Gly inserted between Cys, and Cys, f) one amino acid is inserted Cys (SEQ ID NO: 524) between Cys, and Cys, and one amino acid is inserted between Cys, and Cys, or g) one amino acid is inserted Cys Cys His Trp Cys Cys Asn Pro Ala Cys Thr Gly between Cys, and Cys, one amino acid is inserted between Cys (SEQ ID NO: 525) Cys and Cys and one amino acid is inserted between Cys, and Cys. In addition, one or more amino acids can be inserted 0.192 In specific embodiments, the GC-C agonist peptide preceding Cys and/or one or more amino acids can be comprises, consists, or consists essentially of the amino acid inserted following Cys. In some embodiments, the insertion sequence Cys CysGlu Tyr Cys Cys Asn Pro Ala Cys Thr Gly variants are peptides that bind to and/or agonize the GC-C Cys Tyr (SEQID NO:4). receptor. 0193 Also included are deletion variants of any of the GC-C agonist peptides described herein. Examples include 0.195 Examples of insertion variants of Cys CysGlu Tyr deletion variants where one, two, three or four amino acids (or Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:4) non-natural amino acids or natural or non-natural amino acid include those in which up to four amino acids (i.e., 0, 1, 2, 3 analogs), other than a Cys (or an amino acid Substituted for or 4) are inserted after each amino acid. Thus, included are Cys, e.g., an amino acid capable of forming a covalent bond peptides having the sequence: CyS Xaagola. CyS Xaaola Glu to another amino acid), are deleted. Specific examples Xaaco.4 Tyr Xaaco 4. CyS Xaaco 4. CyS Xaaco 4 ASn Xaaco a include where two (or more) amino acids are deleted and the Pro Xaaco 4 AlaXaaco-CyS Xaaco. ThrXaao. 4 Gly Xaaco peptide comprises the sequence: Cys, Cys, Xaa Xaa Cys, 4) Cys Xaa, a Tyr Xaa, (SEQID NO:527). The inserted Cys, XaaXaaXaa Cys, XaaXaa Cys,(SEQID NO:526). In amino acids can be any amino acid or amino acid analog some of these and related embodiments, two or more dele (natural or non-natural) and can be the same or different. In tions can be located between Cys, and Cys and/or between certain embodiments, the inserted amino acids are all Gly or Cys, and Cys, and/or between Cys, and Cys. However, in all Ala or a combination of Gly and Ala. other embodiments there is at most one deletion between each 0196. Also included are GC-C agonist peptides compris of Cys, and Cys, or between Cys and Cys or between Cys, ing or consisting of the sequence Xaa, Xaa-Xaa, Xaa-Xaas and Cys. Thus, included are any of the GC-Cagonist peptides CyS CyS7 Xaas Xaao CySo CyS Xaa12 Xaa, Xaa1a Cysis described hereincomprising the sequence Cys, Cys, XaaXaa Xaa, Xaa, Cysis Xaao Xaao Xaa, (SEQID NO:46), and Cys. Cys-XaaXaaXaa Cys, XaaXaa Cys, (SEQID NO:526) including, for example, variants of Cys Cys Glu Tyr Cys Cys where: a) one amino acid between Cys, and Cysis deleted; b) ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:4), in which one amino acid between Cys and Cys is deleted; c) one up to four amino acids are deleted and/or up to four amino amino acid between Cys, and Cys, is deleted; d) one amino acids are inserted. In some instances, the insertions and/or acid between Cys, and Cys is deleted and one amino acid deletions can be between Cys and Cysis or they can beamino between Cys and Cys is deleted; e) one amino acid between terminal to Cys and/or carboxy terminal to Cyss. Cys and Cys is deleted and one amino acid between Cys, and Cys, is deleted: f) one amino acid between Cys, and Cys. 0.197 In certain embodiments, a GC-C agonist peptide is is deleted and one amino acid between Cys, and Cys, is based on the core sequence: CyS CyS Glu Leu. CyS CyS ASn deleted or g) one amino acid between Cys, and Cys is Pro Ala Cys Thr Gly Cys Tyr (SEQID NO:528). To create a deleted, one amino acid between Cys and Cys is deleted and variant having a potentially functional chymotrypsin cleav one amino acid between Cys, and Cys, is deleted. In certain age site capable of inactivating the peptide, either the Leu embodiments, the deletion variants are peptides that bind to (underlined) or the Thr (underlined) can be replaced by Trp, and/or agonize the GC-C receptor. Phe or Tyr; or both the Leu and the Thr can be replaced by 0194 Also included are insertion variants of any of the (independently) Trp, Phe, or Tyr. The core sequence can be GC-C agonist peptides described herein. Examples include optionally be preceded by ASn Ser Ser Asn Tyr or Asn. Spe insertion variants where one, two, three or four amino acids cific examples of GC-C agonist peptides based on the core (e.g., Gly or Ala) are inserted before or after any amino acid sequence include those in Table A4 below. in the peptide. In some embodiments, no more than one amino acid is inserted between two Cys residues. Particular TABLE A4 examples include where two or more amino acids are inserted SEQ and the peptide comprises the sequence Cys, Cys, Xaa Xaa ID NO : Cys. Cys, Xaa Xaa Xaa Cys, Xaa Xaa Cys, (SEQ ID NO:526). In some of these and related embodiments, two or Asn Ser Ser Asn Tyr Cys Cys Glu Lieu. Cys 529 more insertions can be located between Cys, and Cys, or Cys Asn Pro Ala Cys Thr Gly Cys Tyr between Cys, and Cys, or between Cys, and Cys. However, Asn Ser Ser Asn Tyr Cys Cys Glu Lieu. Cys 53 O in other embodiments, no more than one insertion is located Cys Asn Pro Ala Cys Trp Gly Cys Tyr between Cys, and Cys, or between Cys and Cys, or between Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys 531 Cys, and Cys, Thus, included are any of the GC-C agonist Cys Asn Pro Ala Cys Thr Gly Cys Tyr peptides described herein comprising the sequence Cys, Cys, XaaXaa Cys. Cys, XaaXaaXaa Cys. XaaXaa Cys,(SEQID Cys Cys Glu Lieu. Cys Cys Asn Pro Ala Cys 528 NO:526) where: a) one amino acid is inserted between Cys, Thr Gly Cys Tyr and Cys; b) one amino acid is inserted between Cys and Cys Cys Glu Lieu. Cys Cys Asn Pro Ala Cys 532 Cys; c) one amino acid is inserted between Cys, and Cys, d) Thr Gly Cys Tyr one amino acid is inserted between Cys, and Cys, and one

US 2016/O 184387 A1 Jun. 30, 2016 27

0214 Xaa is any natural or non-natural amino acid or - Continued amino acid analog (optionally any of the 20 naturally-occur ring amino acids); Glin Glu Asp; 0215 Xaa, is Thr, Val or Gly: Asn Asp Asp; (SEO ID NO: 659) 0216 Xaa, is Gly or Ala; Arg Thir Ile Ala Asn Asp Asp; 0217 Xaas is Cys; and (SEQ ID NO: 660) 0218 Xaa is any natural or non-natural amino acid or Thir Ile Ala Asn Asp Asp; amino acid analog (optionally any of the 20 naturally-occur Asp Asp; ring amino acids) or is missing. (SEQ ID NO: 661) 0219. In certain embodiments:Xaa, is ASn; Xaa, is Ala or Arg Thr Met Asp Asn Asp Glu; Thr, Xaas is missing; and Xaa is Tyr. (SEQ ID NO: 662) 0220. In some embodiments Xaa, is immediately pre Arg Thir Ile Ala Gly Asp Asp; ceded by an amino acid sequence selected from: (SEQ ID NO: 663) Arg Thir Ile Ala Asn Asp; Ser His Thr; Asp;

Pro Ser Thr; Glu Asp; (SEQ ID NO: 664) Thir; Arg Ser Ile Ser Glin Glu Asp;

Pro Asp Pro; Thir Asp Glu; (SEQ ID NO: 665) (SEQ ID NO: 651) Arg Thir Ile Ala Thr Asp Glu; Ile Ala Glu Asp Ser His Thr; Glu; (SEQ ID NO: 652) Ile Ala Glin Asp Pro Ser Thr; (SEQ ID NO: 666) Ile Ile Thr Pro Pro Asp Pro; Ala Asn. Thir; Glin Glu Lleu;

Asn Thr; Lys Asp Asp;

(SEO ID NO : 653) Glin Glu Glu; Asp Pro Asn Thr; (SEO ID NO: 667) Arg Tyr Ile Asn Glin Glu Glu; Lys Asn Thr; (SEQ ID NO: 668) Pro Asn Thr; Ala Ser Ser Tyr Ala Ser; and (SEQ ID NO: 654) (SEQ ID NO: 669) Ile Ala Glin Asp Pro Asn Thr; Thir Ser Ser Tyr Ala Ser. (SEO ID NO : 655) 0221. In particular embodiments, the GC-C agonist pep Llys Pro Asn Thr; tide of formula III is defined as follows: 0222 Xaa, is: a) Ser, ASn, Tyr, Ala, Gln, Pro, Lys, Gly, or (SEQ ID NO: 656) Thr, or is missing; b) preceded by Lys or Tyr; c) any amino Asp Pro Gly Thr; acid; d) missing; e) any amino acid other than Cys; or f) Lys (SEO ID NO : 657) or Arg; Glu Asp Pro Gly Thr; 0223 Xaa, is: a) His, Asp, Glu, Ala, Ser, Asn., Gly, or is missing; b) His, Asp, Glu, Ala, Ser, ASn, Gly, Pro or is miss Pro Gly Thr; ing; c) Asp, Glu, any amino acid or is missing; d) Asp or Glu; e) any amino acid other than Cys; e) Glu, f) missing; g) Trp, Pro Ala Thr; Tyr or Phe; or h) Lys or Arg; 0224 Xaa- is: a) Thr, Asp, Ser, Glu, Pro, Valor Leu: Asp or (SEO ID NO : 658) Glu; b) any amino acid other than Cys; c) Glu; d) Thr; e) Thr, Val Ala Ala Arg Ala Asp Lleu; Asp, Ser, Glu, Pro, Val or Leu or is missing: f) Trp, Tyr or Phe: Gly Asp Asp; org) Lys or Arg; 0225 Cys is optionally Xaa, and is Cys, Mpt (mercapto Asn Asp Glu; proline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, or Glu; US 2016/O 184387 A1 Jun. 30, 2016 28

0226 Xaas is: a) any amino acid; b) Glu, Asp, Gln, Gly or 0241 Xaa- is any natural or non-natural amino acid or Pro; c) Glu; d) Glu or Asp; e) Asp, Ile or Glu: f) any amino amino acid analog or is missing: acid; org) any amino acid other than Cys; 0242 Xaa, is Cys, Mpt (mercaptoproline), Pen (penicil 0227 Xaa is: a) Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or lamine), Dpr (diaminopropionic acid), Asp or Glu; Phe, b) Leu, Ile, Val, Lys, Arg, Trp, Tyr or Phe; Leu, Ile, Lys, 0243 Xaas is Glu; Arg, Trp, Tyr or Phe; c) Leu, Ile, Val, Trp, Tyr or Phe; d) Trp, 0244 Xaa is Tyr, Trp, Phe or Leu: Tyr, Phe or Leu; e) Leu, Ile or Val: f). Ile, Trp or Leu; g) Trp, 0245 Xaa, is Cys, Mpt (mercaptoproline), Pen (penicil Tyr or Phe, h) Ile or Leu: i) Tyr; j) any amino acid; k) any lamine), Dpr (diaminopropionic acid), Asp or Glu; amino acid except Leu; 1) any natural or non-natural aromatic 0246 Xaas is any natural or non-natural amino acid or amino acid; or m) any amino acid other than Cys; amino acid analog other than CyS or is missing: 0228 Xaa, is: a) Cys, Ser, or Tyr; Cys; b) Cys, Mpt (mer 0247 Xaa, is any amino acid; captoproline), Pen (penicillamine), Dpr (diaminopropionic 0248 Xaa is Pro or Gly; acid), Asp or Glu; c) Ser; ord) an amino acid other than Cys; 0249 Xaa is any amino acid; 0229 Xaas is: a) Ala, Val, or Ile; b) Ala, Val, Thr, Ile, Met 0250 Xaa, is Cys, Mpt (mercaptoproline), Pen (penicil or is missing; c) any amino acid; d) Val; e) any amino acid other than Cys; or f) missing: lamine), Dpr (diaminopropionic acid), Asp or Glu; 0230 Xaa, is: a) any amino acid; b) any amino acid other (0251 Xaa, is Thr, Val or Gly: than Phe and Tyr; c) any amino acid other than Phe, Tyr, and 0252 Xaa, is Gly or Ala; Trp; d) any amino acid other than Phe, Tyr, Trp, Ile, Leu and 0253) Xaas is Cys, Mpt (mercaptoproline), Pen (penicil Val; e) any amino acid other than Phe, Tyr, Trp, Ile, Leu, Val, lamine), Dpr (diaminopropionic acid), Asp or Glu, and and His: i) any amino acid other than Gln, g) any amino acid 0254 Xaa is any amino acid or is missing. other than Lys, Arg, Phe, Tyr, and Trp; h) any amino acid other 0255. In particular embodiments, the GC-C agonist pep than Lys, Arg, Phe, Tyr, Trp, Ile, Leu and Val: i) any amino tide of formula III is defined as follows: acid other than Lys, Arg, Phe, Tyr, Trp, Ile, Leu, Val, and His: 0256 Xaa is ASn, any amino acid or is missing: j) any non-aromatic amino acid; k) missing: 1) Phe, Tyr, ASn, 0257 Xaa is Asp, Glu, any amino acid or is missing: or Trp; m) Asn., Tyr, Asp or Ala; n) Asn., Gln, or Tyro) Phe or 0258 Xaa is Asp or Glu; Tyr, p) ASn; or q) any amino acid other than Cys; 0259 Xaas is any amino acid or Glu; 0231. Xaa, is: a) Ala, Pro or Gly; b) Pro or Gly; c) Pro; d) 0260 Xaa is any amino acid or Leu: Ala, Val, Met, Thr or Ile; e) any amino acid: f) Val; g) Val or 0261 Xaa, is Cys: Pro; h) Ala or Val: i) any amino acid other than Cys;j) Pro; or 0262 Xaas is any amino acid or Val; k) Gly: 0263 Xaa, is Asn., Gln, or Tyr; 0232 Xaa, is: a) any amino acid; b) Ala, Leu, Ser, Gly, 0264 Xaao is any amino acid or Val; Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; c) Ala or Gly; d) Ala; 0265 Xaa, is any amino acid or Ala; e) Ala or Val; f) any amino acid, g) Ala or Aib (alpha-ami 0266 Xaa is any amino acid or Thr: noisobutyric acid); h) any amino acid other than Cys: i) Ala or 0267 Xaa, is any amino acid or Gly: Thr; orj) Thr: 0268 Xaas is Cys; 0233 Cys is optionally Xaa, and is a) Cys, Mpt (mer 0269 Xaa is any amino acid, Leu or missing captoproline), Pen (penicillamine), Dpr (diaminopropionic 0270. In some embodiments, the GC-C agonist peptide of acid), Asp, or Glu, or b) any amino acid other than Cys; formula III is not cleaved after Xaa, by chymotrypsin, and is 0234 Xaa, is: a) Thr, Ala, Asn. Lys, Arg, or Trp; b) Thr, defined as follows: Ala, Lys, Arg, or Trp; c) any amino acid; d) any non-aromatic (0271 Xaa, is Ser, ASn, Tyr, Ala, Gln, Pro, Lys, Gly or Thr, amino acid; e) Thr, Ala, or Trp: f) Trp, Tyr or Phe, g) Thr or or is missing: Ala; h) any amino acid: i) Thr, j) any amino acid other than 0272 Xaa, is His, Asp, Glu, Ala, Ser, Asn, or Gly or is Cys; k) Thr, Val, or Gly: 1) Thr or Val, m) Thror Gly, n)Val or missing: Thro) Val; p) Thr; or q) Gly; 0273 Xaa is Thr, Asp, Ser, Glu, Pro, Val or Leu or is 0235 Xaa, is: a) Gly, Pro or Ala; b) Gly; c) any amino missing: acid; d) Gly, Ala or Ser; e) Gly or Ala: f) any amino acid other 0274 Xaas is Asp, Ile or Glu; than Cys; or g) Ala; (0275 Xaa is Ile, Trp or Leu: 0236 Xaas is: a) Cys, Tyr or is missing; b) Cys; c) Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diamino (0276 Xaa, is Cys, Ser, or Tyr; propionic acid), Asp, Glu, ord) any amino acid other than Cys 0277 Xaas is Ala, Val, Thr, Ile, or Met or is missing: or is missing; and 0278 Xaa, is either: a) any amino acid other than Phe and 0237 Xaa is: a) Trp, Tyr, Phe, ASn, lie, Val, His or Leu: Tyr, b) any amino acid other than Phe, Tyr, and Trp, c) any b) Trp, Tyr, Phe, Asin or Leu; c) Tip, Tyr, Phe or Leu; d) Trp, amino acid other than Phe, Tyr, Trp, Ile, Leu and Val; d) any Tyr, or Phe; e) Leu, Ile or Val: f). His, Leu or Serg) Tyror Leu: amino acid other than Phe, Tyr, Trp, Ile, Leu, Val, and His; d) Lys or Arg; h) His: i) any amino acid, j) Leu, or missing, k) any non-aromatic amino acid ore) is missing: Trp, Tyr, Phe, Lys, Arg or is missing: 1) missing; m) any (0279 Xaa is Ala, Val, Met, Thr or Ile: amino acid other than Cys; or n) Tyr. 0280 Xaa is Ala or Val; 0238. In some embodiments, the GC-C agonist peptide of (0281 Xaa, is Ala or Thr; formula III is defined as follows: 0282 Xaa, is Gly, Ala or Ser; 0239 Xaa is any natural or non-natural amino acid or 0283 Xaas is Cys, Tyr or is missing; and amino acid analog or is missing: 0284. Xaa, is: a) Trp, Tyr or Phe to create a chymotrypsin 0240 Xaa is any natural or non-natural amino acid or cleavage site; b) Lys or Arg to create a trypsin cleavage site; c) amino acid analog or is missing: is missing ord) His or Leu or Ser.

US 2016/O 184387 A1 Jun. 30, 2016 32 nonpolar side chains (e.g., alanine, Valine, leucine, isoleu 0303. In some aspects, the GC-C agonist peptide com cine, proline, phenylalanine, methionine, tryptophan), beta prises or consists of about, at least about, or less than about branched side chains (e.g., threonine, Valine, isoleucine) and 150, 140,130, 120, 110, 100,90, 80, 70, 60, 50, 40, 30, 29, 28, aromatic side chains (e.g., tyrosine, phenylalanine, tryp 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, tophan, histidine). A conservative Substitution can Substitute 10, 8, 7, 6, or 5 amino acids. In some aspects, the peptide a naturally-occurring amino acid for a non-naturally-occur comprises no more than 5 amino acids that are N-terminal of ring amino acid or an amino acid analog. The insertions Cys (of Formula I or II) In some aspects, the peptide com and/or deletions can beat the N-terminus, C-terminus, and/or prises no more than 20, 15, 10, or 5 amino acids that are the internal regions of the peptide (e.g., an insertion or dele C-terminal of Cyss (of Formula I or II). tion of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids at the 0304. In some aspects, the peptides are purified. A purified C-terminus, N-terminus, and/or within about 2,3,4,5,6,7,8, peptides is separated from other proteins, lipids, and nucleic 9, or 10 amino acids of the N-terminus and/or C-terminus). In acids or from the compounds from which is it synthesized or Some instances it can be desirable to use a variant peptide that otherwise prepared. A purified peptide can constitute at least binds to and agonizes the intestinal GC-C receptor, but is less about 50, 60, 70, 80, 85,90, 95, 96.97, or 98% by dry weight active than the non-variant form the peptide. This reduced of the purified preparation. activity can arise from reduced affinity for the receptor or a reduced ability to activate the receptor once bound or reduced 0305 As noted above, certain peptides described herein stability of the peptide. can include one or more or all non-natural amino acids or amino acid analogs. Further to those described elsewhere 0299 The GC-Cagonist peptides can be cyclic peptides or herein (e.g., Supra), examples include: a non-natural analogue linear peptides. In addition, multiple copies of the same pep of tyrosine; a non-natural analogue of glutamine; a non-natu tide can be incorporated into a single cyclic or linear peptide. ral analogue of phenylalanine; a non-natural analogue of Cyclic peptides can be prepared by methods known in the art. serine; a non-natural analogue of threonine; an alkyl, aryl, For example, macrocyclization is often accomplished by acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alk forming an amide bond between the peptide N- and C-ter enyl, alkynl, ether, thiol, Sulfonyl, seleno, ester, thioacid, mini, between a side chain and the N- or C-terminus e.g., borate, boronate, phospho, phosphono, phosphine, heterocy with KFe(CN) at pH -8.5 (Samson et al., Endocrinology, clic, enone, imine, aldehyde, hydroxylamine, keto, or amino 137:5182-5185, 1996), or between two amino acid side Substituted amino acid, or any combination thereof an amino chains, such as cysteine (DeGrado, Adv Protein Chem, 39:51 acid with a photoactivatable cross-linker; a spin-labeled 124, 1988). amino acid; a fluorescent amino acid; an amino acid with a 0300. The peptides can include the amino acid sequence of novel functional group; an amino acid that covalently or a peptide that occurs naturally in a vertebrate (e.g., mamma noncovalently interacts with another molecule; a metal bind lian) species or in a bacterial species. In addition, the peptides ing amino acid; a metal-containing amino acid; a radioactive can be partially or completely non-naturally occurring pep amino acid; a photocaged and/or photoisomerizable amino tides. acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto 0301 Also included are peptide analogs corresponding to containing amino acid; amino acids comprising polyethylene the GC-C agonist peptides described herein. Peptide analogs glycol or polyether; a heavy atom Substituted amino acid are commonly used in the pharmaceutical industry as non (e.g., an amino acid containing deuterium, tritium, C, N, peptide drugs with properties analogous to those of the tem or 'O); a chemically cleavable orphotocleavable amino acid; plate peptide. These types of non-peptide compound are an amino acid with an elongated side chain; an amino acid termed "peptide mimetics' or “peptidomimetics’ (Luthman, containing a toxic group; a Sugar Substituted amino acid, e.g., et al., A Textbook of Drug Design and Development, 14:386 a Sugar Substituted serine or the like; a carbon-linked Sugar 406, 2nd Ed., Harwood Academic Publishers, 1996; Joachim containing amino acid; a redox-active amino acid; an O.-hy Grante, Angew. Chem. Int. Ed. Engl., 33:1699-1720, 1994: droxy containing acid; an amino thio acid containing amino Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber and Fre acid; an O.C. disubstituted amino acid; a 3-amino acid; a idinger TINS, p. 392 (1985); and Evans et al., J. Med. Chem. cyclic amino acid other than proline; an O-methyl-L-ty 30:229, 1987). A peptidomimetic is a molecule that mimics rosine; an L-3-(2-naphthyl)alanine; a 3-methyl-phenylala the biological activity of a peptide but is no longer peptidic in nine; a p-acetyl-L-phenylalanine; an 0-4-allyl-L-tyrosine; a chemical nature. Peptidomimetic compounds are known in 4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAcf-serine; an the art and are described, for example, in U.S. Pat. No. 6,245, L-Dopa; a fluorinated phenylalanine; an isopropyl-L-pheny 886. lalanine; a p-azido-L-phenylalanine; a p-acyl-L-phenylala 0302) The present invention also includes peptoids. Pep nine; a p-benzoyl-L-phenylalanine; an L-phosphoserine; a toid derivatives of peptides represent another form of modi phosphonoSerine; a phosphonotyrosine; a p-iodo-phenylala fied peptides that retain the important structural determinants nine; a 4-fluorophenylglycine; a p-bromophenylalanine; a for biological activity, yet eliminate the peptide bonds, p-amino-L-phenylalanine; a isopropyl-L-phenylalanine; thereby conferring resistance to proteolysis (see, e.g., Simon L-3-(2-naphthyl)alanine; an amino-, isopropyl-, or O-allyl et al., PNAS USA. 89:9367-9371, 1992). Peptoids are oligo containing phenylalanine analogue; a dopa, O-methyl-L-ty mers of N-substituted glycines. A number of N-alkyl groups rosine; a glycosylated amino acid; a p-(propargyloxy)pheny have been described, each corresponding to the side chain of lalanine; dimethyl-Lysine; hydroxy-proline; a natural amino acid. The peptoids of the present invention mercaptopropionic acid; methyl-lysine, 3-nitro-tyrosine; include compounds in which at least one amino acid, a few norleucine; pyro-glutamic acid; Z (Carbobenzoxyl); amino acids, or all amino acid residues are replaced by the e-Acetyl-Lysine; B-alanine; aminobenzoyl derivative; ami corresponding N-substituted glycines. Peptoid libraries are nobutyric acid (Abu); citrulline; aminohexanoic acid; ami described, for example, in U.S. Pat. No. 5,811,387. noisobutyric acid; cyclohexylalanine; d-cyclohexylalanine; US 2016/O 184387 A1 Jun. 30, 2016

hydroxyproline; nitro-arginine; nitro-phenylalanine; nitro 0312. In some embodiments, one or more disulfide bonds tyrosine; norvaline; octahydroindole carboxylate; ornithine; can be replaced by alternative covalent cross-linkages, e.g., penicillamine; tetrahydroisoquinoline; acetamidomethyl an amide linkage (-CHCH(O)NHCH or -CH-NHCH protected amino acids and pegylated amino acids. Further (O)CH ), an ester linkage, a thioester linkage, a lactam examples of non-natural amino acids and amino acid analogs bridge, a carbamoyl linkage, a urea linkage, a thiourea link can be found in U.S. Application Nos. 2003/0108885 and age, a phosphonate ester linkage, an alkyl linkage 2003/0082575, and the references cited therein. (—CH2CH2CHCH ), al alkenyl linkage(- 0306. In some embodiments, an amino acid can be CH-CH=CHCH ), an ether linkage ( CHCHOCH2— replaced by a naturally-occurring, non-essential amino acid, or —CHOCHCH ), a thioether linkage e.g., taurine. (—CHCH-SCH or —CH-SCHCH ), an amine link age ( CHCH-NHCH or —CH-NHCHCH ) or a 0307. In some embodiments, 1,2,3,4, 5, or 6 cysteines are thioamide linkage ( CH-CH(S)HNHCH O deleted or replaced with a different amino acid. In particular —CH-NHCH(S)CH, ). For example, Ledu et al. (PNAS. aspects, the most N-terminal and/or C-terminal cysteine resi 100: 11263-78, 2003) describe methods for preparing lactam due or residues are deleted or replaced with a different amino and amide cross-links. Schafmeister et al. (J. Am. Chem. Soc. acid. In certain embodiments, the different amino acid is 122:5891, 2000) describe stable, hydrocarbon cross-links. alanine or serine. Hydrocarbon cross links can be produced via metathesis (or 0308 Peptides can be polymers of L-amino acids, methathesis followed by hydrogenation in the case of Satu D-amino acids, or a combination thereof. For example, in rated hydrocarbons cross-links) using one or another of the certain embodiments, the peptides are D retro-inverso pep Grubbs catalysts (available from Materia, Inc. and Sigma tides. The term “retro-inverso isomer' refers to an isomer of Aldrich and described, for example, in U.S. Pat. Nos. 5,831, a linear peptide in which the direction of the sequence is 108 and 6,111,121). In some instances, the generation of Such reversed and the chirality of each amino acid residue is alternative cross-links requires replacing the Cys residues inverted. See, e.g., Jameson et al., Nature. 368:744-746, with other residues such as Lys or Glu or non-naturally occur 1994; Brady et al., Nature. 368:692-693, 1994. The net result ring amino acids. In addition, the lactam, amide and hydro of combining D-enantiomers and reverse synthesis is that the carbon cross-linkages can be used to stabilize the peptide positions of carbonyl and amino groups in each amide bond even if they link amino acids at positions other than those are exchanged, while the position of the side-chain groups at occupied by CyS. Such cross-linkages can occur, for example, each alpha carbon is preserved. Unless specifically stated between two amino acids that are separated by two amino otherwise, any given L-amino acid sequence of the invention acids or between two amino acids that are separated by six can be made into a D retro-inverso peptide by synthesizing a amino acids (see, e.g., Schafmeister et al., J. Am. Chem. Soc. reverse of the sequence for the corresponding native L-amino 122:5891, 2000). acid sequence 0313 The GC-C agonist peptides can be modified using 0309 Methods of manufacturing peptides containing non standard modifications. Modifications may occur at the natural amino acids can be found, for example, in U.S. Appli amino (N ), carboxy (C ) terminus, internally or a com cation Nos. 2003/0108885 and 2003/0082575, Deiters et al., bination of any of the foregoing. In some aspects, there may JAm Chem. Soc. 125:11782-3, 2003; Chin et al., Science. be more than one type of modification of the peptide. Modi 301:964-7, 2003, and the references cited therein. fications include but are not limited to: acetylation, amida 0310. In some aspects, the GC-C agonist peptides can tion, biotinylation, cinnamoylation, farnesylation, formyla have one or more conventional peptide bonds replaced by an tion, myristoylation, palmitoylation, phosphorylation (Ser, alternative bond. Such replacements can increase the stability Tyr or Thr), Stearoylation, Succinylation, Sulfurylation and of the peptide. For example, replacement of the peptide bond cyclisation (via disulfide bridges or amide cyclisation), and between Cyss and Xaa, (of Formula I or II) with an alter modification by Cy3 or Cy5. The peptides of the invention native bond can reduce cleavage by carboxypeptidases and may also be modified by 2,4-dinitrophenyl (DNP). DNP may increase half-life in the digestive tract. Bonds that can lysin, modification by 7-Amino-4-methyl-coumarin (AMC), replace peptide bonds include without limitation: a retro fluorescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro inverso bonds (C(O)—NH instead of NH COO); a reduced anilide, rhodamine B, EDANS (5-((2-aminoethyl)amino) amide bond (NH-CH-); a thiomethylene bond (S-CH or naphthalene-1-sulfonic acid), dabcyl, dabsyl, dansyl, Texas CH-S); an oxomethylene bond (O CH or CH O); an red, FMOC, and Tamra (Tetramethylrhodamine). The pep ethylene bond (CH, CH,); a thioamide bond (C(S)- NH); tides of the invention may also be conjugated to, for example, a trans-olefine bond (CH=CH); an fluoro substituted trans polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 olefine bond (CF–CH); a ketomethylene bond (C(O) CHR straight or branched alkyl groups); fatty acid radicals; com or CHR C(O) where R is H or CH; and a fluoro-ketometh binations of PEG, alkyl groups and fatty acid radicals (see ylene bond (C(O) CFR or CFR C(O) where R is Hor For U.S. Pat. No. 6,309,633; Soltero et al., Innovations in Phar CH. maceutical Technology. 106-1 10, 2001); BSA and KLH 0311. In some GC-C agonist peptides, one or both mem (Keyhole Limpet Hemocyanin). For instance, in certain bers of one or more pairs of Cys residues which normally embodiments, the N-terminal amino acid, C-terminal amino form a disulfide bond are replaced by homocysteine, penicil acid, or both, is conjuated to a PEG molecule. lamine, 3-mercaptoproline (see, e.g., Kolodzie et al., Int J 0314. In certain embodiments, the GC-C agonist peptides Pept Protein Res. 48:274, 1996); BB dimethylcysteine (see, described herein can be present with a counterion. Exemplary e.g., Hunt et al., Int J Pept Protein Res. 42:249, 1993) or counterions include Salts of acetate, benzenesulfonate, ben diaminopropionic acid (see, e.g., Smith et al., J Med Chem. Zoate, calcium edetate, camsylate, carbonate, citrate, edetate 21:117, 1978), to form alternative internal cross-links at the (EDTA), edisylate, embonate, esylate, fumarate, gluceptate, positions of the normal disulfide bonds. gluconate, glutamate, glycolylarsanilate, hexylresorcinate, US 2016/O 184387 A1 Jun. 30, 2016 34 iodide, bromide, chloride, hydroxynaphthoate, isethionate, lational fusion is generated. Expression of the fusion gene lactate, lactobionate, estolate, maleate, malate, mandelate, results intranslation of a single polypeptide that includes both mesylate, mucate, napsylate, nitrate, pantothenate, phos the peptide of interest and the affinity tag. In some instances phate, Salicylate, Stearate. Succinate, Sulfate, tartarate, theo where affinity tags are utilized, DNA sequence encoding a clate, acetamidobenzoate, adipate, alginate, aminosalicylate, protease recognition site will be fused between the reading anhydromethylenecitrate, ascorbate, aspartate, camphorate, frames for the affinity tag and the peptide of interest. caprate, caproate, caprylate, cinnamate, cyclamate, dichloro 0318 Genetic constructs and methods suitable for produc acetate, formate, gentisate, glucuronate, glycerophosphate, tion of immature and mature forms of the peptides and vari glycolate, hippurate, fluoride, malonate, napadisylate, nico ants of the invention in protein expression systems other than bacteria, and well knownto those skilled in the art, can also be tinate, oleate, orotate, oxalate, oxoglutarate, palmitate, pecti used to produce peptides in a biological system. nate, pectinate polymer, phenylethylbarbiturate, picrate, pro 0319 Peptides and variants thereof can be synthesized by pionate, pidolate, sebacate, rhodanide, tosylate, and tannate. the Solid-phase chemical synthesis. For example, the peptide 0315 GC-C agonist peptides can be produced according can be synthesized on Cyc(4-CH.Bxl)-OCH-4-(oxym to a variety of techniques. For instance, peptides can be pro ethyl)-phenylacetamidomethyl resin using a double coupling duced in bacteria including, without limitation, E. coli, or in program. Protecting groups must be used appropriately to other systems for peptide or protein production (e.g., Bacillus create the correct disulfide bond pattern. For example, the subtilis, baculovirus expression systems using Drosophila following protecting groups can be used: t-butyloxycarbonyl Sf9 cells, yeast or filamentous fungal expression systems, (alpha-amino groups); acetamidomethyl (thiol groups of Cys mammalian cell expression systems), or they can be chemi residues B and E): 4-methylbenzyl (thiol groups of Cys resi cally synthesized. If the peptide or variant peptide is to be dues C and F); benzyl (Y-carboxyl of glutamic acid and the produced in bacteria, e.g., E. coli, the nucleic acid molecule hydroxyl group of threonine, if present); and bromobenzyl (phenolic group of tyrosine, if present). Coupling is effected encoding the peptide may optionally encode a leader with symmetrical anhydride oft-butoxylcarbonylamino acids sequence that permits the secretion of the mature peptide or hydroxybenzotriazole ester (for asparagine or glutamine from the cell. Thus, the sequence encoding the peptide can residues), and the peptide is deprotected and cleaved from the include the pre sequence and the pro sequence of for Solid Support in hydrogen fluoride, dimethyl sulfide, anisole, example, a naturally-occurring bacterial ST peptide. The and p-thiocresol using 8/1/1/0.5 ratio (v/v/v/w) at 0°C. for 60 secreted, mature peptide can be purified from the culture min. After removal of hydrogen fluoride and dimethylsulfide medium. by reduced pressure and anisole and p-thiocresol by extrac 0316. In some instances, the sequence encoding a peptide tion with ethyl ether and ethyl acetate sequentially, crude of the invention is inserted into a vector capable of delivering peptides are extracted with a mixture of 0.5M sodium phos and maintaining the nucleic acid molecule in a bacterial cell. phate buffer, pH 8.0 and N,N-dimethylformamide using 1/1 The DNA molecule may be inserted into an autonomously ratio, V/v. The disulfide bond for Cys residues B and E is the replicating vector (Suitable vectors include, for example, formed using dimethyl sulfoxide (Tam et al., J. Am. Chem. pGEM37 and pcDNA3, and derivatives thereof). The vector Soc. 113:6657-62, 1991). The resulting peptide can be puri nucleic acid may be a bacterial or bacteriophage DNA such as fied by reverse-phase chromatography. The disulfide bond bacteriophage lambda or M13 and derivatives thereof. Con between Cys residues Cand F is formed by first dissolving the struction of a vector containing a nucleic acid described peptide in 50% acetic acid in water. Saturated iodine solution hereincan be followed by transformation of a host cell such as in glacial acetic acid is added (1 ml iodine solution per 100 ml a bacterium. Suitable bacterial hosts include but are not lim Solution). After incubation at room temperature for 2 days in an enclosed glass container, the Solution is diluted five-fold ited to, E. coli B. subtilis, Pseudomonas, Salmonella. The with deionized water and extracted with ethyl ether four times genetic construct may also include, in addition to the encod for removal of unreacted iodine. After removal of the residual ing nucleic acid molecule, elements that allow expression, amount of ethyl ether by rotary evaporation the solution of Such as a promoter and regulatory sequences. The expression crude product is lyophilized and purified by successive vectors may contain transcriptional control sequences that reverse-phase chromatography. control transcriptional initiation, Such as promoter, enhancer, 0320 Peptides can also be synthesized by many other operator, and repressor sequences. A variety of transcrip methods including solid phase synthesis using traditional tional control sequences are well known to those in the art. FMOC protection (i.e., coupling with DCC-HOBt and depro The expression vector can also include a translation regula tection with piperidine in DMF). Cys thiol groups can be trityl tory sequence (e.g., an untranslated 5' sequence, an untrans protected. Treatment with TFA can be used for final depro lated 3' sequence, or an internal ribosome entry site). The tection of the peptide and release of the peptide from the vector can be capable of autonomous replication or it can Solid-state resin. In many cases air oxidation is sufficient to integrate into host DNA to ensure stability during peptide achieve proper disulfide bond formation. production. In some embodiments, the vectors, expression 0321. The ability of peptides and other agents to bind systems and methods described in U.S. Pat. No. 5,395.490 and/or agonize to the intestinal GC-C receptor can be tested, can be used to produce the GC-C agonist peptides described for example, in assays such as intestinal GC-C receptor bind herein. ing assays. In one example, cells of the T84 human colon carcinoma cell line (American Type Culture Collection (Be 0317. The protein coding sequence that includes a peptide thesda, Md.)) are grown to confluence in 24-well culture of the invention can also be fused to a nucleic acid encoding plates with a 1:1 mixture of Hams F12 medium and Dulbec a polypeptide affinity tag, e.g., glutathione S-transferase co's modified Eagle's medium (DMEM), supplemented with (GST), maltose E binding protein, protein A, FLAG tag, 5% fetal calf serum. Cells used in the assay are optionally hexa-histidine, myc tag or the influenza HA tag, in order to between passages 54-60. Briefly, T84 cell monolayers in facilitate purification. The affinity tag or reporter fusion joins 24-well plates are washed twice with 1 ml of binding buffer the reading frame of the peptide of interest to the reading (DMEM containing 0.05% bovine serum albuminand 25 mM frame of the gene encoding the affinity tag such that a trans HEPES, pH 7.2), then incubated for 30 min at 37° C. in the US 2016/O 184387 A1 Jun. 30, 2016 presence of mature radioactively labeled E. coli ST peptide 0331 Adenylyl cyclase is the effector molecule of one of and the test material at various concentrations. The cells are the most widely utilized signal transduction pathways. Its then washed four times with 1 ml of DMEM and solubilized product, cAMP modulates cell growth and differentiation in with 0.5 ml/well 1N NaOH. The level of radioactivity in the organisms from bacteria to higher eukaryotes. In animals, solubilized material is determined using standard methods. there are transmembrane adenylyl-cyclases (tmACs) and 0322. Additional examples of GC-C agonist peptides are soluble adenylate cyclase (SAC). See, e.g., Tresguerres et al., described, for instance, in U.S. Pat. Nos. 7,041,786; 7,304, Kidney Int. 79:1277-1288, 2011. Unlike timACs, SACs are not 036; 7,371,727; 7,494,979; 7,704,947; 7,799,897; 7,745,409; transmembrane proteins and are found distributed throughout 7,772,188; 7.879,802; 7,910,546; 8,034,782; 8,080,526; the cytoplasm and in specific organelles where they are 8,101,579; 8,114,831; 8,110,553; 8,357,775; and 8,367,800; thought to be the source of second messenger mediating the U.S. Application Nos. 2013/0096071; 2013/0190238; 2012/ intracellular functions of cAMP. See, e.g., Buck and Levin, 0040892; 2012/004.0025; 2012/0213846; 2012/0289460; Sensors. (Basel) 11:2112-2128, 2011. Thus, tmACs are 2011/0118184; 2010/0152118; 2010/0048489; 2010/ directly modulated by G proteins which transduce extracel 0120694; 2010/0261877; 2009/0253634; 2009/0192083: lular signals into intracellular cAMP changes. In contrast, 2009/0305993; and PCT Publication Nos. WO 2006/086653 SAC isoforms are regulated by intracellular signals, including and WO 2002/098.912, each of which is incorporated by bicarbonate, calcium, and ATP. reference in its entirety. 0332 Cystic fibrosis transmembrane regulator (CFTR) is 0323 D. Soluble Guanylate Cyclase Agonists a chloride and bicarbonate ion channel that functions at the 0324. In certain embodiments, the compound is a soluble epithelium of multiple tissues. This channel has been shown guanylate cyclase (sGC) agonist. Guanine nucleotidyl (gua to be in charge of HCO' secretion in the small intestine, nylyl; guanylate) cyclases (GCs) are widely distributed signal where said bicarbonate determines the pH on the surface of transduction enzymes that, in response to various cellular the mucosa. See, e.g., Kunzelmann and Mall, Physiol Rev. stimuli, convert GTP into the second messenger cyclic GMP 82:245-289, 2002. CFTR is regulated by cAMP: phosphory (cGMP). There are both membrane-associated and soluble lation of the CFTR regulatory domain by cAMP-dependent guanylate cyclases, both of which can increase the intracel protein kinase A (PKA) increases its activity. Selective acti lular concentrations of c(CMP. Vation of this ion channel can thus result in alkalinization of 0325 In the enterocytes of the intestine, increased coMP the luminal membrane and thereby reduce or decrease the production inhibits intestinal Na+/H+ exchange activity, CEPG. According to one non-limiting theory, selective stimu resulting in alkalinization of the intestinal mucosa. See, e.g., lation of timACs in the intestinal tract should therefore Fawcus et al., Comp Biochem. Physiol A Physiol. 118:291 increase intracellular cAMP, stimulate PKA, increase CFTR 295, 1997. Without being bound by any one mechanism, in activity and thereby inhibit the uptake of Pivia CEPG effects. certain aspects a soluble guanylate cyclase activator inhibits In specific aspects, the compound selectively activates or reduces phosphate uptake in the gastrointestinal tract tmACs, for instance, relative to SACs. increasing c(GMP production and thereby increasing the alka 0333 Adenylate cyclase agonists such as forskolin have linization of the intestinal mucosa. been shown to increase cAMP-mediated duodenal bicarbon ate secretion (without increasing gastric bicarbonate secre 0326 General examples of sGC agonists include heme tion), optionally via signaling of CFTR. See, e.g., Takeuchi et dependent and heme-independent activators. See, e.g., al., Am. J. Physiol. 272(3Pt 1):G646-53, 1997. Without being Evgenov et al., Nat. Rev. Drug Discov. 5:755-768, 2006. bound by any one mechanism, in certain aspects an adenylate According to one non-limiting theory, these and other sGC cyclase agonist inhibits or reduces phosphate uptake in the activators can be used to selectively activate sGC in the intes gastrointestinal tract by Stimulating bicarbonate Secretion tine, increase concentrations of c(GMP, and thereby inhibit into the Small intestine. In some embodiments, and without phosphate uptake as described herein. being bound by any one mechanism, an adenylate cyclase 0327. In some embodiments, and without being bound by agonist inhibits or reduces phosphate uptake in the gas any one mechanism, a sGC agonist inhibits or reduces phos trointestinal tract by decreasing water absorption in the Small phate uptake in the gastrointestinal tract by decreasing water intestine. absorption in the Small intestine. 0334. In particular embodiments, the compound is an ago 0328 Non-limiting examples of sGC agonists include Bay nist of adenylate cyclase III (AC-III), optionally an agonist of 41-2271, Bay 58-2667, and the compounds shown in FIGS. one or more of the AC-III isoforms ADCY1, ADCY2, 9A-9L. Additional structures of exemplary sGC agonists are ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, disclosed, together with methods for their synthesis, in U.S. ADCY9, and/or ADCY10. Pat. No. 7,087,644 and PCT Publication No. WO 2013/ 0335 Particular examples of adenylate cyclase agonists 101830, each of which is incorporated by reference in its include labdane diterpenes such as forskolin and analogs/ entirety. derivatives thereof, including water-soluble forskolin analogs 0329 E. Adenylate Cyclase Agonists such as colforsin (NKH477). Forskolin is a diterpene com 0330. In certain embodiments, the compound is an adeny pound isolated from plants that activates all mammalian late cyclase agonist, optionally a selective agonist. Adenylate tmACs with the exception of timAC IX (mammalian SAC is cyclase (or adenylyl cyclase) refers to a class of enzymes that insensitive to forskolin). See, e.g., Kamenetsky et al., J. Mol. catalyze the conversion of ATP to 3',5'-cyclic AMP (cAMP) Biol. 362:623-639, 2006. Forskolin stimulation can produce and pyrophosphate. Divalent cations (e.g., Mg) are often potent and prolonged cAMP changes. See, e.g., Tresguerres involved in this enzymatic activity. The cAMP produced by et al., Kidney Int. 79:1277-1288, 2011. The structure of for adenylate cyclase serves as a regulatory signal via specific skolin and several forskolin analogs is illustrated in FIG. 10. cAMP-binding proteins, including transcription factors or Water soluble derivatives of forskolin include those acylated other enzymes (e.g., cAMP-dependent kinases). at C-6 or C-7 with a polar aliphatic amine. These derivatives US 2016/O 184387 A1 Jun. 30, 2016 36 are typically more selective for ACs, with fewer off-target Some aspects an imidazoline-1 receptor agonist inhibits or activities. See, e.g., Hartzell and Budnitz, Molecular Phar reduces phosphate uptake in the gastrointestinal tract by macology 41:880-888, 1992. Thus, certain aspects include increasing DBS. the use of soluble forskolin analogs that selectively activate 0341. According to another non-limiting theory, imidazo adenylate cyclases in the cells lining the gastrointestinal tract. line-1 receptor agonists such as moXonidine have also been 0336 Particular examples offorskolin analogs/derivatives shown to decrease acid secretion in the gastrointestinal tract. include aminoalkylcarbamyl derivatives of forskolin, includ See, e.g., Glavin and Smyth, Br J Pharmacol. 114:751-4, ing 1-aminoalkylcarbamates, 9-aminoalkylcarbamates, 1995. Hence, and without being bound by any one mecha 7-aminoalkylcarbamates, 6-aminoalkycarbamates, 6,7-di nism, in certain aspects an imidazoline-1 receptor agonist aminoalkylcarbamates, 1,6-diaminoalkylcarbamates, 1,7-di inhibits or reduces phosphate uptake in the gastrointestinal aminoalkylcarbamates, and 1,6,7-triaminoalkylcarbamates tract by inhibiting or reducing acid secretion in the gas of forskolin, which can be used as intermediates in the Syn trointestinal tract, e.g., the Small intestine. In specific aspects, thesis of forskolin derivatives. See U.S. Pat. No. 5,350,864. and without being bound by any one mechanism, an imida Additional examples of forskolin analogs/derivatives include Zoline-1 receptor agonist inhibits or reduces phosphate 12-halogenated forskolin derivatives, including 12-chlorode uptake in the gastrointestinal tract by increasing DBS and sacetylforskolin, 12-chloroforskolin, 12-bromodesacetylfor reducing acid secretion in the Small intestine. skolin, 12-bromodesacetylforskolin, 12-fluorodesacetylfor 0342. In some embodiments, and without being bound by skolin, and 12-fluoroforskolin. See U.S. Pat. No. 4,871,764. any one mechanism, an imidazoline-2 receptoragonist inhib 0337. In some embodiments, the forskolin analog/deriva its or reduces phosphate uptake in the gastrointestinal tract by tive is 6-acetyl-7-deacetyl-forskolin, 7-deacetyl-forskolin, decreasing water absorption in the Small intestine. 7-deacetyl-6-(N-acetylglycyl)-forskolin, 7-deacetyl-7-B- 0343 Non-limiting examples of imidazoline-1 receptor hemisuccunyl-forskolin, 7-deacetyl-7-(O N-methylpiper agonists include agmatine, apraclonidine, clonidine, efar azino)-y-butryl-dihydrochlonde-forskolin, 7-HPP-forskolin, oxan, moxonidine, rillmenidine, S-23515, S-23757, LNP 6-HPP-forskolin, or colforsin daropate hydrochloride 509, LNP-911, LNP-509, S-23515, PMS-812, PMS-847, (NKH477). See, e.g., U.S. Application Nos. 2011/0171195, BU-98008 and TVP1022 (S-enantiomer of rasagiline). See 2006/0004090, and 2011/0077292; Laurenza et al., Mol also Head and Mayorov, Cardiovasc Hematol Agents Med Pharmacol. 32:133-9, 1987; Lal et al., Bioorg Med Chem. Chem. 4:17-32, 2006, incorporated by reference in its 6:2075-83, 1998; Mori et al., J. Cardiovasc. Pharmacol. entirety. 24:310-6, 2004. See also Levin, Tetrahedon Letters. 37:3079 3082, 1996 for exemplary methods of synthesizing forskolin 0344 Structures of exemplary imidazoline receptor ago analogs, and Lal et al., Indian J. Chemistry. 45B:232-246, nists are shown in FIG. 11, and are further disclosed, together 2006, for additional examples of water soluble forskolin ana with methods for their synthesis, in U.S. Pat. Nos. 4.323.570; logs and methods of synthesizing the same. Additional struc 5,686,477; 3,988,464; 6,300,366; 5,492,912; 5,492,912; and tures of exemplary adenylate cyclase agonists are disclosed, PCT Publication No. WO200141764, each of which is incor together with methods for their synthesis, in U.S. Pat. No. porated by reference in its entirety. 4,954,642 and Khandelwal et al., J Med Chem. 31:1872-9, 0345 Additional examples of imidazoline receptor ago 1988. See also Cunliffe et al., Electrophoresis. 28:1913-20, nists include those described in U.S. Pat. No. 7,309,706; U.S. 2007 for exemplary methods/assays of detecting agonist Pat. No.5,686,477 (EP710,658); U.S. Pat. No.5,925,665 (EP stimulated adenylate cyclase activity. These references are 846,688); WO 2001/41764; and WO 2000/02878. The incorporated by reference in their entireties. 5-(aryloxymethyl)-oxazoline derivatives described in U.S. 0338 F. Imidazoline-1 Receptor Agonists Pat. No. 5,686,477 are characterized by a selective affinity for 0339. In certain embodiments, the compound is an imida the imidazoline-1 receptor. The imidazoline derivatives Zoline-1 receptoragonist, optionally a selective agonist. Imi described in U.S. Pat. No. 5,925,665 bind to imidazoline dazoline receptors include a family of nonadrenergic high receptors without significantly binding to adrenergic recep affinity binding sites for clonidine, idaZOXan, and other tors. WO 2001/41764 describes isoquinoline and quinoline imidazoles. There are three classes of imidazoline receptors: derivatives which possess an affinity for imidazoline recep the I1 receptor, which mediates the sympatho-inhibitory tors. WO 2000/02878 describes exemplary B-carboline actions of imidazolines to lower blood pressure; the I2 recep derivatives as potential ligands for imidazoline receptors. tor, an allosteric binding site of monoamine oxidase and is These references are incorporated by reference in their entire involved in pain modulation and neuroprotection; and the I3 ties. receptor, which regulates insulin secretion from pancreatic 0346) G. Cholinergic Agonists beta cells. In some aspects, the compound is a selective imi 0347 In certain embodiments, the compound is a cholin dazoline-1 receptoragonist, for instance, relative to imidazo ergic agonist, optionally a selective cholinergic agonist. line-2 and/or imidazoline-3 receptors. Examples of cholinergicagonists include indirect cholinergic 0340. The subclass of imidazoline-1 receptors mediate in agonists, which stimulate the production or release of acetyl part the central hypotensive effects of clonidine-like drugs. choline (e.g., actetylcholinesterase inhibitors), and direct According to one non-limiting theory, activated imidazo cholinergic agonists, which bind to and stimulate one or more line-1 receptors trigger the hydrolysis of phosphatidylcholine acetylcholine receptors. The neurotransmitter acetylcholine into DAG, which then triggers the synthesis of second mes (2-acetoxy-N,N,N-trimethylethanaminium) is an ester of sengers such as arachidonic acid and downstream acetic acid and choline. General examples of acetylcholine eicosanoids such as PGE. See, e.g., Ernsberger, Ann. NY receptors include nicotinic acetylcholine receptors and mus Acad. Sci. 881:35-53 1999. PGE is an endogenous inducer of carinic acetylcholine receptors. Nicotinic acetylcholine DBS. See, e.g., Takeuchi et al., Gastroenterology. 113:1553 receptors are ligand-gated ion channels composed of five 1559, 1997). Without being bound by any one mechanism, in protein subunits. US 2016/O 184387 A1 Jun. 30, 2016 37

0348 Muscarinic acetylcholine receptors (i.e., M1, M2, of agonists because these structures replace the labile ester M3, M4, and M5) are G-protein-coupled receptors that acti group of ACh with non-hydrolyzable carbamate functional vate other ionic channels via a second messenger cascade. ity. These receptors are expressed in the digestive tract including 0353 Non-limiting examples of indirect-acting cholin the salivary glands and the Smooth muscle and mucosal cells ergic agonists include acetylcholinesterase inhibitors such as in the stomach and the intestine In certain embodiments, the carbamates (e.g., physostigmine, neostigmine, pyridostig compound is a pan-agonist of muscarinic receptor Subtypes. mine), piperidines (e.g., donepizil), edrophonium, huperzine The endogenous agonist of all five muscarinic receptor Sub A, ladostigil, ungeremine, lactucopicrin, tacrine, galan types is acetylcholine, which exerts physiological control by tamine, trans-delta-9-tetrahydrocannabinol, and phosphates (e.g., isoflurophate, echothiophate, parathion, malathion). both hormonal and neuronal mechanisms. See, e.g., Eglen, Preferably, the methods provided herein will employ revers Ann. N. Y. Acad. Sci. 881:35-53, 2012. Several naturally ible acetylcholinesterase inhibitors. occurring compounds also modulate the muscarinic receptors 0354) Non-limiting examples of direct-acting cholinergic (see FIG. 12), including agonists such as muscarine (a toxin agonists include acetylcholine, nicotine. Succinylcholine, from the mushroom Aminita muscaria and from which the methacholine (acetyl-3-methylcholine), McN-A-343, carba receptor family derives its name) and pilocarpine, and antago chol (carbamoylcholine), bethanecol (carbamoyl-f-methly nists such as atropine or (-)-hyoscine (from the Solanaceae choline), muscarine, pilocarpine, oxotremorine, lobeline, and plant family). When administered in Vivo, muscarinic ago dimethylphenylpiparazinium. nists elicit salivation whereas muscarinic antagonists cause 0355 H. Prostaglandin EP4 Receptor Agonists dry mouth. 0356. In certain embodiments, the compound is E-type 0349. In some embodiments, the compound is a relatively prostanoid receptor 4 (EP4) agonist (or prostaglandin EP4 selective agonist of the M3 muscarinic receptor. The secre receptor agonist), optionally a selective agonist. The EP4 tory response of M3 is stimulated physiologically by acetyl receptor was initially described as a G protein-coupled choline (ACh). Specifically, ACh binds to the G protein receptor leading to stimulation of adenylate cyclase and linked M3 muscarinic ACh receptor, which causes elevation of intracellular cAMP levels. When first cloned as a phospholipase C to generate inositol 1,4,5-trisphosphate prostaglandin E2 (PGE) receptor that stimulated cAMP for (InP3). InP3 binds to and opens the InP3 receptor on the mation, the EP4 receptor was designated as “EP2. However, endoplasmic reticulum, which, according to one non-limiting after another cAMP-stimulating PGE receptor had been dis theory, releases Ca". Increased Ca"), activates the apical covered which was sensitive to butaprost, the butaprost-in membrane Cl- channel and the basolateral K+ channel. sensitive receptor which mediated vasorelaxation was Efflux of Clinto the acinar lumen draws Na+ across the cells, renamed “EP4. It is one of four receptors identified for and the osmotic gradient generates fluid secretion. See, e.g., PGE. Tobin et al., J. Physiol Pharmacol. 60:3-21, 2009. This fluid 0357 According to one non-limiting theory, prostaglan is bicarbonate rich. din EP4 receptor agonists have been shown to stimulate 0350 Muscarinic receptor control of bicarbonate secre duodenalbicarbonate secretion, for instance, by a mechanism tion has been demonstrated repeatedly: intravascularly or that is mediated by cAMP. See, e.g., Aoi et al., Am J Physiol Subcutaneously administered muscarinic agonists increase Gastrointest Liver Physiol. 287:G96-103, 2004; Lundgren, bicarbonate release into the intestinal lumen, a response Acta Physiol Scand. 185:87, 2005: Takeuchi et al., Gastroen blocked by muscarinicantagonists. For instance, according to terology. 113:1553-1559, 1997. Hence, and without being one non-limiting theory, cholinergic agonists such as bound by any one mechanism, in certain aspects a prostag bethanechol (muscarinic receptor selective agonist), carba landin EP4 receptor agonist inhibits or reduces phosphate chol (muscarinic and nicotinic acetylcholine receptor ago uptake in the gastrointestinal tract by stimulating bicarbonate nist), and McN-A-343 (M1 receptor-selective agonist) have secretion into the Small intestine. been shown to increase duodenal bicarbonate secretion. See, 0358. In some embodiments, and without being bound by e.g., Säfsten et al., Am J Physiol. 267(1 Pt 1):G10-7, 1994. any one mechanism, an EP4 agonist inhibits or reduces phos Without being bound by any one mechanism, in certain phate uptake in the gastrointestinal tract by decreasing water aspects a cholinergic agonist inhibits or reduces phosphate absorption in the Small intestine. uptake in the gastrointestinal tract by stimulating bicarbonate 0359 Non-limiting examples of prostaglandin EP4 recep secretion into the Small intestine. tor agonists include PGE, PGE analogs, AE1-329, AGN205203, APS-999 Na, Cay 10598 (19a), CP-044519-02, 0351. In some embodiments, and without being bound by CJ-023,423, EP4RAG, ER-819762, L-902688, lubiprostone, any one mechanism, a cholinergic agonist inhibits or reduces ONO-4819CD, ONO AE1-329, ONO AE1-734, PGE-OH, phosphate uptake in the gastrointestinal tract by decreasing TCS2510, Y-Lactam PGE analog 3, 11-Deoxy-PGE, Y-Lac water absorption in the Small intestine. tam PGE analog 2a, Y-Lactam PGE analog 4. See, e.g., Konya 0352. In some aspects, a muscarinic receptor agonist pos et al., Pharmacol Ther. 138:485-502, 2013. sesses a conformationally-constrained structure with respect 0360. Non-limiting examples of PGE analogs include to the endogenous ligand acetylcholine. Such as the cis-trim 16, 16-dimethyl PGE2, 16-16 dimethylPGE p-(p-acetamido ethyl-(2-methyl-1,3dioxolan-4-ylmethyl)ammonium benzamido)phenyl ester, 11-deoxy-16, 16-dimethyl PGE, iodide structure in FIG. 12. See, e.g., Piergentili et al., Bioor 9-deoxy-9-methylene-16, 16-dimethyl PGE 9-deoxy-9- ganic & Medicinal Chemistry 15:886-896, 2007. This struc methylene PGE 9-keto fluprostenol, 5-trans PGE, 17-phe ture contains a ketal in place of the labile ester of acetylcho nyl-omega-trinor PGE, PGE serinol amide, PGE methyl line, which is a bioisostere that retains both hydrogen bond ester, 16-phenyl tetranor PGE, 15(S)-15-methyl PGE, acceptors of ACh but is much more stable. Similarly, car 15(R)-15-methyl PGE, 8-iso-15-keto PGE, 8-iso PGE iso bechol and bethanechol (also shown in FIG. 12) are examples propyl ester, 20-hydroxy PGE, 11-deoxy PGEi, nocloprost, US 2016/O 184387 A1 Jun. 30, 2016 sulprostone, butaprost, 15-keto PGE2, and 19(R) hydrox 0367 Based on one non-limiting theory, dopamine D1 yyPGE2. See, e.g., U.S. Application No. 2012/0202288. receptoragonists and the peripherally acting catechol-O-me 0361 Additional examples of prostaglandin EP4 receptor thyl-transferase (COMT) inhibitor nitecapone (COMT agonists include those described in U.S. Application Nos. inhibitors decrease tissue degradation of catecholamines, 2001/0056060, 2002/0040149, 2005/0164949, and 2011/ including dopamine) have been shown to stimulate bicarbon 0098481. Also included are prostaglandin EP4 receptor ago ate secretion in the gut and increase the production of cyclic nists described (along with related methods of synthesis) in AMP in isolated duodenal enterocytes. See, e.g., Flemstrom U.S. Pat. Nos. 4,219,479; 4,049,582: 4,423,067; 4,474,802: and Safsten, Dig Dis Sci. 39:1839-42, 1994; and Knutson et 4,692,464; 4,708,963; 5,010,065; 5,013,758; 6,747,037; and al., Gastroenterology. 104: 1409-13 1993: Iwatsuki et al., Eur 7,776,896: European Patent No. EP0084856; Canadian JPharmacol. 218:237-41, 1992; and Fraga et al., Cell Physiol Patent No. 1248.525; U.S. Application Nos. 2004/0102499, Biochem. 18:347-60, 2006. Without being bound by any one 2005/049227, 2005/228185, 2006/106088, 2006/111430, mechanism, in certain aspects a dopamine D1 receptor ago 2007/0010495, 2007/0123568, 2007/0123569, 2005/ nist inhibits or reduces phosphate uptake in the gastrointesti 0020686, 2008/0234337, 2010/0010222, 2010/0216689, nal tract by Stimulating bicarbonate Secretion into the Small 2004/0198701, 2004/0204590, 2005/0227969, 2005/ intestine. 0239872, 2006/0154899, 2006/0167081, 2006/0258726, 0368. In some embodiments, and without being bound by 2006/0270721, 2009/0105234, 2009/0105321, 2009/ any one mechanism, a dopamine D1 agonist inhibits or 0247596, 2009/0258918, 2009/0270395, 2004/0087624, reduces phosphate uptake in the gastrointestinal tract by 2004/0102508, 2006/0252799, 2009/0030061, 2009/ decreasing water absorption in the Small intestine. 0170931, 2010/0022650, 2009/0312388, 2009/0318523, 0369. Non-limiting examples of dopamine D1 receptor 2010/0069457, 2010/0076048, 2007/0066618, 2004/ agonists include dopamine (e.g., dopamine hydrochloride, 0259921, 2005/0065133, and 2007/0191319; and PCT Pub NPEC-caged dopamine), dihydrexidine (e.g., dihydrexidine lication Nos. WO 2004/407 1428, WO 2006/052630, WO hydrochloride), benzazepaine, and analogs/derivatives 2006/047476, WO 2006/058080, WO 2004/065365, WO thereof. Specific examples of dihydrexidine derivatives 2003/047513, WO 2004/085421, WO 2004/085430, WO include A86929, dinapsoline, dinoxyline and doxanthrine, 2005/116010, WO 2005/116010, WO 2007/014454, WO and specific examples of benzazepine derivatives include 2006/080323, and WO 2006/137472, each of which is incor SKF81297, SKF82958, SKF38393, fenoldopam, and 6-Br porated by reference in its entirety. APB. Also included are the dopamine D1 receptor agonists 0362 Particular examples of EP4 receptor agonists are shown in FIG. 14. shown in FIG. 13. 0370 Additional non-limiting examples of dopamine D1 receptor agonists include A68930, A77636, (R)-(-)-apomor 0363. In specific embodiments, the EP4 receptoragonist is phine hydrochloride, CY208-243, SKF89145, SKF89626, lubiprostone (also a calcium-activated chloride channel ago 7,8-Dihydroxy-5-phenyl-octahydrobenzo hisoquinoline, nist). Lubiprostone is a bicyclic fatty acid derived from pros YM435, ABT-431, NNC01-0012, SCH23390, SKF7734, taglandin E1 that acts by specifically activating C1C-2 chlo SKF81297, SKF38322, SKF83959, cabergoline, ride channels on the apical aspect of gastrointestinal fenoldopam (e.g., fenoldapam hydrochloride), bromocrip epithelial cells, producing a chloride-rich fluid secretion. tine, ropinirole, pramipexole, entacapone, tolcapone, dihexa These secretions soften the stool, increase motility, and pro dine, IPX-750, and pergolide. See also Zhanget al., Med Res mote spontaneous bowel movements (SBM). Lubiprostone Rev. 29:272-94, 2009: Yvonne Connolly Martin, Interna stimulates CFTR-dependent duodenal bicarbonate secretion tional Journal of Medicinal Chemistry, vol. 2011, Article ID without changing net Cl— Secretion. See, e.g., Muzimori et 424535, 8 pages, 2011. doi:10.1155/2011/424535; Salmi et al., J Physiol. 573:827-842, 2006. Here, lubiprostone-in al., CNS Drug Rev. 10:23.0-42, 2004; Bourne, CNS Drug Rev. duced duodenal bicarbonate secretion was abolished by the 7:399-414, 2001. Moreover, D1 receptor agonists can be co-perfusion of the potent EP4 receptor antagonist AH23848, identified using standard Screening methods known in the art. whereas an EP1/EP2 receptor antagonist AH6809 had no As a non-limiting example, a cell based functional assay for effect. These results suggest that lubiprostone can increase high-throughput drug screening for dopamine D1 receptor duodenal bicarbonate secretion by agonizing the prostaglan agonists is described in Jiang et al., Acta Pharmacol Sin. din EP4 receptor. Hence, in certain aspects lubiprostone 26:1181-6, 2005. These references are incorporated by refer inhibits or reduces phosphate uptake in the gastrointestinal ence in their entireties. tract by stimulating bicarbonate secretion into the Small intes 0371. As noted above, certain aspects include a dopamine tine. D1 receptor selective agonist. Dopamine D1 selective ago 0364. As noted above, certain aspects include a prostag nists include compounds having an ICs at the D2, D3, D4, landin EP4 receptor selective agonist. EP4 selective agonists and/or D5 receptor subtypes which is at least 5, at least 10, at include compounds having an ICs at the EP1, EP2, and/or least 20, at least 30, at least 40, or at least 50-fold greater than EP3 receptor subtypes which is at least 5, at least 10, at least the ICso at the D1 receptor subtype. 20, at least 30, at least 40, or at least 50-fold greater than the 0372. J. Melatonin Receptor Agonists ICs at the EP4 receptor subtype. 0373) In certain embodiments, the compound is a melato 0365 I. Dopamine D1 Receptor Agonists nin receptoragonist, optionally a selective agonist. Melatonin 0366. In certain embodiments, the compound is a dopam receptors refer to a family of high-affinity G protein-coupled ine D-1 receptor agonist, optionally a selective agonist. The receptors which bind to the pineal hormone melatonin. See dopamine D1 G protein-coupled receptor is the most highly Reppert, Biol Rhythms. 0.12:528-31, 1997. expressed dopamine receptor Subtype among the dopamine 0374 Examples of melatonin receptors include the MT1 receptor family. It stimulates adenylate cyclase and activates and MT2 receptors. In some aspects, the melatonin receptor cyclic AMP-dependent protein kinases. agonists binds to both of the MT1 and MT2 receptors. In US 2016/O 184387 A1 Jun. 30, 2016 39

Some embodiments, the melatonin receptor agonist binds 0383. In some embodiments, and without being bound by selectively to the MT1 or MT2 receptor, e.g., binds to MT2 any one mechanism, a 5HT4agonist inhibits or reduces phos but not significantly to MT1, or binds to MT1 but not signifi phate uptake in the gastrointestinal tract by decreasing water cantly to MT2. absorption in the Small intestine. 0375. According to non non-limiting theory, melatonin 0384. Non-limiting examples of 5HT4 agonists include receptor agonists such as melatonin have been shown to serotonin and its analogs, BIMU-8, cisapride, cleobopride, stimulate duodenal bicarbonate secretion, for example, via CL033466, ML10302, mosapride, prucalopride, renzapride, action at enterocyte MT2-receptors. See, e.g., Sjöblom et al., RS67506, RS67333, SL650155, tegaserod, Zacopride, J Clin Invest. 108:625-33, 2001: Sjöblom and Flemstrom, J. naronopride (ATI-7505), velusetrag (TD-5108). Pineal Res. 34:288-293, 2003. Without being bound by any 0385. In some embodiments, the 5HT4 receptoragonist or one mechanism, in certain aspects a melatonin receptor ago partial agonist is a Substituted benzamide, Such as cisapride, nist inhibits or reduces phosphate uptake in the gastrointesti including individual or combinations of cisapride enanti nal tract by Stimulating bicarbonate Secretion into the Small omers ((+) cisapride and (-) cisapride), mosapride, or renza intestine. pride. In some embodiments, the 5HT4 receptor agonist is a 0376. In some embodiments, and without being bound by benzofuranderivative, Such as prucalopride, an indole Such as any one mechanism, a melatonin receptoragonist inhibits or tegaserod, or a benzimidazolone. Other non-limiting reduces phosphate uptake in the gastrointestinal tract by examples of 5HT4 receptor agonists or partial agonists decreasing water absorption in the Small intestine. include Zacopride (CAS RN 90182-92-6), SC-53116 (CAS 0377 Examples of melatonin receptor agonists include RN 141 196-99-8) and its racemate SC-49518 (CAS RN melatonin (N-acetyl-5-methoxytryptamine) and melatonin 146388-57-0), BIMU1 (CAS RN 127595-43-1), TS-951 analogs which bind to and activate the melatonin receptor. (CAS RN 174486-39-6), ML10302 (CAS RN 148868-55-7), The general structure of melatonin comprises an indole ring metoclopramide, 5-methoxytryptamine, RS67506, 2-1-(4- with methoxy group at position 5 (5-methoxy group) and an piperonyl)piperazinylbenzothiazole, RS66331, BIMU8, SB acylaminoethyl side-chain at position 3; the two side-chains 205149 (the n-butyl quaternary analog of renzapride), and an contribute to binding to and activating the melatonin receptor indole carbazimidamide described in Buchheit et al., J Med. (s). The indole ring has been evaluated at all positions by the Chem. 38:2331-8, 1995. Also included are norcisapride (CAS effect of substitutions. See, e.g., Rivara et al., Curr Top Med RN 102671-04-5), which is the metabolite of cisapride: Chem. 8:954-68, 2008; and Sugen et al., Pigment Cell mosapride citrate; the maleate form of tegaserod (CAS RN Research. 17:454-460, 2004. 189188-57-6): Zacopridehydrochloride (CAS RN996.17-34 0378 Particular examples of melatonin receptor agonists 2); mezacopride (CAS RN 89613-77-4); SK-951 ((+-)-4- include 2-iodomelatonin, 6-chloromelatonin, 6,7-dichloro-2- amino-N-(2-(1-azabicyclo(3.3.0)octan-5-yl)ethyl)-5-chloro methylmelatonin and 8-hydroxymelatonin, all of which con 2,3-dihydro-2-methylbenzobfuran-7-carboxamide tain the 5-methoxy indole ring as a moiety, in addition to hemifumarate); ATI-7505, a cisapride analog: SDZ-216-454, circadin, agomelatine, ramelteon, tasimelteon, beta-methyl a selective 5HT4 receptor agonist that stimulates cAMP for 6-chloromelatonin (TIK-301 or LY 156735), TAK-375, VEC mation in a concentration dependent manner (see, e.g., Mark 162, GR196429, S20242, S23478, S24268, S25150, stein et al., Naunyn-Schmiedebergs Arch Pharmacol. 359: GW290569, BMS-214778, 8-methoxy-2-chloroacetamidot 454-9, 1999); SC-54750, or aminomethylazaadamantane: etralin, 8-methoxy-2-propionamido-tetralin, N-acetyl Y-36912, or 4-amino-N-1-3-(benzylsulfonyl)propylpip tryptamine, 6-chloromelatonin, 2-iodomelatonin, 8-M- eridin-4-ylmethyl-5-chloro-2-methoxybenzamide (see PDOT, and 2-phenylmelatonin. See, e.g., U.S. Application Sonda et al., Bioorg Med. Chem. 12:2737-47, 2004); No. 2005/0164987, which is incorporated by reference in its TKS159, or 4-amino-5-chloro-2-methoxy-N-(2S,4S)-1- entirety. Also included are the exemplary melatonin receptor ethyl-2-hydroxymethyl-4-pyrrolidinylbenzamide: (MT2) agonists shown in FIG. 15. RS67333, or 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1- 0379 Methods of screening for melatonin receptor ago n-butyl-4-piperidinyl)-1-propanone; KDR-5169, O nists are described, for example, in U.S. Application No. 4-amino-5-chloro-N-1-(3-fluoro-4-methoxybenzyl)piperi 2003/0044909, which is incorporated by reference in its din-4-yl)-2-(2-hydr-oxyethoxy)benzamide hydrochloride entirety. dihydrate (see Tazawa, et al., Eur J Pharmacol. 434: 169-76, 0380 K. 5HT4 Receptor Agonists 2002); SL65.0155, or 5-(8-amino-7-chloro-2,3-dihydro-1,4- 0381. In certain embodiments, the compound is a 5HT4 benzodioxin-5-yl)-3-1-(2-phenylethyl)-4-piperidinyl-1,3, receptor agonist, optionally a selective agonist. The 5-hy 4-oxadiazol-2 (3H)-one monohydrochloride; and Y-34959, droxytryptamine receptor 4 (5HT4) is a G protein-coupled or 4-amino-5-chloro-2-methoxy-N-1-5-(1-methylindol-3- serotonin receptor, which stimulates cAMP production in ylcarbonylamino)pentylpiperidin-4-ylmethylbenzamide. response to serotonin (5-hydroxytryptamine or 5-HT) or 0386. Additional examples of 5HT4 receptoragonists and other agonist. partial agonists metoclopramide (CAS RN 364-62-5), 0382 Based on one non-limiting theory, serotonin has 5-methoxytryptamine (CAS RN 608-07-1), RS67506 (CAS been shown to increases protective duodenal bicarbonate RN 168986-61-6), 2-1-(4-piperonyl)piperazinylbenzothia secretion, for example, via enteric ganglia, cAMP- and Ca2+- zole (CAS RN 155106-73-3), RS66331 (see Buccafusco et dependent signaling pathways, and a 5HT4-dependent path al., Pharmacology. 295:438-446, 2000); BIMU8 (endo-N-8- way. See, e.g., Säfsten et al., Scand.J Gastroenterol. 41: 1279 methyl-8-azabicyclo[3 0.2.1]oct-3-yl)-2,3-dehydro-2-oxo 89, 2006; Tuo et al., Am J Physiol Gastrointest Liver Physiol 3-(prop-2-yl)-1H-benzimid-azole-1-carboxamide), or SB 286:G444-G451, 2004. Without being bound by any one 205149 (the n-butyl quaternary analog of renzapride). Also mechanism, in certain aspects a 5HT4 receptoragonist inhib included are compounds related to metoclopramide, Such as its or reduces phosphate uptake in the gastrointestinal tract by metoclopramide dihydrochloride (CAS RN 2576-84-3), stimulating bicarbonate secretion into the Small intestine. metoclopramide dihydrochloride (CAS RN 5581-45-3), and US 2016/O 184387 A1 Jun. 30, 2016 40 metoclopramide hydrochloride (CAS RN 7232-21-5 or 0396 There are at least 15 isoforms of carbonic anhy 54143-57-6). See, e.g., U.S. Application No. 2009/0325949; drase. Carbonic anhydrase IV (CAIV) is a membrane-bound De Maeyer et al., Neurogastroenterology and Motility. 20:99 isoform, while CAII is cytosolic, ubiquitous and highly active 112, 2008; Manabe et al., Expert Opin Investig Drugs. (turnover rate ~10's). See, e.g., Shandro and Casey, 2007. 19:765-75, 2010; Tack et al., Alimentary Pharmacology & Carbonic anhydrase II appears to be functionally coupled— Ther:35:745-767, 2012. These references are incorporated by directly and indirectly—with bicarbonate transporting pro reference in their entireties. teins such as CFTR, SLC26A6 and DRA. See, e.g., Seidler 0387 L. Atrial Natriuretic Peptide Receptor Agonists and Sjöblom, 2012. In general, the COOH-terminal tail of all 0388. In some embodiments, the compound is an atrial bicarbonate transport proteins, with the exception of DRA, natriuretic peptide (NP) receptor agonist. NP receptors are possesses a consensus carbonic anhydrase II-binding motif. single transmembrane catalytic receptors with intracellular See, e.g., Dudeja and Ramaswamy, 2006. guanylyl cyclase (GC) activity. There are three isoforms of 0397 Carbonic anhydrases are involved in several physi NP receptors: NPR1, NPR2 and NPR3. These receptors have ological processes, including pH homeostasis. The classical conserved catalytic and regulatory domains and divergent carbonic anhydrase inhibitors, such as acetazolamide and ligand binding domains. benzolamide, have been shown to inhibit multiple CA iso 0389 Natriuretic peptide receptors are found in the brain, forms, including CAII and CAIV. See, e.g., ScoZZafava et al., vasculature kidney, and gastrointestinal tract and bind J. Med. Chem. 45:1466-1476, 2002. According to one non C.-atrial natriuretic peptide, brain natriuretic peptide, and type limiting theory, inhibition of carbonic anhydrase would be C-natriuretic peptide with varying affinities. The main physi expected to decrease subapical intracellular pH. Without ological role of NP receptors is the homeostasis of body fluid being bound by any one mechanism, selective inhibition of Volume. According to one non-limiting theory, exogenous CA in the enterocytes of the duodenum could thereby natriuretic peptide stimulates GC activity in the gastrointes decrease the CEPG, resulting in a decrease in phosphate tinal tract. See, e.g., Rambotti et al. Histochem. J. 29:117 transport. 126, 1997. 0398. In some embodiments, and without being bound by 0390. Without being bound by any one mechanism, in any one mechanism, a carbonic anhydrase inhibitor inhibits certain aspects an NP receptor agonist inhibits or reduces or reduces phosphate uptake in the gastrointestinal tract by phosphate uptake in the gastrointestinal tract by stimulating decreasing water absorption in the Small intestine. bicarbonate secretion and/or inhibiting acid secretion in the 0399 FIG. 17 shows the structures of exemplary carbonic small intestine. anhydrase inhibitors, including dorzolamide and brinzola mide, among others. In certain aspects, carbonic anhydrase 0391. In some embodiments, and without being bound by inhibitors can be used in combination with classes of com any one mechanism, an NP receptor agonist inhibits or pounds capable of elevating cAMP. c6MP calcium or other reduces phosphate uptake in the gastrointestinal tract by second messengers in apical mucosal cells of the gastrointes decreasing water absorption in the Small intestine. tinal tract. 0392 The structures of exemplary peptide agonists of the (0400 N. Phosphodiesterase Inhibitors NP receptor(s) are shown in FIG. 16, and described, for 04.01. In some embodiments, the compound is a phos example, in von Geldern et al., J. Med. Chem. 35:808–816, phodiesterase inhibitor. Phosphodiesterases (PDEs) are a 1992, which is incorporated by reference in its entirety. family of related phosphohydrolyases that selectively cata 0393. In certain embodiments, the NP receptor agonist lyze the hydrolysis of 3' cyclic phosphate bonds in adenosine comprises, consists, or consists essentially of the atrial natri and/or guanine 3',5' cyclic monophosphate (cAMP and/or uretic peptide amino acid sequence: Ser Leu Arg Arg Ser Ser cGMP). They regulate the cellular levels, localization and Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu duration of action of these second messengers by controlling Gly Cys Asn Ser Phe Arg Tyr (SEQ ID NO:7), including the rate of their degradation. active variants thereofhaving 1,2,3,4,5,6,7,8,9, 10, 11, or (0402. There are 11 subtypes of PDEs, named PDE1-11: 12 deletions, insertions, and/or Substitutions. Specific PDE4, 7 and 8 selectively degrade cAMP, PDE5, 6 and 9 examples of deletion mutants include those having the selectively degrade c0MP and PDE 1, 2, 3, 10 and 11 degrade sequence: Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln both cyclic nucleotides. PDEs are expressed ubiquitously, Ser Gly Leu Gly Cys (SEQ ID NO:8); and Ser Ser Cys Phe with each subtype having a specific tissue distribution. FIG. Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys 18 shows the structures of exemplary phosphodiesterase Asn Ser Phe Arg (SEQ ID NO:9). As described elsewhere inhibitors with varied subtype specificity, including theophyl herein, Such peptides can be composed of any combination of line, cilostazol, Vinpocetine, amrinone, EHNA, trequinsin, naturally-occurring and non-naturally-occurring amino drotaverine, roflumilast, and sildenafil. acids. 0403. According to one non-limiting theory, phosphodi 0394 M. Carbonic Anhydrase Inhibitors esterase inhibitors are capable of modulating duodenal bicar 0395. In some embodiments, the compound is a carbonic bonate secretion (DBS) alone and in combination with agents anhydrase inhibitor. Bicarbonate uptake into epithelial cells that increase cytosolic cAMP and coMP by maintaining the occurs by CO diffusion with subsequent conversion to level of these second messengers in enterocytes. PDE1 and HCO, and H" by cellular carbonic anhydrase (CA). Bicar PDE3 inhibitors are specifically implicated in modulating bonate is then secreted across the apical membrane by anion DBS. See, e.g., Hayashi, Biochem. Pharmacol. 74: 1507 exchange. CA is the enzyme that hydrates CO to produce 1513, 2007. Without being bound by any one mechanism, in HCO and H and is present in most tissues, including certain embodiments a phosphodiesterase inhibitor inhibits duodenal epithelial cells. See, e.g., Kaunitz and Akiba, 2006. or reduces phosphate uptake in the gastrointestinal tract by This endogenously produced HCOT is a significant source of stimulating bicarbonate secretion into the Small intestine or transported bicarbonate. DBS. US 2016/O 184387 A1 Jun. 30, 2016

04.04. In some embodiments, and without being bound by ZOL, QUAZINONE, and N-(3,5-dichloropyrid-4-yl)-3-cy any one mechanism, a phosphodiesterase inhibitor inhibits or clopropylmethoxy-4-difluoromethoxybenzamide. Examples reduces phosphate uptake in the gastrointestinal tract by of PDE3 inhibitors include SULMAZOLE, AMPIZONE, decreasing water absorption in the Small intestine. CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMA 0405. In certain embodiments, PDE inhibitors slow the ZODAN, CI-930, SIGUAZODAN, ADIBENDAN, degradation of cyclic AMP (cAMP) and/or cyclic GMP SATERINONE, SKF-95654, SDZ-MKS-492, 349-U-85, (cGMP), which can then lead to a relative increase in the EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP intracellular concentration of cAMP and/or cGMP General 307, REVIZINONE, NM-702, WIN-62582 and WIN-63291, examples include PDE1 inhibitors, PDE3 inhibitors, PDE4 ENOXIMONE, and MILRINONE. Examples of PDE3/4 inhibitors, PDE5 inhibitors, PDE3/4 inhibitors, and PDE3/ inhibitors include BENAFENTRINE, TREQUINSIN, ORG 4/5 inhibitors. Merely by way of non-limiting example, PDE 3.0029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG inhibitors may include those disclosed in the following patent 20241, EMD-54622, and TOLAFENTRINE. Otherexamples applications and patents: DE 1470341, DE2108438, of PDE inhibitors include cilomilast, pentoxifylline, roflumi DE2123328, DE2305339, DE2305575, DE2315801, last, tadalafil (Cialis(R), theophylline, Vardenafil (Levitra(R), DE2402908, DE2413935, DE2451417, DE2459090, and Zaprinast (PDE5 specific). DE2646469, DE2727481, DE2825048, DE2837161, 0407. In certain aspects, phosphodiesterase inhibitors can DE2845220, DE2847621, DE2934747, DE3021792, be used in combination with classes of compounds capable of DE3038166, DE3044568, DE3142982, DE1116676, elevating cAMP. c6MP calcium or other second messengers DE2162096, EP000718, EP0008.408, EP0010759, in apical mucosal cells of the gastrointestinal tract. EP0059948, EP0075436, EP00965.17, EPO112987, (0408 O. Agonists of DRA (SLC26A3) EP0116948, EPO150937, EP0158380, EPO161632, 04.09. In certain embodiments, the compound is an agonist EPO161918, EP0167121, EP019.9127, EP0220044, of the chloride/bicarbonate antiporter SLC26A3, also EP0247725, EP0258191 EP0272910, EP0272914, referred to as Down-Regulated in Adenoma (DRA). One EP0294647, EP0300726, EP0335386, EP0357788, non-limiting function of DRA in the gut is to absorb luminal EP0389282, EP0406958, EP0426180, EP0428302, chloride and secrete bicarbonate ions. Pharmacological EP0435811, EP0470805, EP0482208, EP0490823, stimulation of DRA is expected reduce pHi for instance, by EP0506194, EP0511865, EP0527117, EP0626939, increasing the pH of the UWL, and provide a phosphate EP0664289, EP0671389, EP0685474, EP0685475, lowering effect as described herein. EP0685479, EP0293063, EP0463756, EP0482208, 0410 Examples of DRA agonists include lysophosphatic EP0579496, EP0667345, EPO163965, EP0393500, acid (LPA) and structurally related compounds. This class of EP0510562, EP0553174, JP92234389, JP94329652, compounds is thought to be acting on DRA activity via stimu JP950.10875, U.S. Pat. Nos. 4,963,561; 5,141,931; and 6,331, lation of LPA receptor (for instance LPA2) signaling through 543; International Patent Application Publication Nos. the Pi3K/AKT pathway, which is thought to not only activate WO9117991, WO9200968, WO9212961, WO9307146, DRA gene transcription but also increase DRA Surface accu WO9315044, WO9315045, WO9318024, WO9319068, mulation (Singla et al. Am. J. Physiol Gastrointest. Liver WO9319720, WO931.9747, WO9319749, WO9319751, Physiol. 298: G182-G189, 2010: Singla et al. Am. J. Physiol. WO9325517, WO9402465, WO9406423, WO9412461, Gastrointest. Liver Physiol. 302: G618-G627, 2012). WO9420455, WO9422852, WO9425437, WO9427947, Examples of LPA related compounds with potential role in WO9500516, WO950.1980, WO9503794, WO9504045, DRA stimulation are described in Jiang et al., Bioorg. Med. WO9504046, WO9505386, WO9508534, WO9509623, Chem. Lett. 23:1865-1869, 2013; Kiss et al., Molecular Phar WO9509624, WO9509627, WO9509836, WO9514667, macology 82:1162-1173, 2012: Kozian et al., Bioorg. Med. WO9514680, WO9514681, WO9517392, WO9517399, Chem. Lett. 22:5239-5243, 2012: Parrill, Expert. Opin. Ther. WO9519362, WO9522520, WO9524381, WO9527692, Pat. 21:281-286, 2011; Gupte et al., Bioorg. Med. Chem. Lett. WO9528926, WO953.5281, WO9535282, WO9600218, 20: 7525-7528, 2010; Liliom et al., Biochim. Biophys. Acta WO9601825, WO9602541, WO961 1917, WO9307124, 1761:1506-1514, 2006; and Durgam et al., Journal of Medici WO950 1338 and WO9603399; and U.S. Application No. nal Chemistry 48: 4919-4930, 2005. 2005/0004222 (including those disclosed in formulas I-XIII 0411. According to one non-limiting theory, protein and paragraphs 37-39, 85-0545 and 557-577), each of which Kinase C inhibitors may also increase DRA activity and is incorporated by reference in its entirety. similarly create a cross-epithelial pH gradient. For example, 04.06 Examples of PDE5 inhibitors include RX-RA-69, phorbol 12-myristate 13-acetate (PMA), an in vitro PKC SCH-51866, KT-734, vesnarinone, Zaprinast, SKF-96231, agonist, was shown to directly inhibit the apical membrane ER-21355, BF/GP-385, NM-702 and sildenafil (Viagra(R). Cl/HCOs activity (Gillet al., Physiology of the Gastrointes Examples of PDE4 inhibitors include RO-20-1724, MEM tinal Tract, Chapter 67, 2012). Without being bound by any 1414 (R1533/R1500; Pharmacia Roche), DENBUFYL one mechanism, inhibition of the appropriate PKC isoforms LINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE, could conversely increase Cl/HCO, activity and thereby Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZI inhibit phosphate uptake via the mechanisms described in the NONE, INDOLIDAN, OLPRINONE, ATIZORAM, disclosure. KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, 0412 FIGS. 21A-B (Mochly-Rosenet al., Nature Reviews AROFYLLINE, FILAMINAST, PDB-093, UCB-29646, Drug Discovery 11, 937-957, 2012) depict representative CDP-840, SKF-107806, PICLAMILAST, RS-17597, examples of subtype selective PKC inhibitors with the poten RS-25344-000, SB-207499, TIBENELAST, SB-210667, tial to increase Cl/HCOs activity, among other potential SB-211572, SB-211600, SB-212066, SB-212179, mechanisms of action. Other potential DRA agonists include GW-3600, CDP-840, MOPIDAMOL, ANAGRELIDE, All-trans-retinoic acid (ATRA) and related compounds, more IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTA generally compounds activating the retinoic acid receptors US 2016/O 184387 A1 Jun. 30, 2016 42

(RAR's) C. Bandy, preferably the RAR-13. RAR-13 agonists cally non-bioavailable' includes embodiments where no are believed to induce DRA at the transcriptional level (All detectable amount of absorption or permeation or systemic Trans-Retinoic Acid Increases SLC26A3 (DRA) Expression exposure of the compound is detected, using means generally via HNF-1 (Priyamvada et al., DDW 2013, Orlando). Another known in the art. exemplary compound is 520787, which was shown to stimu 0417. In this regard it is to be further noted, however, that late the activity of human DRA expressed in oocytes (Cher in alternative embodiments “substantially impermeable' or nova et al., J. Physiol., 549, 1, 3-19, 2003). Agonists of “substantially systemically non-bioavailable' provides or neuropeptide Y1 and Y2 receptor stimulate DRA activity in allows for some limited absorption in the GI tract, and more caco2 monolayers. Stimulation DRA by NPY was found to be particularly the gut epithelium, to occur (e.g., Some detect independent of membrane trafficking and associated with able amount of absorption, Such as for example at least about localization of DRA to lipid rafts (Saksena et al. Am. J. 0.1%, 0.5%, 1% or more and less than about 30%, 20%, 10%, Physiol Gastrointest Liver Physiol. 299: G1334-G1343, 5%, etc., the range of absorption being for example between 2010). Examples of representative NPY1 and NPY2 agonists about 1% and 30%, or 5% and 20%, etc.); stated another way, include NPY, Leu31, Pro34-NPY, NPY 13-36, Peptide YY “substantially impermeable' or “substantially systemically (3-36) and GR 2311 18. non-bioavailable' may refer to compounds that exhibit some detectable permeability to an epithelial layer of cells in the GI II. Substantially Systemically Non-Bioavailable Compounds tract of less than about 20% of the administered compound 0413 A. Physical and Performance Properties of Com (e.g., less than about 15%, about 10%, or even about 5%, 4%, pounds Localizable to the GI Tract 3%, or 2%, and for example greater than about 0.5%, or 1%), 0414 Certain of the compounds described herein are but then are cleared by the liver (i.e., hepatic extraction) designed to be substantially active or localized in the gas and/or the kidney (i.e., renal excretion). trointestinal lumen of a human or animal Subject. The term 0418. In this regard it is to be further noted, that in certain 'gastrointestinal lumen’ is used interchangeably herein with embodiments, due to the substantial impermeability and/or the term “lumen.” to refer to the space or cavity within a Substantial systemic non-bioavailability of the compounds of gastrointestinal tract (GI tract, which can also be referred to as the present invention, greater than about 50%, 60%, 70%, the gut), delimited by the apical membrane of GI epithelial 80%, 90%, or 95% of a compound of the invention is recov cells of the Subject. In some embodiments, the compounds are erable from the feces over, for example, a 24, 36, 48, 60, 72, not absorbed through the layer of epithelial cells of the GI 84, or 96 hour period following administration to a subject in tract (also known as the GI epithelium). "Gastrointestinal need thereof. In this respect, it is understood that a recovered mucosa” refers to the layer(s) of cells separating the gas compound can include the Sum of the parent compound and trointestinal lumen from the rest of the body and includes its metabolites derived from the parent compound, e.g., by gastric and intestinal mucosa, such as the mucosa of the Small means of hydrolysis, conjugation, reduction, oxidation, intestine. A “gastrointestinal epithelial cell' or a “gut epithe N-alkylation, glucuronidation, acetylation, methylation, Sul lial cell as used herein refers to any epithelial cell on the fation, phosphorylation, or any other modification that adds Surface of the gastrointestinal mucosa that faces the lumen of atoms to or removes atoms from the parent compound, where the gastrointestinal tract, including, for example, an epithelial the metabolites are generated via the action of any enzyme or cell of the stomach, an intestinal epithelial cell, a colonic exposure to any physiological environment including, pH, epithelial cell, and the like. temperature, pressure, or interactions with foodstuffs as they 0415 "Substantially systemically non-bioavailable' and/ exist in the digestive milieu. or “substantially impermeable' as used herein (as well as 0419 Measurement of fecal recovery of compound and variations thereof) generally refer to situations in which a metabolites can be carried out using standard methodology. statistically significant amount, and in some embodiments For example, a compound can be administered orally at a essentially all of the compound remains in the gastrointestinal Suitable dose (e.g., 10 mg/kg) and feces are then collected at lumen. For example, inaccordance with one or more embodi predetermined times after dosing (e.g., 24 hours, 36 hours, 48 ments of the present disclosure, preferably at least about 60%, hours, 60 hours, 72 hours, 96 hours). Parent compound and about 70%, about 75%, about 80%, about 85%, about 90%, metabolites can be extracted with organic solvent and ana about 95%, about 96%, about 97%, about 98%, about 99%, or lyzed quantitatively using mass spectrometry. A mass balance even about 99.5%, of the compound remains in the gas analysis of the parent compound and metabolites (including, trointestinal lumen. In Such cases, localization to the gas parent=M, metabolite 1 M+16, and metabolite 2 M+32) trointestinal lumen refers to reducing net movement of a can be used to determine the percent recovery in the feces. compound across a gastrointestinal layer of epithelial cells, 0420 (i) Permeability for example, by way of both transcellular and paracellular 0421. In this regard it is to be noted that, in various transport, as well as by active and/or passive transport. The embodiments, the ability of the compound to be substantially compound in Such embodiments is hindered from net perme systemically non-bioavailable is based on the compound ation of a layer of gastrointestinal epithelial cells in transcel charge, size, and/or other physicochemical parameters (e.g., lular transport, for example, through an apical membrane of polar Surface area, number of hydrogen bond donors and/or an epithelial cell of the small intestine. The compound in acceptors therein, number of freely rotatable bonds, etc.). these embodiments is also hindered from net permeation More specifically, it is to be noted that the absorption char through the "tight junctions' in paracellular transport acter of a compound can be selected by applying principles of between gastrointestinal epithelial cells lining the lumen. pharmacokinetics, for example, by applying Lipinski's rule, 0416) In this regard it is to be noted that, in one particular also known as “the rule offive.” Although not a rule, but rather embodiment, the compound is essentially not absorbed at all a set of guidelines, Lipinski shows that Small molecule drugs by the GI tract orgastrointestinal lumen. As used herein, the with (i) a molecular weight, (ii) a number of hydrogen bond terms “substantially impermeable' or “substantially systemi donors, (iii) a number of hydrogenbond acceptors, and/or (iv) US 2016/O 184387 A1 Jun. 30, 2016 a water/octanol partition coefficient (Moriguchi Log P), cal graphic tools packages, such as ChemDraw. The term greater than a certain threshold value, generally do not show “topological PSA (tPSA) has been coined for this fast-cal significant systemic concentration (i.e., are generally not culation method. thSA is well correlated with human absorp absorbed to any significant degree). (See, e.g., Lipinski et al., tion data with common drugs (see Table 1, from Ertlet al., J. Advanced Drug Delivery Reviews, 46:3-26, 2001 incorpo Med. Chem. 43:3714-3717, 2000): rated herein by reference.) Accordingly, Substantially sys temically non-bioavailable compounds can be designed to TABLE 1 have molecular structures exceeding one or more of Lipins ki's threshold values. (See also Lipinski et al., Experimental l8le 96 FA TPSA and Computational Approaches to Estimate Solubility and metoprolol 102 50.7 Permeability in Drug Discovery and Development Settings, nordiazepam 99 41.5 diazepam 97 32.7 Adv. Drug Delivery Reviews, 46:3-26, 2001; and Lipinski, oXprenolol 97 50.7 Drug-like Properties and the Causes of Poor Solubility and phenazone 97 26.9 Poor Permeability, J. Pharm. & Toxicol. Methods, 44:23.5- oxazepam 97 61.7 249, 2000, which are incorporated by reference in their alprenolol 96 41.9 entireties. practolol 95 70.6 pindolol 92 57.3 0422. In some embodiments, for example, a substantially ciprofloxacin 69 74.6 impermeable or substantially systemically non-bioavailable metolazone 64 92.5 tranexamic acid 55 6.3.3 compound of the present disclosure can be constructed to altenolol S4 846 feature one or more of the following characteristics: (i) a MW Sulpiride 36 101.7 greater than about 500 Da, about 600 Da, about 700 Da, about mannitol 26 121.4 800 Da, about 900 Da, about 1000 Da, about 1200 Da, about foscarnet 17 94.8 SulfaSalazine 12 1413 1300 Da, about 1400 Da, about 1500 Da, about 1600 Da, olsalazine 2.3 139.8 about 1800 Da, about 2000 Da, about 2500 Da, about 3000 lactulose O6 1974 Da, about 4000 Da, about 5000 Da, about 7500 Da, about raffinose O.3 268.7 10,000 Da or more (in the non-salt form of the compound); (ii) a total number of NH and/or OH and/or other potential hydrogen bond donors greater than about 5, about 6, about 7. 0424. Accordingly, in some embodiments, the compounds about 8, about 9, about 10, about 11, about 12, about 13, about of the present disclosure may be constructed to exhibitatPSA 14, about 15, about 20 or more; (iii) a total number of Oatoms value greater than about 100A, about 116 A, about 120 A, and/or Natoms and/or other potential hydrogen bond accep about 130 A, or about 140 A, and in some instances about tors greater than about 5, about 6, about 7, about 8, about 9, 150 A, about 160A, about 170 A, about 180A, about 190 about 10, about 11, about 12, about 13, about 14, about 15, A, about 200 A, about 225A, about 250A, about 270 A, about 20 or more; (iv) a Moriguchi partition coefficient about 300 A, about 350 A, about 400 A, about 450 A. greater than about 10 (i.e., Log P greater than about 5, about about 500 A, about 750 A, or even about 1000 A, or in the 6, about 7, about 8, about 9, about 10 etc.), or alternatively less range of about 100-120 A, 100-130 A, 100-140 A, 100 than about 10 (i.e., a Log P of less than 1, or even 0); and/or 150 A, 100-160A, 100-170A, 100-170A, 100-190 A, (v) a total number of rotatable bonds greater than about 5, 100-200 A, 100-225A, 100-250A, 100-300 A, 100-400 about 10 or about 15, or more. In specific embodiments, the A, 100-500 A, 100-750 A, 100-1000 A, 116-120 A, compound has a Log P that is not 14 or is less than about 14, 116-130A, 116-140A, 116-150 A, 116-160A, 116-170 for instance, a Log P that is in the range of about 6-7, 6-8, 6-9, A, 116-170A, 116-190A, 116-200 A, 116-225A, 116 6-10, 6-11, 6-12, 6-13, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 8-9, 250A, 116-300 A, 116-400A, 116-500 A, 116-750 A, 8-10, 8-11, 8-12, 8-13, 9-10, 9-11, 9-12, 9-13, 10-11, 10-12, 116-1000A, 120-130A, 120-140A, 120-150A, 120-160 10-13, 11-12, 11-13, or 12-13. A, 120-170A, 120-170A, 120-190A, 120-200 A, 120 0423. In addition to the parameters noted above, the 225A, 120-250A, 120-300 A, 120-400A, 120-500 A, molecular polar surface area (i.e., “PSA), which may be 120-750A, 120-1000A, 130-140A, 130-150A, 130-160 characterized as the Surface belonging to polar atoms, is a A°, 130-170A, 130-170A, 130-190A, 130-200 A, 130 descriptor that has also been shown to correlate well with 225A, 130-250A, 130-300 A, 130-400A, 130-500 A, passive transport through membranes and, therefore, allows 130-750A, 130-1000A, 140-150A, 140-160A, 140-170 prediction of transport properties of drugs. It has been Suc A, 140-170A, 140-190A, 140-200 A, 140-225A, 140 cessfully applied for the prediction of intestinal absorption 250A, 140-300 A, 140-400A, 140-500 A, 140-750 A, and Caco2 cell monolayer penetration. For exemplary Caco2 140-1000A, 150-160A, 150-170A, 150-170A, 150-190 cell monolayer penetration test details, see for example the A, 150-200 A, 150-225 A, or 150-250A, 150-300 A, description of the Caco2 Model provided in U.S. Pat. No. 150-400A, 150-500A, 150-750A, 150-1000A, 200-250 6.737,423, incorporated by reference, particularly the text A, 200-300 A, 200-400A, 200-500 A, 200-750 A, 200 describing the Caco2 Model, which may be applied for 1000 A, 250-250A, 250-300 A, 250-400A, 20-500 A, example to the evaluation or testing of the compounds of the 250-750 A, or 250-1000 A, such that the compounds are present invention. PSA is expressed in A (squared ang Substantially impermeable (e.g., cell impermeable) or Sub stroms) and is computed from a three-dimensional molecular stantially systemically non-bioavailable (as defined else representation. A fast calculation method is also available where herein). (see, e.g., Ertl et al., Journal of Medicinal Chem. 43:3714 0425 Because there are exceptions to Lipinski’s “rule,” or 3717, 2000 the entire contents of which are incorporated the tRSA model, the permeability properties of the com herein by reference for all relevant and consistent purposes) pounds of the present disclosure may be screened experimen using a desktop computer and commercially available chemi tally. The permeability coefficient can be determined by US 2016/O 184387 A1 Jun. 30, 2016 44 methods known to those of skill in the art, including for 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4, example by Caco-2 cell permeability assay and/or using an 0.5,0.6, 0.7, 0.8, 0.9x (0.9 times) the ICs for inhibiting Pi artificial membrane as a model of a gastrointestinal epithelial transport or uptake. cell. A synthetic membrane impregnated with, for example, 0430. In certain embodiments, one or more of the substan lecithin and/or dodecane to mimic the net permeability char tially systemically non-bioavailable compounds detailed acteristics of a gastrointestinal mucosa may be utilized as a herein, when administered (e.g., enterally) to a Subject in model of a gastrointestinal mucosa. The membrane can be need thereof, may have a ratio of C:ICs (for inhibiting Pi used to separate a compartment containing the compound of transport or update), where C and ICso are expressed in the present disclosure from a compartment where the rate of terms of the same units, of at about or less than about 0.01, permeation will be monitored. Also, parallel artificial mem 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4, brane permeability assays (PAMPA) can be performed. Such 0.5,0.6, 0.7, 0.8, 0.9, or 1.0, or a range in between about in vitro measurements can reasonably indicate actual perme 0.01-1.0, 0.01-0.9, 0.01-0.8, 0.01-0.7, 0.01-0.6, 0.01-0.5, ability in vivo (see Wohnsland et al., J. Med. Chem. 44:923 0.01-0.4, 0.01-0.3, 0.01-02, or 0.01-0.1, or a range in 930, 2001; Schmidt et al., Millipore Corp. Application Note, between about 0.1-1.0, 0.1-0.9, 0.1-0.8, 0.1-0.7, 0.1-0.6, 0.1- 2002, in AN1725EN00, and in AN1728EN00, incorporated 0.5,0.1-0.4, 0.1-0.3, or 0.1-0.2. herein by reference). 0431. In some embodiments, the substantially systemi 0426. Accordingly, in some embodiments, the compounds cally non-bioavailable compounds detailed herein, when utilized in the methods of the present disclosure may have a administered (e.g., enterally) either alone or in combination permeability coefficient, P., of less than about 100x10 with one or more additional pharmaceutically active com cm/s, or less than about 10x10 cm/s, or less than about pounds or agents to a subject in need thereof, exhibit a maxi 1x10 cm/s, or less than about 0.1x10 cm/s, when mea mum concentration detected in the serum, defined as C, Sured using means known in the art (Such as for example the that is about the same as or less than ECso of the compound for permeability experiment described in Wohnsland et al., 2001, increasing fecal output of phosphate, where fecal output is Supra). increased by about or at least about 5%, 10%, 20%, 30%, 0427 As previously noted, in accordance with the present 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodi disclosure, compounds may be modified to hinder their net ments, for instance, the C is about or at least about 5%, absorption through a layer of gut epithelial cells, rendering 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% them Substantially systemically non-bioavailable. In some less than the ECso for increasing fecal output of phosphate. In particular embodiments, the compounds of the present dis some embodiments, the C is about 0.01, 0.02, 0.03, 0.04, closure comprise a compound that is linked, coupled or oth 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4,0.5,0.6,0.7, 0.8, erwise attached to a non-absorbable moiety, which may be an 0.9x (0.9 times) the ECs for increasing fecal output of phos oligomer moiety, a polymer moiety, a hydrophobic moiety, a phate. hydrophilic moiety, and/or a charged moiety, which renders 0432. In some embodiments, one or more of the substan the overall compound Substantially impermeable or Substan tially systemically non-bioavailable compounds detailed tially systemically non-bioavailable. In some preferred herein, when administered (e.g., enterally) either alone or in embodiments, the compound is coupled to a multimer or combination with one or more additional pharmaceutically polymer portion or moiety, Such that the resulting molecule is active compounds or agents to a Subject in need thereof, or Substantially impermeable or Substantially systemically non measured in an animal model or cell-based assay, may have bioavailable. The multimer or polymer portion or moiety may an ECso for increasing fecal output of phosphate of about or be of a molecular weight greater than about 500 Daltons (Da), less than about 10 uM, 9 uM, 8 uM, 7 uM, 7.5 uM, 6 uM, 5 about 1000 Da, about 2500 Da, about 5000 Da, about 10,000 uM, 4 uM, 3 uM, 2.5uM, 2 uM, 1 uM, 0.5uM, 0.1 uM, 0.05 Da or more, and in particular may have a molecular weight in uM, or 0.01 uM, or less, the ICso being, for example, within the range of about 1000 Daltons (Da) to about 500,000 Da. the range of about 0.01 uM to about 10M, or about 0.01 uM preferably in the range of about 5000 to about 200,000 Da. to about 7.5 uM, or about 0.01 uM to about 5 uM, or about and more preferably may have a molecular weight that is 0.01 uM to about 2.5 LM, or about 0.01 uM to about 1.0, or Sufficiently high to essentially preclude any net absorption about 0.1 uM to about 10 uM, or about 0.1 uM to about 7.5 through a layer of gut epithelial cells of the compound. In uM, or about 0.1 uM to about 5uM, or about 0.1 uM to about these or other particular embodiments, the compound is 2.5 LM, or about 0.1 uM to about 1.0, or about uM 0.5 M to modified to substantially hinder its net absorption through a about 10 uM, or about 0.5uM to about 7.5 uM, or about 0.5 layer of gut epithelial cells. uM to about 5uM, or about 0.5 LM to about 2.5 LM, or about 0.5uM to about 1.0 uM. 0428 (ii) C and ICs or ECso 0433. In particular embodiments, the substantially sys 0429. In some embodiments, the substantially systemi temically non-bioavailable compounds detailed herein, when cally non-bioavailable compounds detailed herein, when administered (e.g., enterally) either alone or in combination administered (e.g., enterally) either alone or in combination with one or more additional pharmaceutically active com with one or more additional pharmaceutically active com pounds or agents to a subject in need thereof, exhibit a maxi pounds or agents to a subject in need thereof, exhibit a maxi mum concentration detected in the serum, defined as C, mum concentration detected in the serum, defined as C. that is about the same as or less than ECs of the compound for that is about the same as or less than the phosphate ion (Pi) reducing urinary output of phosphate, where urinary output is transport or uptake inhibitory concentration ICs of the com reduced by about or at least about 5%, 10%, 20%, 30%, 40%, pound. In some embodiments, for instance, the C is about 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, for instance, the C is about or at least about 5%, 10%, 20%, 80%, 90%, or 100% less than the ICs for inhibiting Pitrans 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% less than the port or uptake. In some embodiments, the C is about 0.01, ECso for reducing urinary output of phosphate. In some