Compounds and methods for inhibiting phosphate transport

Abstract

Non-NHE3 binders are provided that have activity as phosphate transport inhibitors in the gastrointestinal tract, including in the small intestine. A composition for treating hyperphosphatemia in a patient in need thereof, comprising a compound that is substantially active in the gastrointestinal tract to inhibit the transport of phosphate ions (Pi) therein and that is a guanylate cyclase C receptor (GC-C) agonist, wherein the compound does not bind to NHE3.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under 119(e) of the United States Patent Act to U.S. Application No. 61 / 864,215 filed 9 August 2013 and U.S. Application No. 61 / 936,715 filed 6 February 2014, which are incorporated herein by reference in their entirety.

[0002] Description regarding sequence listings The sequence listing relating to this application is provided in text format instead of as a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is "ARDE_017_01WO_ST25.txt". This text file is 193KB in size, was created on August 8, 2014, and filed electronically via EFS-Web.

[0003] The present invention relates to non-NHE3 conjugates having activity as phosphate transport inhibitors in the gastrointestinal tract, including the small intestine, methods for using them as therapeutic or prophylactic agents, and related methods for drug discovery. [Background technology]

[0004] Patients with insufficient renal function, hypoparathyroidism, or certain other medical conditions (e.g., hereditary hyperphosphatemia, Albright's hereditary osteodystrophy, amyloidosis, etc.) often have hyperphosphatemia, i.e., elevated serum phosphorus levels (e.g., above approximately 6 mg / dL). Hyperphosphatemia, especially when present for a long period, causes severe abnormalities in calcium and phosphorus metabolism, often manifesting as secondary hyperparathyroidism, bone disease, and ectopic calcification in the cardiovascular system, joints, lungs, eyes, and other soft tissues. Higher serum phosphorus levels are strongly associated with progression of renal failure, cardiovascular calcification, and mortality in patients with end-stage renal disease (ESRD). Higher-normal serum phosphorus levels are associated with cardiovascular events and mortality among individuals with chronic kidney disease (CKD) and among individuals with normal renal function (see, e.g., Joy et al., J. Manag. Care Pharm., 13:397-411, 2007). The progression of kidney disease can be slowed by reducing phosphate retention. Therefore, therapies that reduce phosphate retention are beneficial for patients with renal failure who have hyperphosphatemia, and for patients with chronic kidney disease who have normal or only slightly elevated serum phosphorus levels.

[0005] Calcium salts are widely used in patients experiencing hyperphosphatemia to bind to phosphates in the intestinal tract and prevent their absorption. Different types of calcium salts, including calcium carbonate, acetate, citrate, alginate, and ketoate, are used for phosphate binding. However, these therapies often cause hypercalcemia, a condition resulting from the absorption of large amounts of ingested calcium. Hypercalcemia can lead to serious side effects such as cardiac arrhythmias, renal failure, and calcification of the skin and blood vessels. Frequent monitoring of serum calcium levels is necessary during therapy with calcium-based phosphate binders. Other calcium and aluminum-free phosphate binders, such as sevelamer, a cross-linked polyamine polymer, have drawbacks, including the amount and frequency of dosage required to be therapeutically active. The relatively mild in vivo phosphate-binding capacity of these drugs forces patients to increase their doses (up to more than 7 grams per day). Such doses have been shown to cause gastrointestinal discomfort, including dyspepsia, abdominal pain, and in some extreme cases, intestinal perforation.

[0006] An alternative approach to prevent phosphate absorption from the intestines in patients with elevated serum phosphate levels involves inhibiting the intraluminal transport system that mediates phosphate uptake within the intestines. It is understood that phosphate absorption in the upper intestines is at least partially mediated by carrier-mediated mechanisms that bind phosphate absorption to sodium absorption. Inhibiting intraluminal phosphate transport reduces the body's phosphate overload. In patients with progressive renal disease (e.g., stages 4 and 5), the body's phosphate overload manifests as serum phosphate concentrations above normal levels, i.e., hyperphosphatemia. Hyperphosphatemia is directly associated with mortality and morbidity. Inhibiting intraluminal phosphate transport reduces serum phosphate concentrations and, consequently, improves the prognosis of these patients. In patients with stage 2 or 3 chronic renal disease, the body's phosphate overload does not necessarily lead to hyperphosphatemia; i.e., some patients maintain a normal phosphate state. However, even in these early stages, there is a need to reduce or prevent the body's phosphate overload to avoid associated bone and vascular damage and ultimately improve mortality. Similarly, inhibiting phosphate transport within the intestinal tract would be particularly beneficial for patients with diseases treatable by inhibiting phosphate uptake from the intestines. Furthermore, inhibiting phosphate transport may slow the progression of renal failure and reduce the risk of cardiovascular events.

[0007] The luminal pole of the intestinal epithelium contains a so-called unagitated water layer (UWL), where transport is inherently diffusive due to the viscosity of the mucus layer. This unagitated layer is defined as a stationary layer adjacent to the apical membrane, acting as a diffusion barrier so that rapidly permeating substances can actually be rate-limited by diffusion. This limited diffusion is H + Applied to this, UWL contributes to the establishment of a pH microclimate due to the outward flux of protons and the diffusion restriction imposed by the mucus layer. The acidic environment near the cell surface maintains a relatively large electrochemical gradient across the epithelial membrane, namely the cross epithelial pH gradient, or CEPG.

[0008] Strong evidence exists regarding the involvement of CEPG in the transport of nutrients mediated by proton cotransporters and -OH- exchange transporters, such as PEPT1, folate / OH- exchange transporters, and β-alanine / H+ cotransporters. See, for example, Ikuma, J Med Chem. 50:1166-1176, 1996. Disturbances of the pH microclimate, such as a decrease in CEPG, can alter nutrient absorption. This has been demonstrated in the case of proton-mediated absorption of peptides mediated by PEPT1. See, for example, Thwaites et al., Gastroenterology. 122:1322-1333, 2002, and Thwaites and Anderson, Exp. Physiol. 92:603-619, 2007. However, the role of CEPG in the absorption of phosphate ions across the mesenteric membrane has not been established.

[0009] Evidence also exists regarding the involvement of water absorption in ion transport across the epithelium of the small intestine, particularly the jejunum (Juan et al., J Clin Endocrinol Metab. 43:517-22, 1976). However, such mechanisms have been largely uninvestigated in the field of phosphate-lowering therapies. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Joy et al.,J.Manag.Care Pharm.,13:397-411,2007 [Non-Patent Document 2] Ikuma, J Med Chem.50:1166-1176,1996 [Non-Patent Document 3] Thwaites et al.,Gastroenterology.122:1322-1333,2002 [Non-Patent Document 4] Thwaites and Anderson,Exp.Physiol.92:603-619,2007 [Non-Patent Document 5] Juan et al.,J Clin Endocrinol Metab.43:517-22,1976 [Overview of the project]

[0011] The present invention relates, in a broad sense, to non-NHE3-binding compounds (including their stereoisomers, pharmaceutically acceptable salts, and prodrugs) having activity as phosphate transport inhibitors in the gastrointestinal tract, particularly in the small intestine, and to methods of using such compounds to inhibit phosphate uptake and thereby to treat any of the various conditions or diseases for which the regulation of phosphate uptake provides therapeutic benefits.

[0012] Accordingly, embodiments of the present invention include a method for inhibiting phosphate uptake in the gastrointestinal tract of a patient requiring phosphate reduction, comprising administering to the patient a compound that does not bind to NHE3, wherein the compound, when administered to the patient requiring it, is substantially active to inhibit the transport of phosphate ions (Pi) within the gastrointestinal tract.

[0013] In certain embodiments, the compound is a guanylate cyclase C receptor (GC-C) agonist compound.

[0014] In certain embodiments, the compound is a pH adjuster. These and related embodiments are methods for inhibiting phosphate uptake in the gastrointestinal tract of a patient requiring phosphate reduction, comprising administering to the patient a compound that reduces the transepithelial pH gradient (CEPG) in the small intestine, where the CEPG is defined as the pH difference between (i) the cytoplasm of epithelial cells on the surface of the small intestine, optionally at the subapical surface of the epithelial cells, and (ii) the unagitated layer at the apical surface of the small intestine, and the compound, when administered to the patient requiring it, is substantially active to inhibit the transport of phosphate ions (Pi) within the gastrointestinal tract, wherein the compound does not bind to NHE3.

[0015] In some embodiments, the compound reduces water absorption in the small intestine and optionally in the jejunum. These and related embodiments include methods for inhibiting phosphate uptake in the gastrointestinal tract of patients requiring phosphate reduction, comprising administering to the patient a compound that reduces water absorption in the small intestine and optionally in the jejunum, wherein the compound does not bind to NHE3 and, when administered to the patient requiring it, is substantially active to inhibit the transport of phosphate ions (Pi) within the gastrointestinal tract.

[0016] In some embodiments, the compound reduces CEPG in the small intestine and also reduces water absorption in the small intestine. In some embodiments, the compound reduces CEPG in the small intestine without significantly reducing water absorption in the small intestine. In other embodiments, the compound reduces water absorption in the small intestine without significantly reducing CEPG in the small intestine (for example, without significantly stimulating bicarbonate secretion and / or inhibiting acid secretion).

[0017] In some embodiments, this method (a) Hyperphosphatemia, methods for the treatment of postprandial hyperphosphatemia, (b) Methods for treating kidney disease, optionally chronic kidney disease (CKD) or end-stage renal disease (ESRD), (c) Methods for reducing serum creatinine levels, (d) Methods for treating proteinuria, (e) Renal replacement therapy (RRT), a method to optionally delay the time until dialysis. (f) Methods for reducing FGF23 levels, (g) Methods for reducing the hyperphosphatemic effect of activated vitamin D, (h) Hyperparathyroidism, methods to alleviate secondary hyperparathyroidism (optional), (i) Methods to reduce serum parathyroid hormone (PTH), (j) A method for improving endothelial damage that is optionally induced by postprandial serum phosphorus. (k) Vascular calcification, methods for arbitrarily reducing localized intimal vascular calcification, (l) Methods for reducing urinary phosphite, (m) Methods for normalizing serum phosphorus levels (n) Methods for reducing phosphorus load in elderly patients, (o) Methods to reduce dietary phosphate uptake, (p) Methods for reducing renal hypertrophy, and (q) A method selected from one or more of the following to reduce cardiac hypertrophy.

[0018] In certain embodiments, the compound reduces the intracellular pH of epithelial cells on the surface of the small intestine, optionally at the subapical surface of the epithelial cells. In certain embodiments, the compound increases the pH of the unagitated layer at the apical surface of the small intestine. In some embodiments, the compound (a) stimulates bicarbonate secretion in the small intestine, (b) inhibits acid secretion in the small intestine, or (c) stimulates bicarbonate secretion and inhibits acid secretion in the small intestine.

[0019] In certain embodiments, the compound increases one or more intracellular secondary messengers in epithelial cells on the surface of the small intestine. In some embodiments, the one or more intracellular secondary messengers are Ca ++ Selected from cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP).

[0020] In certain embodiments, the compound is substantially inavailable to the patient systemically when administered enterally. In certain embodiments, the compound is substantially impermeable to the epithelium of the gastrointestinal tract. In some embodiments, the compound is substantially permeable to the epithelium of the gastrointestinal tract.

[0021] In certain embodiments, administration to a patient who needs it reduces the serum phosphorus concentration or level to about 150% or less of the normal serum phosphorus level and / or reduces the dietary phosphite uptake by at least about 10% compared to the untreated state. In some embodiments, administration to a patient who needs it increases the phosphate level in fecal excretion by at least about 10% compared to the untreated state. In some embodiments, administration to a patient who needs it reduces the urinary phosphate concentration or level by at least about 10% compared to the untreated state.

[0022] In some embodiments, the patient who needs it has ESRD, and administration to the patient reduces the serum phosphorus concentration or level by at least about 10% compared to the untreated state.

[0023] In some embodiments, the patient who needs it has CKD, and administration to the patient reduces the FGF23 level and the serum intact parathyroid hormone (iPTH) level by at least about 10% compared to the untreated state.

[0024] In certain embodiments, the compound is selected from one or more of guanylate cyclase C receptor (GC-C) agonists, P2Y agonists, adenosine A2b receptor agonists, soluble guanylate cyclase agonists, adenylate cyclase receptor agonists, imidazoline-1 receptor agonists, cholinergic agonists, prostaglandin EP4 receptor agonists, dopamine D1 agonists, melatonin receptor agonists, 5HT4 agonists, atrial natriuretic peptide receptor agonists, carbonic anhydrase inhibitors, phosphodiesterase inhibitors, and Down-Regulated in Adenoma (DRA or SLC26A3) agonists.

[0025] In some embodiments, the GC-C agonist is a peptide, optionally a bacterial heat-stable enterotoxin, guanylin, proguanylin, uroguanylin, prouroguanylin, lymphoguanylin, or a variant or analog of any of the foregoing.

[0026] In some embodiments, the GC-C agonist peptide has the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO: 1), wherein Xaa1Xaa2Xaa3Xaa4Xaa5 is Asn Ser Ser Asn Tyr (SEQ ID NO: 2), or is absent, or Xaa1Xaa2Xaa3Xaa4 is absent.

[0027] In certain embodiments, Xaa5 is Asn, Trp, Tyr, Asp, or Phe.

[0028] In certain embodiments, Xaa5 is Thr or Ile.

[0029] In certain embodiments, Xaa5 is Tyr, Asp, or Trp.

[0030] In certain embodiments, Xaa8 is Glu, Asp, Gln, Gly, or Pro.

[0031] In certain embodiments, Xaa9 is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr, or Phe.

[0032] In certain embodiments , Xaa9 is Leu, Ile, Val, Lys, Arg, Trp, Tyr, or Phe.

[0033] In certain embodiments, Xaa 12 is Asn, Tyr, Asp, or Ala.

[0034] In a particular embodiment, Xaa 13 These are Ala, Pro, or Gly.

[0035] In a particular embodiment, Xaa 14 These are Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp.

[0036] In a particular embodiment, Xaa 16 These are Thr, Ala, Asn, Lys, Arg, or Trp.

[0037] In a particular embodiment, Xaa 17 These are Gly, Pro, or Ala.

[0038] In a particular embodiment, Xaa 19 These are Trp, Tyr, Phe, Asn, or Leu.

[0039] In a particular embodiment, Xaa 19 It is either Lys or Arg.

[0040] In a particular embodiment, Xaa 20 Xaa 21 Is it AspPhe or Xaa 20 is Asn or Glu, Xaa 21 It is missing. In a particular embodiment, Xaa 19 Xaa 20 Xaa 21 It is missing.

[0041] In certain embodiments, the GC-C agonist peptide includes the amino acid sequence Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 3), or variants thereof having one, two, three, four, or five deletions, insertions, and / or substitutions. In certain embodiments, the peptide includes the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 4), or variants thereof having one, two, three, four, or five deletions, insertions, and / or substitutions.

[0042] In a particular embodiment, the GC-C agonist peptide has the amino acid sequence (III): Xaa1Xaa2Xaa3Cys4Xaa5Xaa6Xaa7Xaa8Xaa9Xaa 10 Xaa 11 Cys 12 Xaa 13 Xaa 14 Xaa 15 Xaa 16 (Sequence ID 5) is included, Xaa1 is Ser, Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is missing, Xaa2 is His, Asp, Glu, Ala, Ser, Asn, Gly, or is missing, Xaa3 is Thr, Asp, Ser, Glu, Pro, Val, or Leu, Xaa5 is Asp, Ile, or Glu, Xaa6 is Ile, Trp, or Leu, Xaa7 is Cys, Ser, or Tyr, Xaa8 is Ala, Val, Thr, Ile, Met, or is missing, Xaa9 is Phe, Tyr, Asn, or Trp, and Xaa 10 is Ala, Val, Met, Thr, or Ile, and Xaa 11 is Ala or Val, Xaa 13 is Thr or Ala, Xaa 14 is Gly, Ala, or Ser, and Xaa 15It is either Cys, Tyr, or missing, Xaa 16 It is His, Leu, or Ser.

[0043] In some embodiments, the peptide includes the amino acid sequence Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu (SEQ ID NO: 6), or variants thereof having one, two, three, four, or five deletions, insertions, and / or substitutions.

[0044] In certain embodiments, the P2Y agonist is selected from the compounds in Figure 4 or Figures 5A-5C. In certain embodiments, the adenosine A2b receptor agonist is selected from the compounds in Figures 6A-6C. In some embodiments, the soluble guanylate cyclase agonist is selected from the compounds in Figures 9A-9L. In certain embodiments, the adenylate cyclase receptor agonist is selected from the compounds in Figure 10. In some embodiments, the imidazoline-1 receptor agonist is selected from moxonidine and the compounds in Figure 11. In certain embodiments, the cholinergic agonist is selected from the compounds in Figure 12. In certain embodiments, the prostaglandin EP4 receptor agonist is selected from PGE2 or its analogs / derivatives and the compounds in Figure 7 or Figure 13. In certain embodiments, the dopamine D1 agonist is selected from the compounds in Figure 14. In some embodiments, the melatonin receptor agonist is selected from melatonin and the compounds in Figure 15. In some embodiments, the 5HT4 agonist is selected from serotonin and its analogues, prcalopride, metoclopramide, cleovoprid, mosapride, prcalopride, lenzaprid, tegaserod, zacoprid, norcisapride, naronopride, and bercetrag.

[0045] In some embodiments, the atrial natriuretic peptide receptor agonist comprises or consists of an amino acid sequence selected from Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr (SEQ ID NO: 7), Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys (SEQ ID NO: 8), and Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg (SEQ ID NO: 9) (including variants thereof having 1, 2, 3, 4, or 5 deletions, insertions, and / or substitutions).

[0046] In certain embodiments, the carbonic anhydrase inhibitor is selected from the compounds in FIG. 17. In certain embodiments, the phosphodiesterase inhibitor is selected from the compounds in FIG. XVIII. In some embodiments, the DRA agonist is selected from FIGS. 21A - B.

[0047] In some embodiments, the compound is substantially systemically bio - unavailable when administered enterally to the patient and (i) has a tPSA of at least about 200 Å 2 In certain embodiments, the compound has a tPSA of at least about 250 Å 2 In certain embodiments, the compound has a tPSA of at least about 270 Å 2 In certain embodiments, the compound has a tPSA of at least about 300 Å 2 In certain embodiments, the compound has a tPSA of at least about 350 Å 2 In certain embodiments, the compound has a tPSA of at least about 400 Å 2 In certain embodiments, the compound has a tPSA of at least about 500 Å 2It has tPSA. In certain embodiments, the compound has a molecular weight of at least about 500 Da, at least about 1000 Da, at least about 2500 Da, or at least about 5000 Da or more. In some embodiments, the compound has (i) a total number of about 5 or more NH and / or OH and / or other possible hydrogen bond donor moieties, (ii) a total number of about 10 or more O atoms and / or N atoms and / or other possible hydrogen bond acceptors, and / or (iii) about 10 5 It has a Moriguchi partition coefficient greater than or less than approximately 10. In some embodiments, the compound has a partition coefficient of approximately 100 × 10⁻⁶. -6 Less than cm / s, or approximately 10 × 10 -6 Less than cm / s, or approximately 1 × 10⁻⁶ -6 Less than cm / s, or approximately 0.1 × 10⁻⁶ -6 Transmission coefficient P less than cm / s app It holds.

[0048] Certain methods further include administering one or more additional bioactive agents. In some embodiments, the compound and one or more additional bioactive agents are administered as part of a single pharmaceutical composition. In certain embodiments, the compound and one or more additional bioactive agents are administered as separate pharmaceutical compositions. In some embodiments, the separate pharmaceutical compositions are administered sequentially. In some embodiments, the separate pharmaceutical compositions are administered simultaneously.

[0049] In certain embodiments, additional bioactive agents are selected from vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), active vitamin D (calcitriol), and active vitamin D analogs (e.g., doxelcalciferol, paricalcitol).

[0050] In certain embodiments, the additional bioactive agent is a phosphate binder. In some embodiments, the phosphate binder is selected from the group consisting of sevelamers (e.g., Renvela® (cevelamer carbonate), Renagel® (cevelamer hydrochloride)), lanthanum carbonate (e.g., Fosrenol®), calcium carbonate (e.g., Calcichew®, Titralac®), calcium acetate (e.g., PhosLo®, Phosex®), calcium acetate / magnesium carbonate (e.g., Renepho®, OsvaRen®), MCI-196, ferric citrate (e.g., Zerenex®), magnesium iron carbonate hydroxide (e.g., Fermagate®), aluminum hydroxide (e.g., Alucaps®, Basaljel®), APS1585, SBR-759, and PA-21.

[0051] In certain embodiments, the additional bioactive agent is a NaPi2b inhibitor. In some embodiments, the additional bioactive agent is niacin or nicotinamide.

[0052] In certain embodiments, the subject has CKD, and the active biological agent is selected from one or more of the following: ACE inhibitors, antiogensin II receptor blockers, β-blockers, calcium channel blockers, direct renin inhibitors, diuretics, vasodilators, erythropoietin therapy, iron supplementation therapy, advanced glycation end product inhibitors, vitamin D, and statins.

[0053] In certain embodiments, the compound or composition is administered orally, and optionally, the compound or composition is administered orally once daily.

[0054] A method for screening phosphate uptake inhibitors, comprising: (a) culturing intestinal cells; (b) contacting the cultured intestinal cells with a test compound; (c) measuring (i) the pH at the apical surface of the intestinal cells, (ii) the intracellular pH of the intestinal cells, and / or (iii) phosphate uptake by the intestinal cells; and (d) identifying the test compound as a phosphate uptake inhibitor if the pH in (c)(i) increases compared to a control, the intracellular pH in (c)(ii) decreases compared to a control, and / or phosphate uptake in (c)(iii) decreases compared to a control.

[0055] In some embodiments, step (a) includes culturing intestinal cells in a monolayer. In certain embodiments, step (a) includes isolating the cells from the intestinal crypts and culturing them under conditions sufficient to form an enteroid. In certain embodiments, step (a) includes culturing the isolated embryonic stem cells, endodermal cells, or pluripotent stem cells under conditions sufficient to form an organoid. In some embodiments, step (a) includes culturing the intestinal section(s) in a Ussing chamber.

[0056] In certain embodiments, step (c)(i) includes contacting the cells with a pH-sensitive fluorescent dye and measuring the fluorescence of the dye. In some embodiments, step (c)(ii) includes contacting the cells 33 This includes contacting a P-labeled phosphate ion with the phosphate ion and measuring the uptake of the labeled phosphate ion.

[0057] In some embodiments, the increase and / or decrease of (d) is statistically significant.

[0058] In certain embodiments, the test compound is a small molecule or peptide known or suspected to stimulate bicarbonate secretion and / or inhibit acid secretion in the small intestine.

[0059] In certain embodiments, the test compound is selected from one or more of the following, as described herein and / or known in the art: P2Y agonists, adenosine A2b receptor agonists, guanylate cyclase C receptor agonists, soluble guanylate cyclase agonists, adenylyl cyclase receptor agonists, imidazoline-1 receptor agonists, cholinergic agonists, prostaglandin EP4 receptor agonists, dopamine D1 agonists, melatonin receptor agonists, 5HT4 agonists, atrial natriuretic peptide receptor agonists, carbonic anhydrase inhibitors, phosphodiesterase inhibitors, and exchange transporter (DRA or SLC26A3) agonists that are downregulated in adenomas.

[0060] These and other aspects of the present invention will become apparent from the following detailed description. [Brief explanation of the drawing]

[0061] [Figure 1] Figures 1A-1B show that linaclotide (a GC-C receptor agonist) reduces phosphate uptake in the gastrointestinal tract of rats. [Figure 2] Figures 2A-2B show that moxonidine (an imidazoline subtype 1 (I1) receptor agonist) and colforsin (an adenylyl cyclase agonist), a water-soluble forskolin analog, reduce phosphate uptake in the gastrointestinal tract of rats. [Figure 3] This study demonstrates that the P2Y2 receptor agonist Up4U reduces phosphate uptake in the gastrointestinal tract of rats. [Figure 4-1] An example of a small molecule P2Y receptor agonist is shown. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5A] An example of a small molecule P2Y receptor agonist is shown. [Figure 5B] This is a continuation of Figure 5A. [Figure 5C] This is a continuation of Figure 5B. [Figure 6A-1]This shows exemplary small molecule adenosine A2b receptor agonists, including representative adenosine-like A2b agonists (6B) and representative dicyanopyridine A2b agonists (6C). [Figure 6A-2] This is a continuation of Figure 6A-1. [Figure 6B] This is a continuation of Figure 6A. [Figure 6C] This is a continuation of Figure 6B. [Figure 7] A list of exemplary prostaglandin EP4 receptor agonists is shown below. [Figure 8A] The photophysical properties of an exemplary pH indicator near neutral (8A) and an acidic pH indicator (8B) are shown. [Figure 8B] This is a continuation of Figure 8A. [Figure 9-1] This document describes exemplary soluble guanylate cyclase (sGC) agonists, including heme-dependent and heme-independent agonists (9A). [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. [Figure 9-4] This is a continuation of Figure 9-3. [Figure 9-5] This is a continuation of Figure 9-4. [Figure 9-6] This is a continuation of Figure 9-5. [Figure 9-7] This is a continuation of Figure 9-6. [Figure 9-8] This is a continuation of Figure 9-7. [Figure 9-9] This is a continuation of Figure 9-8. [Figure 9-10] This is a continuation of Figure 9-9. [Figure 9-11] This is a continuation of Figure 9-10. [Figure 9-12] This is a continuation of Figure 9-11. [Figure 9-13] This is a continuation of Figure 9-12. [Figure 9-14] This is a continuation of Figure 9-13. [Figure 9-15] This is a continuation of Figure 9-14. [Figure 9-16] This is a continuation of Figure 9-15. [Figure 9-17] This is a continuation of Figure 9-16. [Figure 9-18] This is a continuation of Figure 9-17. [Figure 9-19] This is a continuation of Figure 9-18. [Figure 9-20] This is a continuation of Figure 9-19. [Figure 9-21] This is a continuation of Figure 9-20. [Figure 9-22] This is a continuation of Figure 9-21. [Figure 10-1] An example of adenylyl cyclase receptor agonists is shown. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11] An example of an imidazoline receptor agonist is shown. [Figure 12-1] Exemplary cholinergic agonists and antagonists, atropine and (-)-hyosine, are shown. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 13-1] An exemplary EP4 receptor agonist is shown. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14] An example of a dopamine D1 receptor agonist is shown. [Figure 15] An example of a melatonin (MT2) receptor agonist is shown. [Figure 16] The structures of exemplary peptide agonists that act as NP receptors (SEQ ID NOs: 7, 8, and 9) are shown. [Figure 17-1] An example of a carbonic anhydrase inhibitor is shown. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18] An exemplary phosphodiesterase inhibitor is shown. [Figure 19] The diagram illustrates the pH gradient observed within the intestinal tract, including the pH gradient across the cell membrane, as well as the pH gradients in the immediate vicinity of the epithelial membrane and within the intestinal lumen. [Figure 20] This diagram shows the phase diagrams of solubility of calcium and phosphate ions in an aqueous environment (at room temperature) across a certain range of pH values. [Figure 21-1] Representative examples of subtype-selective PKC inhibitors are shown below. [Figure 21-2] This is a continuation of Figure 21-1. [Figure 22] Figures 22A-22C show that acidification within HEK-293 cells caused a significant reduction in phosphate uptake, as measured by the uptake of 33P-labeled Pi. [Modes for carrying out the invention]

[0062] The following description includes certain specific details in order to provide a complete understanding of the various embodiments of the present invention. However, those skilled in the art will understand that the present invention can be practiced without using these details.

[0063] Unless otherwise required by context, throughout this specification and the claims, “comprise” and its variations, such as “comprises” and “comprising,” shall be interpreted in a non-restrictive and comprehensive sense, that is, “comprises but not limited to.”

[0064] Throughout this specification, any reference to “one embodiment” means that any particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, the appearance of the phrase “in one embodiment” in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, any particular feature, structure, or characteristic may be combined in any preferred manner in one or more embodiments.

[0065] Embodiments of the present invention, in a broad sense, relate to the discovery that non-NHE3-binding compounds, such as guanylate cyclase agonist compounds, can inhibit phosphate uptake in the gastrointestinal tract, for example, in the small intestine.

[0066] According to one non-limiting theory, cellular uptake of phosphate ions (Pi) may be influenced by changes in intracellular pH and / or the pH of the adjacent extracellular environment. For example, as shown in the accompanying example, intracellular acidification of human fetal kidney (HEK-293) cells (while maintaining extracellular pH at approximately 7.4) 33 The uptake of P-labeled Pi resulted in a significant reduction in phosphate uptake as measured.

[0067] The same phenomenon was observed in related experiments in which the phosphate transporter NaPi2b(SLC34A2) was transiently expressed in HEK-293 cells. Since the endogenous Pi transporters Pit-1 and / or Pit-2(SLC20A2) are responsible for Pi uptake in untransformed HEK-293 cells (to meet cellular metabolic needs), it was concluded that the effect of the decrease in intracellular pH on Pi uptake is a general decrease that is not necessarily related to a specific phosphate transporter. Pit-1 and Pit-2 are single-nucleotide forms of phosphate, NaH2PO3 - It transports NaHPO3, while NaPi2b is in a dinucleotide form. 2- It transports phosphate. The observation that cellular acidification affects phosphate uptake using both transporters suggests that H + This contradicts a mechanism based solely on changes in the electrochemical gradient.

[0068] These observations are entirely counterintuitive, since an increase in Pi uptake could have been expected. For example, a decrease in intracellular pH (e.g., without any corresponding change in extracellular pH) could lead to a decrease in the dinucleotide form of phosphate (NaPO3). 2- It could be predicted that this would generate a driving force for the incorporation of basic anions such as ).

[0069] Nevertheless, a reduction in phosphate uptake has been observed, and the possibility of using direct or indirect pH modifiers, particularly those with pH modifier activity in the gastrointestinal tract (e.g., small intestine), to reduce phosphate uptake in patients requiring phosphate reduction has been suggested. This possibility is supported by the observation that various pH modifiers can reduce phosphate uptake in the gastrointestinal tract of mammals (see accompanying examples). As used herein, the term "pH modifier" refers to the introduction of bicarbonate (HCO3) into the lumen of the gastrointestinal tract, e.g., the small intestine or duodenum. - ) Directly or indirectly increases secretion and / or acid / proton (e.g., H + ) Contains drugs or compounds that can reduce secretion. Some pH-adjusting compounds, for example, target certain intracellular secondary messengers in the epithelial cells of the gastrointestinal tract, such as Ca ++ These compounds can act by regulating (e.g., increasing) cAMP, cGMP, and others. Therefore, some exemplary compounds directly or indirectly stimulate bicarbonate secretion into the lumen of the small intestine, inhibit acid secretion into the lumen of the small intestine, or stimulate bicarbonate secretion into the lumen of the small intestine and inhibit acid secretion. In some embodiments, the compounds reduce the cytoplasmic or intracellular pH of epithelial cells on the surface of the small intestine, optionally at the subapical surface of the epithelial cells, with or without adjustment of the pH of the adjacent extracellular environment. In certain embodiments, the compounds do not bind to or inhibit sodium-hydrogen exchange transporter 3 (NHE3).

[0070] In some embodiments, the compound reduces the pH of the "unagitated layer" at the apical surface of the small intestine. The "unagitated layer" is permeable to rapidly moving substances (e.g., 1 H +This refers to a stationary layer adjacent to the apical membrane (e.g., approximately 600 μm deep) that acts as a diffusion barrier, allowing the rate of diffusion to be limited. While we do not wish to be bound by theory, such a technique would induce the flow of bicarbonate across the epithelial cells of the gastrointestinal tract, increasing the pH in the immediate vicinity of the extracellular space (UWL) and consequently decreasing the pH gradient on the mucosal surface. Due to the continuous exchange of proton ions and bicarbonate ions at the apical surface of intestinal cells via cotransporters, exchange transporters, and channels, a pH gradient is maintained across the cell membrane. As a result of the undisturbed layer, another pH gradient is established between the immediate vicinity of the epithelial membrane and the intestinal lumen. The two pH gradients are schematically represented in Figure 19.

[0071] Therefore, in some embodiments, certain compounds reduce the transepithelial pH gradient (CEPG) within the gastrointestinal tract. The term "CEPG" includes the difference between (i) the cytoplasmic pH (i.e., intracellular pH) of epithelial cells on the surface of the small intestine, optionally at the subapical surface of the epithelial cells, and (ii) the pH of the unagitated layer at the apical surface of the small intestine. Certain embodiments exclude compounds (e.g., antacids) that simply increase the pH of the lumen of the gastrointestinal tract without regulating the secretion of bicarbonates and / or acids, or without altering the pH of the unagitated layer or UWL.

[0072] While we do not wish to be bound by any one theory, in some embodiments, free calcium ions in the lumen may contribute to the inhibition of Pi uptake induced by a decrease in CEPG. The phase diagram of calcium and phosphate ions in an aqueous environment at room temperature shows that the solubility of calcium (and thus phosphate) is pH-dependent, meaning that the solubility of phosphate decreases as the pH increases. See Figure 20. This phenomenon suggests that, all other than the present, a drug-inducible increase in pH in the mucosal surface microenvironment minimizes the availability of free Pi and, consequently, reduces its uptake by cells in the gastrointestinal tract.

[0073] According to another non-limiting theory, phosphate ion uptake may be affected by water absorption in the small intestine, primarily in the jejunum. Specifically, increased water absorption in the small intestine is associated with increased phosphate uptake, and vice versa. In such cases, non-NHE3-binding compounds that reduce water absorption in the small intestine may be used to inhibit phosphate uptake. Thus, a particular embodiment relates to a method for inhibiting phosphate uptake in the gastrointestinal tract of a patient requiring phosphate reduction, comprising administering to the patient a compound that reduces water absorption in the small intestine, wherein the compound does not bind to NHE3, and when administered to the patient requiring it, the compound is substantially active to inhibit the transport of phosphate ions (Pi) within the gastrointestinal tract. In a particular embodiment, the compound reduces "net" water absorption, for example, by regulating the balance between secretion and absorption, for example, by reducing absorption, increasing secretion, or both. In some embodiments, the compound reduces water absorption in the jejunum.

[0074] In some embodiments, inhibition of phosphate uptake in the gastrointestinal tract can be achieved by administering certain compounds and / or pharmaceutical compositions containing them, which can be advantageously designed so that the compounds are absorbed little or substantially no into the bloodstream (i.e., designed to be non-systemic or substantially non-systemic). In this respect, the compounds are characterized by producing little or substantially no systemic availability when administered enterally, including orally. In other words, the compounds are not absorbed into the bloodstream at a meaningful level and therefore have no activity in the bloodstream, but instead their activity is substantially localized in the gastrointestinal tract.

[0075] Accordingly, in certain exemplary embodiments further described herein, the compounds of the present invention generally require a combination of structural and / or functional features that relate to or contribute to their activity in the gastrointestinal tract and / or their substantial non-systemic bioavailability. Such features include, for example, (i) specific tPSA values ​​and / or MW values ​​(e.g., at least about 190 Å each).2 (i) a specific level of fecal recovery rate of the compound and / or its metabolites after administration (e.g., greater than 50% in 72 hours), (iii) a specific number of NH and / or OH and / or potentially hydrogen bond donor moieties (e.g., greater than about 5), (iv) a specific number of rotatable bonds (e.g., greater than about 5), (iv) a specific permeability characteristic (e.g., about 100 × 10⁻¹⁶). -6 P less than cm / s app ), and / or one or more of the other features and characteristics described herein.

[0076] In patients with progressive kidney disease (e.g., stages 4 and 5), phosphate overload in the body manifests as serum phosphorus levels exceeding normal levels, i.e., hyperphosphatemia. Hyperphosphatemia is directly associated with mortality and morbidity. Inhibition of phosphate transport in the intestinal tract reduces serum phosphorus levels and, consequently, improves the prognosis of these patients. In patients with chronic kidney disease in stages 2 and 3, phosphate overload in the body does not necessarily lead to hyperphosphatemia; that is, the patient remains orthophosphomic, which does not induce an increase in FGF-23, a risk factor for mortality and morbidity in these patients. Therefore, it is necessary to reduce or prevent phosphate overload in the body even in these early stages to avoid associated bone and vascular damage and ultimately improve mortality.

[0077] Inhibition of phosphate transport within the intestinal tract would be particularly beneficial for patients with diseases treatable by inhibiting phosphate uptake from the intestines. Furthermore, inhibition of phosphate transport may slow the progression of renal failure and reduce the risk of cardiovascular events, among other diseases or conditions associated with the need for phosphate reduction.

[0078] I. Phosphate transport inhibitor compounds Embodiments of the present invention relate to compounds that can inhibit or reduce phosphate transport / uptake in the gastrointestinal tract, for example, by adjusting the pH in or adjacent to the epithelial membrane of the gastrointestinal lumen, reducing water absorption in the small intestine, or both. Examples of pH-adjusting compounds include those that stimulate bicarbonate secretion in the small intestine (i.e., duodenal bicarbonate secretion, i.e., DBS), inhibit acid / proton secretion in the small intestine, or both.

[0079] The compounds provided herein may include small molecules of synthetic or bio-derived origin, and peptides or polypeptides. The terms “peptide” and “polypeptide” are used interchangeably herein, but in certain instances, the term “peptide” may refer to shorter polypeptides, e.g., polypeptides consisting of approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, including all integers and ranges between them (e.g., 5–10, 8–12, 10–15). Polypeptides and peptides may consist of spontaneously occurring and / or non-spontaneous amino acids. Antibodies are also included as polypeptides.

[0080] In some embodiments, the compound is selected from one or more of the following: P2Y receptor agonists, adenosine A2b receptor agonists, guanylate cyclase C receptor agonists, soluble guanylate cyclase agonists, adenylyl cyclase receptor agonists, imidazoline-1 receptor agonists, cholinergic agonists, prostaglandin EP4 receptor agonists, dopamine D1 agonists, melatonin receptor agonists, 5HT4 agonists, atrial natriuretic peptide receptor agonists, carbonic anhydrase inhibitors, phosphodiesterase inhibitors, or exchange transporter (DRA or SLC26A3) agonists that are downregulated in adenomas. As described above, in some embodiments, such agonist compounds induce bicarbonate secretion and / or inhibit acid secretion in the upper gastrointestinal tract, including the duodenum and proximal jejunum. In some embodiments, this mechanism of action directly or indirectly modulates apical proton and bicarbonate transporters, resulting in a reduction of CEPG or a relatively basic microenvironment on the mucosal surface, thereby reducing phosphate uptake / absorption.

[0081] In certain embodiments, the compound directly or indirectly stimulates duodenal bicarbonate secretion (DBS). DBS is a natural defense mechanism of the mucosa that functions in the duodenal and proximal jejunal regions of the intestine to neutralize acidic gastric juice. DBS can be stimulated by several biological pathways, including, in particular, those that regulate the activity of chloride and bicarbonate exchange transporters such as SLC26A3(DRA) and SLC26A3(PAT-1), chloride and bicarbonate channels mediated by CFTR, and calcium-activated chloride channels. In some embodiments, these pathways are mediated by intracellular Ca ++ It is stimulated by an increase in one or more secondary messengers such as cAMP and / or cGMP.

[0082] In some embodiments, the compound directly or indirectly reduces water absorption in the small intestine. In certain embodiments, the compound reduces water absorption in the jejunum. In certain embodiments, the compound increases water absorption in the small intestine by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control compound or no compound.

[0083] The term "agonist" includes compounds that bind to target molecules, such as receptors, and induce or stimulate a cellular response by those target molecules. These include super agonists, full agonists, partial agonists, and selective agonists. Super agonists produce a greater maximal response to a target molecule than endogenous agonists; full agonists produce a comparable response to a target molecule compared to endogenous agonists; and partial agonists produce a significantly smaller (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%) maximal response to a target molecule than endogenous agonists.

[0084] In addition to its activity as an agonist, in certain embodiments, a compound may also be characterized by its "specific binding" to a target. For example, in some embodiments, a compound (e.g., a direct-acting compound) has a binding affinity (K) to the targets described herein of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM. d) can be specifically bound to. In certain embodiments, the target is selected from one or more of the following, as described herein: P2Y receptor, adenosine A2b receptor, guanylate cyclase C receptor, adenylyl cyclase receptor, imidazoline-1 receptor, acetylcholine receptor, prostaglandin EP4 receptor, dopamine D1 receptor, melatonin receptor, 5HT4, atrial natriuretic peptide receptor, carbonic anhydrase, phosphodiesterase, and exchange transporters (DRA or SLC26A3) that are downregulated in adenomas.

[0085] A. P2Y agonist In certain embodiments, the compound is a P2Y agonist (or P2Y receptor agonist). P2Y receptors refer to a family of purinergic G protein-binding receptors. Examples of human P2Y receptors include P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y8, P2Y9, and P2Y 10 P2Y 11 P2Y 12 P2Y 13 , and P2Y 14 These include: The main natural or endogenous ligands for the P2Y receptor are adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), uridine 5'-diphosphate (UDP), and UDP-glucose (or other UDP sugars). Dinucleotides such as Ap4U are also naturally occurring P2Y agonists.

[0086] P2Y receptors are located in duodenal cells. ++ It has been shown to mediate signaling and contribute to duodenal bicarbonate secretion. See, for example, Dong et al., Am J Physiol Gastrointest Liver Physiol 296:G424-G432, 2009. Although not limited to one mechanism, in certain aspects, P2Y receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine (also known as duodenal bicarbonate secretion or DBS).

[0087] Although not limited to any one mechanism, in some embodiments, P2Y receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0088] Some P2Y receptors are selectively activated by adenine nucleotides, such as ATP and ADP, while others are selectively activated by uracil nucleotides or UDP-glucose. The P2Y1 receptor is the primary component of the functionality of defined P2Y-purine receptors. It functions in platelets as well as in various tissues, including smooth muscle, endothelium, and nerve tissue. The P2Y1 receptor is selective for adenine nucleotides. ADP is the most potent physiological agonist. In some embodiments, the compound is a P2Y1 receptor agonist, optionally a P2Y1 receptor agonist selective to other P2Y receptors. An example of a P2Y1 receptor agonist is 2-methylthio-ADP.

[0089] In certain embodiments, the compound is a P2Y2 and / or P2Y4 receptor agonist, optionally a selective P2Y2 receptor agonist compared to other P2Y receptors. These two receptors exhibit the highest identity (66.8%) in the sequence of the TM domain of all their P2Y receptor subtypes. The P2Y2 receptor can be activated by adenine nucleotides such as uracil nucleotides, UDP sugar derivatives, and ATP. The P2Y2 receptor is expressed in many tissues, including the lungs, heart, skeletal muscle, spleen, kidneys, liver, and epithelium. These receptors play a crucial role in regulating ion transport within epithelial cells. Triphosphate nucleotides, including UTP, ATP, UTPγS, and ATPγS, act as complete agonists of the P2Y2 receptor. In addition to the above agonists, the P2Y2 receptor also responds to diadenosine tetraphosphate (AP4A) and Up4U (diquafosol, INS365, used to treat dry eye). The analog P-(uridine 5')-P4-(2'-deoxycytidine 5')tetraphosphate (INS37217) is a potent agonist at the P2Y2 receptor with several agonist effects on the P2Y4 receptor. Denufosol ((3S,5R)-5-(4-amino-2-oxopyrimidine-1-yl)-3-hydroxyoxolan-2-yl)methoxy-hydroxyphosphoryl [[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidine-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogen phosphate (including its tetrasodium salt) is also an exemplary P2Y2 receptor agonist. PSB1114 is also included.

[0090] In ribose and uracil modifications, both 2'-deoxy-2'-amino-UTP and 2'-thio-UTP maintain the agonist efficacy of UTP at the P2Y2 receptor. The combination of these two modifications works synergistically to increase efficacy (8 nM EC2). 50 This results in 2'-amino-2-thio-UTP, which improves both the 5-position modification, for example, 5-bromo-UTP (EC 50 =0.75μM) and 5-iodine-UTP(EC50 The results (e.g., 0.83 μM) suggest that the introduction of small hydrophobic groups may be beneficial in the P2Y2 receptor.

[0091] The P2Y receptor agonists provided herein include mononucleotides, dinucleotides, and nucleotide sugars, among other agonists known in the art. For example, U.S. Patent No. 6,624,150, European Patent No. 1196396, International Publication No. 2008 / 060632, Cosyn et al., Bioorg Med Chem Lett. 19:3002-5, 2009 (describes uridine 5'-(phospho)phosphonate and 5'-methylenephosphonate equivalent of UMP), Ko et al., Bioorg Med. Chem. 16:6319-32, 2008 (for example, describing α,β-methylene-UDP, a P2Y6 receptor agonist; Up(4)-phenyl ester and Up(4)-[1]glucose, selective P2Y2 receptor agonists; dihalomethylenephosphonate analogs, selective P2Y2 receptor agonists; and the 2-thio analog of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y2 receptor agonist; Ivanov et al., J Med Chem. 50:1166-76, 2007; Brookings et al., Bioorg Med Chem Lett. 17:562-5, 2007 (describing the synthesis and P2Y2 agonist activity of a series of nucleoside triphosphates); and Jacobson et al. See al., Purinergic Signal. 5:75-89, 2009.

[0092] Additional examples of P2Y receptor agonists include P1,P4-diadenosine tetraphosphate (A2P4), uridine-5'-diphosphate (UDP), uridine-5'-O-(2-thiodiphosphate) (UDPβS), 5-bromouridine-5'-triphosphate (5-BrUTP), 5-(1-phenylethynyl)-uridine-5'-triphosphate (5-(1-phenylethynyl)UTP), 5-methyluridine-5'-diphosphate (5-methylUDP), and 4-hexylthiouridine-5'-triphosphate (4-hexylthiouridine). XylthioUTP), 4-thiouridine-5'-triphosphate (4-thioUTP), 2-methoxyuridine-5'-triphosphate (2-methoxyUTP), 4-(1-morpholino)uridine-5'-tetraphosphate (4-(1-morpholino))UP4, 4-hexyloxyuridine-5'-diphosphate (4-hexyloxyUDP), 4-(N,N-dimethyl)cytidine-5'-triphosphate (N,N-dimethylCTP), 4-(N-hexyl)cytidine-5'-triphosphate (N-hexylCTP), P 1 -(Citidine-5')-P 4 -(uridine-5'-)tetraphosphate (CP4U), P 1 -O-(methyl)-P 4 Examples include those described in International Publication No. 1999 / 09998 and U.S. Applications No. 2002 / 0052336 and 2003 / 0027785, which include -(uridine-5'-)tetraphosphate (MeP4U) and 4-(N-cyclopentyl)thymidine-5'-triphosphate (N-cyclopentylCTP).

[0093] 5'-adenosine triphosphate (ATP), 5'-uridine triphosphate (UTP), uridine-5'-O-(3-thiotriphosphate) (UTPγS), P 1This also includes -(uridine-5')-P.sup.4-(uridine-5'-)tetraphosphate (U2P4), 5'-[4-(thiouridine)]-triphosphate (4-thioUTP), and P1-(cytidine-5')-P4-(uridine-5'-)tetraphosphate (CP4U). The identification and preparation of certain thiophosphate analogs of nucleoside diphosphates (e.g., UDP-β-S) are described in U.S. Patent No. 3,846,402 and Goody and Eckstein (J.Am.Chem.Soc.93:6252-6257.1971). Alternatively, UTP and other analogs of UTP are also marketed by distributors such as Sigma (St. Louis, Mo.) and Pharmacia (Uppsala, Sweden). An exemplary method for identifying P2Y receptor agonists is described, for example, in U.S. Patent Application No. 2003 / 0175810.

[0094] In some embodiments, the P2Y receptor agonist is a non-endogenous small molecule agonist. Examples of additional P2Y receptor agonists are shown in Figures 4 and 5A-5C.

[0095] B. Adenosine A2b receptor agonists In certain embodiments, the compound is an adenosine A2b receptor agonist, optionally a selective agonist. Adenosine exerts most of its physiological functions by acting as a localization regulator in four receptor subtypes, named A1, A2A, A2B, and A3 adenosine receptors (ARs). The adenosine A2b receptor (i.e., ADORA2B) is a G protein-bound adenosine receptor, an endogenous membrane protein that stimulates adenylate cyclase activity in the presence of adenosine.

[0096] The A2b receptor is expressed in various tissues, with high concentrations suggested in the cecum and large intestine on both the mucosal and basal surfaces of colonic epithelial cells. See Baraldi et al., Purinergic Signal. 5:3-19, 2009. Activation at any site leads to Cl- secretion via direct activation of cAMP-activated Cl- channel transmembrane conductance regulator (CFTR). CFTR regulates the secretion of both chloride and bicarbonate ions. For example, in rats, the A2B receptor is immunolocalized to the brush border membrane of duodenal villi, where luminal adenosine has been shown to stimulate bicarbonate secretion via the A2B receptor and CFTR. See, for example, Ham et al., J Pharmacol Exp Ther. 335:607-13, 2010. While not limited to any single mechanism, in certain aspects adenosine A2b receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract, for example, by reducing CEPG or by stimulating bicarbonate secretion into the small intestine.

[0097] Although not limited to any one mechanism, in some embodiments, adenosine A2b receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0098] Common examples of adenosine A2b receptor agonists include adenosine, adenosine-like compounds, and non-adenosine compounds. In some embodiments, nucleoside adenosine A2b receptor agonists include modified adenosine compounds, such as adenosine compounds substituted at the N(6) position of the purine heterocycle, substituted at the C(2) position of the purine heterocycle, substituted at the 5' position of the ribose moiety, and any combination thereof. Non-ribose ligands such as substituted dicarbonitriliary pyridines are also included, one example being 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridine-2-ylsulfanil]acetamide. For example, see Baraldi et al., Purinergic Signal. 4:287-303, 2008 and Baraldi et al., Purinergic Signal. 5:3-19, 2009, which are incorporated herein by reference in their entirety.

[0099] Additional non-limiting examples of adenosine A2b receptor agonists include BAY 60-6583, CV 1808, AMP579, NECA (N-ethylcarboxamide adenosine), (S)-PHPNECA, LUF-5835, 6-guanylNECA, and LUF-584. See also Beukers et al., J.Med.Chem.47:3707-3709, 2004 (for example, describing non-adenosine agonists such as LUF5834 (2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazole-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile) and LUF5835 (3-hydroxyphenyl analog)), Beukers et al., Med Res Rev.26:667-98, 2006 (for example, describing (S)PHPNECA and certain non-ribose ligands as adenosine A2b receptor agonists), and Liu et al., Basic Res Cardiol.105:129-37, 2010. This also includes the A2b receptor agonists described in U.S. Patent Application No. 2002 / 0156076. These references are incorporated herein by reference in their entirety.

[0100] Examples of adenosine A2b receptor agonists are shown in Figures 6A–6C, and together with methods for their synthesis, are further disclosed in U.S. Patent Application No. 2009 / 0221649 and PCT Publications International Publications No. 2006 / 027142, 2007 / 101531, and 2003 / 008384, which are incorporated herein by reference in their entirety.

[0101] C. Guanylate cyclase C receptor agonists In certain embodiments, the compound is a guanylyl cyclase C (GC-C) agonist, optionally a selective agonist. GC-C is an isoform of the guanylate cyclase family that is highly concentrated in the apical membrane of intestinal epithelial cells. It is also a target receptor for bacterial secretory thermostable enterotoxins that cause acute secretory diarrhea. GC-C is also known as guanylate cyclase 2C, intestinal guanylate cyclase, guanylate cyclase C receptor, and thermostable enterotoxin receptor (hSTAR).

[0102] GC-C possesses an extracellular ligand-binding domain, a single transmembrane domain, a protein kinase-like domain, and a C-terminal guanylate cyclase domain. Its tyrosine kinase activity mediates the intracellular GC-C signaling pathway. Guanylin and uroguanilin are endogenous peptide ligands for GC-C. Activation of GC-C triggers other downstream signals, such as increased intracellular cGMP, PKGII-dependent phosphate chlorination of cystic fibrosis transmembrane regulator (CFTR), and increased endocrine secretion of chloride and bicarbonate ions (via CFTR, and possibly DRA or PAT-1).

[0103] GC-C agonists such as linaclotide, guanylin, and heat-stable enterotoxin (STa) of Escherichia coli have been shown to stimulate duodenal bicarbonate secretion. See, for example, Rao et al., Am J Physiol Gastrointest Liver Physiol 286:G95-G101, 2004; Busby et al., Eur J Pharmacol. 649:328-35, 2010; and Bryant et al., Life Sci. 86:760-5, 2010. While not limited to any single mechanism, in certain aspects, GC-C agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine.

[0104] Although not limited to any one mechanism, in some embodiments, GC-C agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0105] General examples of GC-C agonists include peptide agonists and their analogues, including synthetic analogues of endogenous GC-C peptide agonists. Specific examples of GC-C agonists include, but are not limited to, heat-stable enterotoxins (ST or STa peptides), including those derived from E. coli, guaniline, proguaniline, uroguaniline, prouroguaniline, lymphoguaniline, linaclotide (Linzess), SP-333, and precanatide. See, for example, Drug Des Devel Ther. 7:351-60, 2013. Linaclotide is a synthetic STa analogue marketed for the treatment of irritable bowel syndrome (constipation type) (IBS-C). See, for example, Bryant et al., Life Sci. 86:760-5, 2010. Precanatide is a synthetic analogue of uroguaniline developed for the treatment of IBS-C. For example, see Pitari (above) and Shailubhai et al., Dig Dis Sci. 2013 Apr 27. [Epub ahead of print]. Examples of additional GC-C agonists are described in U.S. Patent Applications 2012 / 0064039, 2004 / 0258687, 2005 / 0287067, 2006 / 0281682, 2006 / 0258593, 2006 / 0094658, 2008 / 0025966, 2003 / 0073628, 2004 / 0121961, and 2004 / 0152868, as well as U.S. Patents 5,140,102, 7,041,786, and 7,304,036. These references are incorporated herein by reference in their entirety.

[0106] In some embodiments, the GC-C agonist is a bacterial ST (or STa) peptide, or a variant, analog, or derivative thereof. Within bacteria, ST or STa peptides are generally derived from preproproteins having at least 70 amino acids. The pre and pro regions are cleaved as part of the secretory process, and the resulting mature protein generally contains fewer than about 20 amino acids and is biologically active.

[0107] Examples of bacterial ST peptides include: E. coli ST Ib with the mature amino acid sequence Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 10) (Moseley et al., Infect.Immun.39:1167,1983); E. coli ST Ia with the mature amino acid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr (SEQ ID NO: 11) (So and McCarthy, PNAS USA.77:4011,1980); E. coli ST I with the mature amino acid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Tyr Pro Ala Cys Ala Gly Cys Asn (SEQ ID NO: 12) (Chan and Giannella, J.Biol.Chem.256:7744,1981); and E. coli ST I with the mature amino acid sequence Asn Citrobacter freundii ST peptide containing Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr (SEQ ID NO: 13) (Guarino et al., Infect. Immun. 57:649, 1989); and the following pro-form amino acid sequences: Gln Ala Cys Asp Pro Pro Ser Pro Pro Ala Glu Val Ser Ser Asp Trp Asp Cys Cys Asp Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO: 14) (and Ser-7~Leu-7 variants of Y-STa (SEQ ID NO: 15), (Takao et al., Eur. J. Biochem.)Y-ST (Y-STa), Y-STh, and Y-STc (reviewed in Huang et al., Microb. Pathog. 22:89, 1997) are Y.enterocolitica ST peptides having the following sequences: Lys Ala Cys Asp Thr Gln Thr Pro Ser Pro Ser Glu Glu Asn Asp Asp Trp Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO: 16), Gln Glu Thr Ala Ser Gly Gln Val Gly Asp Val Ser Ser Ser Thr Ile Ala Thr Glu Val Ser Glu Ala Glu Cys Gly Thr Gln Ser Ala Thr Thr Gln Gly Glu Asn Asp Trp Asp Tip Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Phe Gly Cys (SEQ ID NO: 17); mature amino acid sequence Ser Asp Examples include the Y. kristensenii ST peptide with the sequence Trp Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO: 18); the V. cholerae non-01 ST peptide with the mature amino acid sequence Ile Asp Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys Phe Gly Cys Leu Asn (SEQ ID NO: 19) (Takao et al., FEBS Lett. 193:250, 1985); and the V. mimicus ST peptide with the mature amino acid sequence Ile Asp Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys Phe Gly Cys Leu Asn (SEQ ID NO: 20) (Arita et al., FEMS Microbiol. Lett. 79:105, 1991). Table A1 below shows the sequences of exemplary mature ST peptides. [Table 1]

[0108] The immature E. coli ST-IA (ST-P) protein (including the pre and pro regions) has the sequence mkklmlaifisvlsfpsfsqstesldsskekitletkkcdvvknnsekksenmnntfyccelccnpacagcy (SEQ ID NO: 41) (see GenBank® registration number P01559 (gi:123711)). The pre sequence extends from residues 1 to 19. The pro sequence extends from residues 20 to 54. The mature protein extends from residues 55 to 72. The immature E. coli ST-1B (ST-H) protein (including the pre and pro regions) has the sequence mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmnssnyccelccnpactgcy (SEQ ID NO: 42) (see GenBank® registration number P07965 (gi:3915589)). The immature Y. enterocolitica ST protein (including the pre and pro regions) has the sequence mkkivfvlylmlssfgafgqetvsgqfsdalstpitaevykqacdpplppaevssdwdccdvccnpacagc (SEQ ID NO: 43) (see GenBank® registration number S25659 (gi:282047)). Therefore, the GC-C agonist peptide may contain or consist of one or more of the bacterial ST peptide sequences (including their variants) described herein.

[0109] Bacterial ST peptides typically have six Cys residues. These six Cys residues form three disulfide bonds within the mature and active peptide. If the six Cys residues are identified as A, B, C, D, E, and F from the amino terminus to the carboxyl terminus of the peptide, the disulfide bonds are typically: AD, BE, and CF. The formation of these bonds is thought to contribute to GC-C receptor binding. Therefore, in certain embodiments, GC-C agonist peptides have at least one, two, or three disulfide bonds selected from any combination of AD, BE, and CF as shown above. However, in some embodiments, one or more cysteines in the GC-C peptide agonists described herein are deleted or substituted with different amino acids. In some embodiments, one, two, three, four, five, or six cysteines are deleted or substituted with different amino acids. In certain embodiments, the N-terminal cysteine ​​residue (e.g., A, B, or A and B) and / or the C-terminal cysteine ​​residue (one or more) (e.g., E, F, or E and F) are either deleted or substituted with a different amino acid. In certain embodiments, the different amino acid is alanine or serine.

[0110] Certain GC-C agonist peptides contain potentially functional chymotrypsin cleavage sites, such as Trp, Tyr, or Phe located either between Cys B / Cys D or Cys E / Cys F. Cleavage at any of these chymotrypsin cleavage sites can reduce the peptide's ability to bind to the GC-C receptor. In the human body, chymotrypsinogen, the inactive form of chymotrypsin, is produced in the pancreas. When this inactive enzyme reaches the small intestine, it is converted to active chymotrypsin by the removal of two dipeptides. Active chymotrypsin can cleave peptides at the carboxyl-terminal peptide bond of Trp, Tyr, or Phe. The presence of active chymotrypsin in the intestinal tract can cause cleavage of certain GC-C peptide agonists that have appropriately located functional chymotrypsin cleavage sites. In some cases, chymotrypsin cleavage is expected to modulate the action of GC-C peptide agonists that have appropriately positioned chymotrypsin cleavage sites as peptides pass through the intestinal tract.

[0111] Certain GC-C agonist peptides contain potentially functional trypsin cleavage sites, such as Lys or Arg. Trypsinogen, like chymotrypsin, is a serine protease produced in the pancreas and present in the gastrointestinal tract. Active trypsin cleaves peptides containing Lys or Arg. The presence of active trypsin in the intestinal tract can cause cleavage of certain GC-C agonist peptides that have appropriately located functional trypsin cleavage sites. In certain cases, trypsin cleavage is expected to modulate the action of GC-C peptide agonists with appropriately located trypsin cleavage sites as the peptide passes through the intestinal tract.

[0112] In certain embodiments, the peptide comprises at least six cysteine ​​molecules capable of forming three disulfide bonds. In certain embodiments, the disulfide bonds are substituted with other covalent crosslinks, and in some cases, the cysteine ​​molecules are substituted with other residues to result in alternative covalent crosslinks (as described elsewhere herein). Certain peptides contain a functional chymotrypsin or trypsin cleavage site located to inactivate the peptide upon cleavage. Certain peptides having a functional cleavage site undergo cleavage and progressive inactivation in the gastrointestinal tract, which is desirable in some situations. In certain peptides, the functional chymotrypsin site is denatured to increase the in vivo stability of the peptide.

[0113] In certain embodiments, the peptide contains, at its carboxyl terminus, one or more adjacent charged amino acids (e.g., Asp or Glu), one or more adjacent positively charged residues (e.g., Lys or Arg), or one or more adjacent positively charged or charged amino acids. In these and related embodiments, all adjacent amino acids at the carboxyl terminus are either positively charged or charged. In some embodiments, the carboxyl-terminated charged amino acid is preceded by Leu. For example, the following amino acid sequences: Asp, Asp Lys, Lys Lys Lys Lys Lys (SEQ ID NO: 44), Asp Lys Lys Lys Lys Lys Lys (SEQ ID NO: 45), Leu Lys Lys, and Leu Asp may be added to the carboxyl terminus of the peptide. In certain embodiments, Leu is added to the carboxyl terminus.

[0114] In some embodiments, the (bacterial ST analog) GC-C agonist peptide comprises, consists of, or essentially consists of the amino acid sequence (I) shown below. Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO: 46)

[0115] In some embodiments, Xaa1Xaa2Xaa3Xaa4Xaa5 is Asn Ser Ser Asn Tyr (SEQ ID NO: 2), or is absent, or Xaa1Xaa2Xaa3Xaa4 is absent. In certain embodiments, Xaa8, Xaa9, Xaa 12 , Xaa 14 , Xaa 16 , Xaa 17 , and Xaa 15 are any amino acid. In certain embodiments, Xaa8, Xaa9, Xaa 12 , Xaa 14 , Xaa 16 , Xaa 17 , and Xaa 19 are any natural or non-natural amino acid or amino acid analog.

[0116] In certain embodiments, Xaa5 is Asn, Trp, Tyr, Asp, or Phe. In other embodiments, Xaa5 is Thr or Ile. In some embodiments, Xaa5 is Tyr, Asp, or Trp. In certain embodiments, Xaa5 is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe. In a particular embodiment, Xaa5 is Asn.

[0117] In certain embodiments, Xaa8 is any natural or non-natural amino acid or amino acid analog. In some embodiments, Xaa8 is Glu, Asp, Gln, Gly, or Pro. In other embodiments, Xaa8 is Glu. In some embodiments, Xaa8 is Glu or Asp. In some embodiments, Xaa8 is Asn, Glu, or Asp. In some embodiments, Xaa8 is Glu, His, Lys, Gln, Asn, or Asp. In some embodiments, Xaa8 is Glu, His, Gln, Asn, or Asp. In some embodiments, Xaa8 is Glu, Asn, His, Gln, Lys, Asp, or Ser. In certain embodiments, Xaa8 is Pro.

[0118] In certain embodiments, Xaa9 is any natural or non-natural amino acid or amino acid analog. In some embodiments, Xaa9 is any natural or non-natural aromatic amino acid or amino acid analog. In some embodiments, Xaa9 is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr, or Phe. In some embodiments, Xaa9 is Leu, Ile, Val, Lys, Arg, Trp, Tyr, or Phe. In some embodiments, Xaa9 is Leu, Ile, Val, Trp, Tyr, or Phe. In some embodiments, Xaa9 is Leu, Ile, or Val. In some embodiments, Xaa9 is Trp, Tyr, or Phe. In some embodiments, Xaa9 is Leu, Ile, Lys, Arg, Trp, Tyr, or Phe. In some embodiments, Xaa9 is Leu, Val, Ile, or Met. In some embodiments, Xaa9 is Leu or Phe. In some embodiments, Xaa9 is Leu, Phe, or Tyr. In some embodiments, Xaa9 is Tyr, Phe, or His. In some embodiments, Xaa9 is Phe, His, Trp, or Tyr. In certain embodiments, Xaa9 is not Leu. In certain embodiments, Xaa9 is Tyr.

[0119] In a particular embodiment, Xaa 12 is any natural or non-natural amino acid or amino acid analog. In certain embodiments, Xaa 12 This is Asn, Tyr, Asp, or Ala. In certain embodiments, Xaa 12 Asn. In a particular embodiment, Xaa 12 is Asn, Met, Arg, Lys, His, or Gln. In certain embodiments, Xaa 12 is Asn, Lys, His, or Gln. In certain embodiments, Xaa 12 is Asn, Asp, Glu, or Gln. In certain embodiments, Xaa 12is Asn, Thr, Ser, Arg, Lys, Gln, or His. In some embodiments, Xaa 12 This is Asn, Ser, or His.

[0120] In a particular embodiment, Xaa 13 is Ala, Pro, or Gly. In certain embodiments, Xaa 13 is Pro or Gly. In certain embodiments, Xaa 13 It is Pro. In certain embodiments, Xaa 13 It is Gly.

[0121] In a particular embodiment, Xaa 14 is any natural or non-natural amino acid or amino acid analog. In certain embodiments, Xaa 14 is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Thr, Lys, Arg, or Asp. In certain embodiments, Xaa 14 is Ala or Gly. In some embodiments, Xaa 14 is Val or Ala. In a particular embodiment, Xaa 14 is Ala or Thr. In certain embodiments, Xaa 14 is Ala. In a particular embodiment, Xaa 14 is Val, Gln, Asn, Glu, Asp, Thr, or Ala. In certain embodiments, Xaa 14 These are Gly, Cys, or Ser.

[0122] In a particular embodiment, Xaa 16 is any natural or non-natural amino acid or amino acid analog. In some embodiments, Xaa 16 is any natural or unnatural non-aromatic amino acid or amino acid analog. In certain embodiments, Xaa 16 Thr, Ala, Asn, Lys, Arg, Trp, Gly, or Val. In certain embodiments, Xaa16 is Thr, Ala, Asn, Lys, Arg, or Trp. In certain embodiments, Xaa 16 is Thr, Ala, Lys, Arg, or Trp. In some embodiments, Xaa 16 is Thr, Ala, or Trp. In some embodiments, Xaa 16 In some embodiments, Xaa 16 is Trp, Tyr, or Phe. In some embodiments, Xaa 16 is Thr or Ala. In certain embodiments, Xaa 16 It is Val. In a particular embodiment, Xaa 16 It is Gly. In some embodiments, Xaa 16 is Thr, Ser, Met, or Val. In some embodiments, Xaa 16 is Val, Ala, or Thr. In some embodiments, Xaa 16 These are Ile, Val, Lys, Asn, Glu, Asp, or Thr.

[0123] In a particular embodiment, Xaa 17 is any natural or non-natural amino acid or amino acid analog. In some embodiments, Xaa 17 This is Gly, Pro, or Ala. In certain embodiments, Xaa 17 is Gly. In certain embodiments, Xaa 17 is Ala. In some embodiments, Xaa 17 is Gly or Ala. In some embodiments, Xaa 17 is Gly, Asn, Ser, or Ala. In some embodiments, Xaa 17 is Asn, Glu, Asp, Thr, Ala, Ser, or Gly. In some embodiments, Xaa 17 These are Asp, Ala, Ser, or Gly.

[0124] In a particular embodiment, Xaa19 is any natural or non-natural amino acid or amino acid analog. In some embodiments, Xaa 19 is Trp, Tyr, Phe, Asn, Ile, Val, His, Leu, or Arg. In some embodiments, Xaa 19 is Trp, Tyr, Asn, or Leu. In some embodiments, Xaa 19 is Trp, Tyr, or Phe. In some embodiments, Xaa 19 is Tyr, Phe, or His. In some embodiments, Xaa 19 is Tyr or Trp. In certain embodiments, Xaa 19 is Tyr. In some embodiments, Xaa 19 is Leu, Ile, or Val. In certain embodiments, Xaa 19 In some embodiments, Xaa 19 is Trp, Tyr, Phe, Asn, Ile, Val, His, or Leu. In some embodiments, Xaa 19 is Trp, Tyr, Phe, or Leu. In some embodiments, Xaa 19 is Tyr or Leu. In some embodiments, Xaa 19 is Lys or Arg. In some embodiments, Xaa 19 is any amino acid other than Pro, Arg, Lys, Asp, or Glu. In some embodiments, Xaa 19 is any amino acid other than Pro. In some embodiments, Xaa 19 It is missing.

[0125] In a particular embodiment, Xaa 20 is either Asp or Asn. In a particular embodiment, Xaa 20 Xaa 21 It is either AspPhe or missing. In some embodiments, Xaa 20 is Asn or Glu, Xaa 21It is missing. In some embodiments, Xaa 19 Xaa 20 Xaa 21 It is missing.

[0126] In some embodiments, the GC-C agonist peptide comprises, consists of, or essentially consists of the amino acid sequence (II) shown below. Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys 10 Cys 11 Asn 12 Pro 13 Ala 14 Cys 15 Xaa 16 Gly 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (Sequence ID 47) [In the formula, Xaa1Xaa2Xaa3Xaa4Xaa5 is either Asn Ser Ser Asn Tyr (sequence number 2) or missing, or Xaa1Xaa2Xaa3Xaa4 is missing and Xaa5 is Asn, Xaa8 is either Glu or Asp, Xaa9 is Leu, Ile, Val, Trp, Tyr, or Phe. Xaa 16 These are Thr, Ala, and Trp, Xaa 19 is either Trp, Tyr, Phe, or Leu, or is missing, Xaa 20 Xaa 21 This is AspPhe.

[0127] In some embodiments, the GC-C agonist peptide has the amino acid sequence (II): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys 10 Cys 11 Asn 12 Pro 13 Ala 14 Cys 15 Xaa16 Gly 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (Sequence ID 48) contains, consists of, or is essentially composed of, Xaa9 is Leu, Ile, or Val, and Xaa 16 is Trp, Tyr, or Phe, and Xaa9 is Trp, Tyr, or Phe, and Xaa 16 is Thr or Ala, Xaa 19 These are Trp, Tyr, Phe, and Xaa 20 Xaa 21 It is AspPhe, Xaa1, Xaa2, Xaa3, and Xaa4 are missing, Xaa5 is Asn, and the peptide contains 50, 40, 30, or fewer than 25 amino acids, or fewer than 5 amino acids precede Cys6.

[0128] In some embodiments, the GC-C agonist peptide has the amino acid sequence Xaa1Xaa2Xaa3Xaa4Xaa5Cys Cys Glu Xaa9Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa 20 Xaa 21 (II) (SEQ ID NO: 49) contains, consists of, or is essentially composed of, Xaa9 is any amino acid, where Xaa9 is any amino acid other than Leu; Xaa9 is selected from Phe, Trp, and Tyr; Xaa9 is selected from any natural or unnatural aromatic amino acid; Xaa9 is Tyr; Xaa9 is Phe; Xaa9 is Trp; Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 are Asn Ser Ser Asn Tyr; Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 are missing; Xaa1, Xaa2, Xaa3, and Xaa4 are missing; Xaa1, Xaa2, and Xaa3 are missing; Xaa1 and Xaa2 are missing; Xaa1 is missing; Xaa 20 Xaa 21It is either AspPhe, or missing, or Xaa 20 is Asn or Glu, Xaa 21 is missing, or Xaa 19 Xaa 20 Xaa 21 It is missing, Xaa1Xaa2Xaa3Xaa4Xaa5 and Tyr Xaa 20 Xaa 21 It is missing. In some embodiments, the GC-C agonist peptide has the amino acid sequence Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (I) Contains, consists of, or essentially consists of (SEQ ID NO: 50), and Xaa1Xaa2Xaa3Xaa4Xaa5 is missing and / or sequence Xaa 19 Xaa 20 Xaa 21 The peptide is missing, and optionally contains additional carboxyl-terminal and / or amino-terminal amino acids. The peptide is Xaa1 or Xaa 21 In cases where one or more terminal amino acids are missing, such as those mentioned above, the peptide may optionally contain additional carboxyl-terminal and / or amino-terminal amino acids.

[0129] In a particular embodiment, the peptide is Cys6 and Cys 11 Between Cys7 and Cys 15 Between and, and Cys 10 and Cys 16 A disulfide bond is included between them. In some embodiments, the peptide is a reduced peptide that does not have a disulfide bond. In yet another embodiment, the peptide is Cys6 and Cys 11 Disulfide bond between Cys7 and Cys15 The disulfide bond between and Cys 10 and Cys 16 It has one or two disulfide bonds, selected from the disulfide bonds between it and the other.

[0130] In certain embodiments, one or more amino acids are substituted with non-spontaneous amino acids, or spontaneously or non-spontaneously occurring amino acid analogs. Many amino acids exist beyond the standard 20. Some are spontaneous, others are non-spontaneous (see, for example, Hunt, The Non-Protein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, Barrett, Chapman and Hall, 1985). For example, aromatic amino acids may be substituted with 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg), or nor-tyrosine (norTyr). Other amino acids, including Phg and norTyr, as well as Phe and Tyr, may be substituted with halogens, -CH3, -OH, -CH2NH3, -C(O)H, -CH2CH3, -CN, -CH2CH2CH3, -SH, or other groups. Any amino acid can be substituted with a D-type amino acid.

[0131] With respect to non-spontaneous amino acids or spontaneously or non-spontaneous amino acid analogs, several substitutions are possible in the peptide of formula I or the peptide of formula II. For example, in some embodiments, Xaa8 may be substituted with γ-hydroxy-Glu or γ-carboxy-Glu. In some embodiments, Xaa9 may be substituted with an α-substituted amino acid such as L-α-methylphenylalanine, or with analogues such as 3-amino-Tyr, Tyr(CH3), Tyr(PO3(CH3)2), Tyr(SO3H), β-cyclohexyl-Ala, β-(1-cyclopentenyl)-Ala, β-cyclopentyl-Ala, β-cyclopropyl-Ala, β-quinolyl-Ala, β-2-thiazolyl)-Ala, β-(triazole-1-yl)-Ala, β-(2-pyridyl)-Ala, β-(3-pyridyl)-Ala, amino-Phe, fluoro-Phe, cyclohexyl-Gly, tBu-Gly, β-(3-benzothienyl)-Ala, β-2-thienyl)-Ala, 5-methyl-Trp, and 4-methyl-Trp.

[0132] In some embodiments, Xaa 13 It may be an N(α)-C(α) cyclized amino acid analog having the following structure. [ka]

[0133] Xaa 13 This may be Homopro (L-pipecholine salt), hydroxy-Pro, 3,4-dehydro-Pro, 4-fluoro-Pro, or α-methyl-Pro.

[0134] Xaa 13 In the mode in which is Gly, Ala, Leu, or Val, Xaa 14 The following are possible: [ka]

[0135] In a certain manner, Xaa 14This could be an α-substituted or N-methylated amino acid such as α-aminoisobutyric acid (aib), L / D-α-ethylalanine (L / D-isovaline), L / D-methylvaline, or L / D-α-methylleucine, or a non-natural amino acid such as β-fluoro-Ala.

[0136] In some embodiments, Xaa 17 This may be α-aminoisobutyric acid (aib) or L / D-α-ethylalanine (L / D-isovaline).

[0137] Additional examples of unnatural amino acids and amino acid analogs are known in the art and are described elsewhere in this specification.

[0138] For example, Xaa9 is Trp, Tyr, or Phe, or Xaa 16 In some cases where is Trp, the peptide has a potentially functional chymotrypsin cleavage site located at a position where cleavage can denature the GC-C receptor binding by the peptide. 16 If the peptide is Lys or Arg, it has a potentially functional trypsin cleavage site located at a position where cleavage can denature the GC-C receptor binding by the peptide.

[0139] For example, Xaa 19 In certain cases where the peptide is Trp, Tyr, or Phe, the peptide is cleaved by the Xaa of the peptide. 19 It has a chymotrypsin cleavage site located in a position that releases the carboxyl-terminal portion. Xaa 19 If is Leu, Ile, or Val, the peptide is cleaved by the peptide Xaa 19 It may have a chymotrypsin cleavage site located at a position that releases the amino-terminal portion. Xaa 19 If it is His, the same effect can be observed at relatively high pH. Xaa 19 If the peptide is Lys or Arg, the cleavage of the peptide is Xaa 19It has a trypsin cleavage site located in a position that releases the carboxyl-terminal portion.

[0140] For example, in some cases where the Xaa1 or amino-terminal amino acid (e.g., Xaa2 or Xaa3) of the peptide is Trp, Tyr, or Phe, the peptide has a chymotrypsin cleavage site located at a position where the cleavage releases the amino-terminal portion of Xaa1 (or Xaa2 or Xaa3) of the peptide, along with Xaa1, Xaa2, or Xaa3. When the Xaa1 or amino-terminal amino acid (e.g., Xaa2 or Xaa3) of the peptide of the present invention is Lys or Arg, the peptide has a trypsin cleavage site located at a position where the cleavage releases the amino-terminal portion of Xaa1 (or Xaa2 or Xaa3) of the peptide, along with Xaa1, Xaa2, or Xaa3. When the Xaa1 or amino-terminal amino acid of the peptide of the present invention is Leu, Ile, or Val, the peptide may have a chymotrypsin cleavage site located at a position where the cleavage releases the amino-terminal portion of Xaa1 of the peptide. If Xaa1 is His, the same effect can be observed at relatively high pH levels.

[0141] When fully folded, the disulfide bond is between Cys6 and Cys 11 Between Cys7 and Cys 15 Between and, and Cys 10 and Cys 18They may exist between the two. In some embodiments, the GC-C agonist peptide is identical to or sequence-similar to the ST peptide. However, in some embodiments, the GC-C agonist peptide includes amino acid changes and / or additions that improve functionality. These changes can, for example, increase or decrease activity (e.g., increase or decrease the peptide's ability to reduce phosphate uptake), denature the peptide's ability to fold correctly, denature the peptide's stability, denature the peptide's ability to bind to the GC-C receptor, and / or reduce toxicity. In some cases, the peptide may function more favorably than the wild-type ST peptide. For example, in certain cases, undesirable side effects such as diarrhea and dehydration are reduced.

[0142] Array(I)Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (Sequence ID 50) or Xaa1Xaa2Xaa3Xaa4Xaa5Cys Cys Glu Xaa9Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa 20 Xaa 21 (II)(Sequence code 49)(Xaa1Xaa2Xaa3Xaa4Xaa5 is missing and / or sequence Xaa 19 Xaa 20 Xaa 21In the case of a peptide containing or consisting of (which is missing), the peptide may optionally contain additional carboxy-terminal and / or amino-terminal amino acids. For example, the peptide may contain an amino-terminal sequence that promotes the recombinant production of the peptide and is cleaved before administration to the patient. The peptide may also contain other amino-terminal or carboxy-terminal amino acids. In some cases, the additional amino acids protect the peptide, stabilize it, and / or denature its activity. In some cases, some or all of the additional amino acids are removed before administration to the patient. A peptide may contain 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids at its amino-terminal and / or carboxy-terminal. The number of adjacent amino acids does not need to be the same. For example, 10 additional amino acids may be at the amino-terminal of the peptide but not at the carboxy-terminal.

[0143] In some embodiments, the peptide has the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (Sequence ID 50) is included, Xaa1, Xaa2, Xaa3, Xaa4, Xaa5 are missing, Xaa8 is Glu, Xaa9 is Leu, Ile, Lys, Arg, Trp, Tyr, or Phe, Xaa 12 is Asn, Xaa 13 It is Pro, Xaa 14 is Ala and Xaa 16 These are Thr, Ala, Lys, Arg, Trp, and Xaa 17 Gly and Xaa 19is Tyr or Leu, Xaa 20 Xaa 21 It is either an Asp Phe or missing. Xaa 20 Xaa 21 In cases where Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 are missing, the peptide may optionally contain additional adjacent amino acids.

[0144] Amino acid sequence Xaa1Xaa2Xaa3Xaa4Xaa5Cys Cys Glu Xaa9Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa 20 Xaa 21 Examples of GC-C agonist peptides containing, consisting of, or essentially consisting of (II)(SEQ ID NO: 49) are shown in Table A2 below. [Table 2] TIFF0007894693000005.tif139156 (In the table, SEQ ID NO: represents the sequence number) Examples of additional GC-C agonist peptides are shown in Table A3 below. [Table 3] TIFF0007894693000007.tif212157TIFF0007894693000008.tif211158TIFF0007894693000009.tif211157TIFF0007894693000010.tif211157TIFF0007894693000011.tif211156TIFF0007894693000012.tif211157TIFF0007894693000013.tif212156TIFF0007894693000014.tif54155 (In the table, SEQ ID NO: represents the sequence number)

[0145] In certain embodiments, the GC-C agonist peptide comprises, consists of, or essentially consists of the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 4).

[0146] This includes any deletion variant of any of the GC-C agonist peptides described herein. Examples include deletion variants in which one, two, three, or four amino acids (or unnatural amino acids or natural or unnatural amino acid analogs) other than Cys (or the amino acid that Cys is substituted for, for example, an amino acid that can form a covalent bond with another amino acid) are deleted. A specific example is a deletion in which two (or more) amino acids are deleted and the peptide has the sequence Cys a Cys b Xaa Xaa Cities c Cys d Xaa Xaa Xaa Publications e Xaa Xaa Cities f Examples include those containing (SEQ ID NO: 526). In some of these and related embodiments, two or more deletions are present in Cys b and Cys c Between and and / or Cys d and Cys e Between and and / or Cys e and Cys f It may be located between. However, in other embodiments, at most one deletion may be in Cys b and Cys c Between each other, or Cys d and Cys e Between or Cys e and Cys f It exists between and . Therefore, the array Cys a Cys b Xaa Xaa Cities c Cys d Xaa Xaa Xaa Publications e Xaa Xaa Cities f (Sequence ID 526) includes, a) Cys b and Cys cEither one amino acid is missing between them, or b) Cys d and Cys e Either one amino acid is missing between them, or c)Cys e and Cys f Either one amino acid is missing between them, or d) Cys b and Cys c One amino acid is missing between them, Cys d and Cys e Either one amino acid is missing between them, or e) Cys d and Cys e One amino acid is missing between them, Cys e and Cys f Either one amino acid is missing between them, or f)Cys b and Cys c One amino acid is missing between them, Cys e and Cys f Either one amino acid is missing between them, or g)Cys b and Cys c One amino acid is missing between them, Cys d and Cys e One amino acid is missing between them, Cys e and Cys f The peptide comprises one of the GC-C agonist peptides described herein, which is missing one amino acid between the two. In certain embodiments, the deletion variant is a peptide that binds to and / or stimulates a GC-C receptor.

[0147] This specification includes any insertion variant of the GC-C agonist peptides described herein. Examples include insertion variants in which one, two, three, or four amino acids (e.g., Gly or Ala) are inserted before or after any amino acid in the peptide. In some embodiments, one or fewer amino acids are inserted between two Cys residues. In specific examples, two or more amino acids are inserted, and the peptide is sequenced Cys a Cys b Xaa Xaa Cities c Cys dXaa Xaa Xaa Publications e Xaa Xaa Cities f Examples include those including (Sequence ID 526). In some of these and related embodiments, two or more insertions are made in Cys b and Cys c Between or Cys d and Cys e Between or Cys e and Cys f It may be located between. However, in other embodiments, one or fewer insertions may be located between Cys b and Cys c Between or Cys d and Cys e Between or Cys e and Cys f It exists between and . Therefore, the array Cys a Cys b Xaa Xaa Cities c Cys d Xaa Xaa Xaa Publications e Xaa Xaa Cities f (Sequence ID 526) includes, a) Cys b and Cys c Either one amino acid is inserted between them, or b) Cys d and Cys e Either one amino acid is inserted between them, or c) Cys e and Cys f Either one amino acid is inserted between them, or d) Cys b and Cys c A single amino acid is inserted between them, and Cys d and Cys e Either one amino acid is inserted between them, or e) Cys d and Cys e A single amino acid is inserted between them, and Cys e and Cys f Either one amino acid is inserted between them, or f) Cys b and Cys c A single amino acid is inserted between them, and Cys e and Cys f Either one amino acid is inserted between them, or g)Cysb and Cys c A single amino acid is inserted between them, and Cys d and Cys e A single amino acid is inserted between them, and Cys e and Cys f It contains one of the GC-C agonist peptides described herein, in which one amino acid is inserted between them. Furthermore, it contains one or more amino acids of Cys a It may be inserted prior to and / or one or more amino acids are Cys f It can be inserted after [the specified part]. In some embodiments, the insertion variant is a peptide that binds to and / or stimulates the GC-C receptor.

[0148] An example of an insertion variant of Cys Cys Glu Tyr, Cys Cys Asn Pro Ala, Cys Thr Gly, or Cys Tyr (SEQ ID NO: 4) is one in which up to four amino acids (i.e., 0, 1, 2, 3, or 4) are inserted after each amino acid. Therefore, the sequence Cys Xaa (0~4) Cys Xaa (0~4) Glu Xaa (0~4) Tyr Xaa (0~4) Cys Xaa (0~4) Cys Xaa (0~4) Asn Xaa (0~4) Pro Xaa (0~4) Ala Xaa (0~4) Cys Xaa (0~4) Thr Xaa (0~4) Gly Xaa (0~4) Cys Xaa (0~4) Tyr Xaa (0~4) The peptide contains (SEQ ID NO: 527). The inserted amino acids can be any amino acid or amino acid analog (natural or unnatural), and may be the same or different. In certain embodiments, the inserted amino acids are all glycerides, all alas, or a combination of glycerides and alas.

[0149] Array Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 GC-C agonist peptides also include variants of Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 4), which contain or consist of (SEQ ID NO: 46) and, for example, have up to four amino acid deletions and / or up to four amino acid insertions. In some cases, the insertions and / or deletions are Cys6 and Cys 18 It may exist between or between the amino terminal side of Cys6 and / or Cys 18 It may also be the carboxyl terminal side.

[0150] In certain embodiments, the GC-C agonist peptide is based on the core sequence Cys Cys Glu[Leu]Cys Cys Asn Pro Ala Cys[Thr]Gly Cys Tyr (SEQ ID NO: 528). To generate variants having potentially functional chymotrypsin cleavage sites that can inactivate the peptide, either Leu (indicated in parentheses) or Thr (indicated in parentheses) may be substituted with Trp, Phe, or Tyr, or both Leu and Thr may be substituted with Trp, Phe, or Tyr independently. The core sequence may optionally be preceded by Asn Ser Ser Asn Tyr or Asn. Specific examples of GC-C agonist peptides based on the core sequence are listed in Table A4 below. [Table 4]

[0151] In certain embodiments, the GC-agonist peptide is guaniline, lymphoguaniline, uroguaniline, or lenoguaniline peptide, optionally a human peptide, or a variant, derivative, or analog thereof. The amino acid sequence of human guaniline is Pro Gly Thr Cys Glu Ile Cys Ala Tyr Ala Ala Cys Thr Gly Cys (SEQ ID NO: 562). Exemplary analogs of the human guaniline sequence are shown in Table A5 below. [Table 5] TIFF0007894693000017.tif86151

[0152] Therefore, in some embodiments, the GC-C agonist peptide comprises, consists of, or essentially consists of, a human guaniline sequence or its variants, derivatives, or analogues.

[0153] The amino acid sequence of lymphoguaniline is Gln-Glu-Glu-Cys-Glu-Leu-Cys-Ile-Asn-Met-Ala-Cys-Thr-Gly-Tyr. (SEQ ID NO: 615). Exemplary analogues of the human lymphoguaniline sequence are shown in Table A6 below. [Table 6]

[0154] Therefore, in some embodiments, the GC-C agonist peptide comprises, consists of, or essentially consists of, a human lynhoguaniline sequence or its variants, derivatives, or analogues.

[0155] The amino acid sequence of human uroganine is Asn Asp Asp Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu (SEQ ID NO: 648). In some embodiments, the GC-C agonist peptide contains, consists of, or essentially consists of the human uroganine sequence or its analogues. In certain embodiments, the human uroganine analogue has the amino acid sequence Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu (SEQ ID NO: 6; precanatide) or Gln Asp Asp Cys Glu Thr Cys Ile Asn Met Ala Cys Thr Gly Tyr (SEQ ID NO: 649). In certain embodiments, the N-terminal Asn of the peptide (e.g., precanatide) is pyroglutamic acid. In some embodiments, the C-terminal Leu of the peptide (e.g., precanatide) is a D-amino acid (d-Leu).

[0156] In certain embodiments, the human uroganine peptide or analogue comprises, consists of, or essentially consists of the amino acid sequence (III) shown below. Xaa1Xaa2Xaa3Cys4Xaa5Xaa6Xaa7Xaa8Xaa9Xaa 10 Xaa 11 Cys 12 Xaa 13 Xaa 14 Xaa 15 Xaa 16 (Sequence ID 650)

[0157] In some embodiments, the GC-C agonist peptide of formula III is defined as follows: Xaa1 is any natural or non-natural amino acid or amino acid analog, or is missing. Xaa2 is any natural or non-natural amino acid or amino acid analog, or is missing. Xaa3 is any natural or non-natural amino acid or amino acid analog, or is missing. Xaa5 is Glu, Xaa6 is Tyr, Trp, Phe, or Leu. Xaa7 is Cys, Xaa8 is any natural or non-natural amino acid or amino acid analog other than Cys (arbitrarily any of the 20 naturally occurring amino acids), or is missing. Xaa9 is any natural or non-natural amino acid or amino acid analog other than Cys (optionally any of the 20 naturally occurring amino acids), Xaa 10 It is Pro or Gly, Xaa 11 is any natural or non-natural amino acid or amino acid analog (any of the 20 naturally occurring amino acids), Xaa 13 These are Thr, Val, or Gly, Xaa 14 It is Gly or Ala, Xaa 15 It is Cys, Xaa 16 It is either any natural or non-natural amino acid or amino acid analog (optionally any of the 20 naturally occurring amino acids), or it is missing.

[0158] In a particular embodiment, Xaa9 is Asn, and Xaa 11 is Ala or Thr, Xaa8 is missing, Xaa 16 It is Tyr.

[0159] In some embodiments, Xaa4 is Ser His Thr;Pro Ser Thr;Thr;Pro Asp Pro;Ile Ala Glu Asp Ser His Thr(SEQ ID NO: 651);Ile Ala Gln Asp Pro Ser Thr(SEQ ID NO: 652);Ala Asn Thr;Asn Thr;Asp Pro Asn Thr(SEQ ID NO: 653);Lys Asn Thr;Pro Asn Thr;Ile Ala Gln Asp Pro Asn Thr(SEQ ID NO: 654);Lys Pro Asn Thr(SEQ ID NO: 655);Asp Pro Gly Thr(SEQ ID NO: 656);Glu Asp Pro Gly Thr(SEQ ID NO: 657);Pro Gly Thr;Pro Ala Thr;Val Ala Ala Arg Ala Asp Leu(SEQ ID NO: 658);Gly Asp Asp;Asn Asp Glu;Gln Glu Asp;Asn Asp Asp;Arg Thr Ile Ala Asn Asp Asp(SEQ ID NO: 659);Thr Ile Ala Asn Asp Asp (SEQ ID NO: 660); Asp Asp; Arg Thr Met Asp Asn Asp Glu (SEQ ID NO: 661); Arg Thr Ile Ala Gly Asp Asp (SEQ ID NO: 662); Arg Thr Ile Ala Asn Asp (SEQ ID NO: 663); Asp; Glu Asp; Arg Ser Ile Ser Gln Glu Asp (SEQ ID NO: 664); Thr Asp Glu; Arg Thr Ile Ala Thr Asp Glu (SEQ ID NO: 665); Glu; Ile Ile Thr Pro Pro Asp Pro (SEQ ID NO: 666); Gln Glu Leu; Lys Asp Asp; Gln Glu Glu; Arg Tyr Ile Asn Gln Glu Glu (SEQ ID NO: 667); Ala Ser Ser Tyr Ala Ser (SEQ ID NO: 668); and Thr Ser Ser Tyr Ala Ser (SEQ ID NO: 669), directly preceding the amino acid sequence.

[0160] In certain embodiments, the GC-C agonist peptide of formula III is defined as follows: Xaa1 is a) Ser, Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is missing; b) preceded by Lys or Tyr; c) any amino acid; d) missing; e) any amino acid other than Cys; or f) Lys or Arg. Xaa2 is a) His, Asp, Glu, Ala, Ser, Asn, Gly, or missing; b) His, Asp, Glu, Ala, Ser, Asn, Gly, Pro, or missing; c) Asp, Glu, any amino acid, or missing; d) Asp or Glu; e) any amino acid other than Cys; e) Glu; f) missing; g) Trp, Tyr, or Phe; or h) Lys or Arg. Xaa3 is a) Thr, Asp, Ser, Glu, Pro, Val, or Leu; or Asp or Glu; b) any amino acid other than Cys; c) Glu; d) Thr; e) Thr, Asp, Ser, Glu, Pro, Val, or Leu, or missing; f) Trp, Tyr, or Phe; or g) Lys or Arg. Cys4 is optionally Xaa4 and is Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, or Glu. Xaa5 is a) any amino acid, b) Glu, Asp, Gln, Gly, or Pro, c) Glu, d) Glu or Asp, e) Asp, Ile, or Glu, f) any amino acid, or g) any amino acid other than Cys. Xaa6 is a) Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr, or Phe; b) Leu, Ile, Val, Lys, Arg, Trp, Tyr, or Phe; c) Leu, Ile, Val, Trp, Tyr, or Phe; d) Trp, Tyr, Phe, or Leu; e) Leu, Ile, or Val; f) Ile, Trp, or Leu; g) Trp, Tyr, or Phe; h) Ile or Leu; i) Tyr; j) any amino acid; k) any amino acid other than Leu; l) any natural or unnatural aromatic amino acid; or m) any amino acid other than Cys. Xaa7 is a) Cys, Ser, or Tyr;Cys, b) Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, or Glu, c) Ser, or d) an amino acid other than Cys. Xaa8 is a) Ala, Val, or Ile, b) Ala, Val, Thr, Ile, Met, or missing, c) any amino acid, d) Val, e) any amino acid other than Cys, or f) missing. Xaa9 is a) any amino acid, b) any amino acid other than Phe and Tyr, c) any amino acid other than Phe, Tyr, and Trp, d) any amino acid other than Phe, Tyr, Trp, Ile, Leu, and Val, e) any amino acid other than Phe, Tyr, Trp, Ile, Leu, Val, and His, i) any amino acid other than Gln, g) any amino acid other than Lys, Arg, Phe, Tyr, and Trp, h) Lys, Arg, Phe, T It is any amino acid other than yr, Trp, Ile, Leu, and Val, i) any amino acid other than Lys, Arg, Phe, Tyr, Trp, Ile, Leu, Val, and His, j) any non-aromatic amino acid, k) missing, l) Phe, Tyr, Asn, or Trp, m) Asn, Tyr, Asp, or Ala, n) Asn, Gln, or Tyr, o) Phe or Tyr, p) Asn, or q) any amino acid other than Cys. Xaa 10 is a) Ala, Pro, or Gly, b) Pro or Gly, c) Pro, d) Ala, Val, Met, Thr, or Ile, e) any amino acid, f) Val, g) Val or Pro, h) Ala or Val, i) any amino acid other than Cys, j) Pro, or k) Gly. Xaa 11 is a) any amino acid, b) Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp, c) Ala or Gly, d) Ala, e) Ala or Val, f) any amino acid, g) Ala or Aib (α-aminoisobutyric acid), h) any amino acid other than Cys, i) Ala or Thr, or j) Thr. Cys 12 Xaa 12It is and a) Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, or Glu, or b) any amino acid other than Cys. Xaa 13 is a) Thr, Ala, Asn, Lys, Arg, or Trp, b) Thr, Ala, Lys, Arg, or Trp, c) any amino acid, d) any non-aromatic amino acid, e) Thr, Ala, or Trp, f) Trp, Tyr, or Phe, g) Thr or Ala, h) any amino acid, i) Thr, j) any amino acid other than Cys, k) Thr, Val, or Gly, l) Thr or Val, m) Thr or Gly, n) Val or Thr, o) Val, p) Thr, or q) Gly. Xaa 14 is a) Gly, Pro, or Ala, b) Gly, c) any amino acid, d) Gly, Ala, or Ser, e) Gly or Ala, f) any amino acid other than Cys, or g) Ala. Xaa 15 It is a) Cys, Tyr, or missing; b) Cys; c) Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, Glu; or d) any amino acid other than Cys, or missing. Xaa 16is a) Trp, Tyr, Phe, Asn, lie, Val, His, or Leu; b) Trp, Tyr, Phe, Asn, or Leu; c) Tip, Tyr, Phe, or Leu; d) Trp, Tyr, or Phe; e) Leu, Ile, or Val; f) His, Leu, or Ser; g) Tyr or Leu; Lys or Arg; h) His; i) any amino acid; j) Leu or missing; k) Trp, Tyr, Phe, Lys, Arg or missing; l) missing; m) any amino acid other than Cys; or n) Tyr.

[0161] In some embodiments, the GC-C agonist peptide of formula III is defined as follows: Xaa1 is any natural or non-natural amino acid or amino acid analog, or is missing. Xaa2 is any natural or non-natural amino acid or amino acid analog, or is missing. Xaa3 is any natural or non-natural amino acid or amino acid analog, or is missing. Xaa4 is Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, or Glu. Xaa5 is Glu, Xaa6 is Tyr, Trp, Phe, or Leu. Xaa7 is Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, or Glu. Xaa8 is any natural or unnatural amino acid or amino acid analog other than Cys, or is missing. Xaa9 is any amino acid, Xaa 10 It is Pro or Gly, Xaa 11 is any amino acid, Xaa12 These are Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, or Glu. Xaa 13 These are Thr, Val, or Gly, Xaa 14 It is Gly or Ala, Xaa 15 These are Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic acid), Asp, or Glu. Xaa 16 It is either any amino acid or it is missing.

[0162] In certain embodiments, the GC-C agonist peptide of formula III is defined as follows: Xaa1 is Asn, any amino acid, or is missing. Xaa2 is either Asp, Glu, any amino acid, or is missing. Xaa3 is either Asp or Glu. Xaa5 is any amino acid or Glu, Xaa6 is any amino acid or Leu, Xaa7 is Cys, Xaa8 is any amino acid or Val, Xaa9 is Asn, Gln, or Tyr. Xaa 10 is any amino acid or Val, Xaa 11 is any amino acid or Ala, Xaa 13 is any amino acid or Thr, Xaa 14 is any amino acid or Gly, Xaa 15 It is Cys, Xaa 16 It is either any amino acid, Leu, or it is missing.

[0163] In some embodiments, the GC-C agonist peptide of formula III is not cleaved after Xaa9 by chymotrypsin and is defined as follows: Xaa1 is either Ser, Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is missing. Xaa2 is either His, Asp, Glu, Ala, Ser, Asn, or Gly, or is missing. Xaa3 is either Thr, Asp, Ser, Glu, Pro, Val, or Leu, or is missing. Xaa5 is Asp, Ile, or Glu. Xaa6 is Ile, Trp, or Leu. Xaa7 is Cys, Ser, or Tyr. Xaa8 is either Ala, Val, Thr, Ile, or Met, or is missing. Xaa9 is one of the following: a) any amino acid other than Phe and Tyr, b) any amino acid other than Phe, Tyr, and Trp, c) any amino acid other than Phe, Tyr, Trp, Ile, Leu, and Val, d) any amino acid other than Phe, Tyr, Trp, Ile, Leu, Val, and His, d) any non-aromatic amino acid, or e) missing. Xaa 10 These are Ala, Val, Met, Thr, or Ile. Xaa 11 It is Ala or Val, Xaa 13 It is Ala or Thr, Xaa 14 It is Gly, Ala, or Ser, Xaa 15 It is either Cys, Tyr, or missing. Xaa 16This is a) Trp, Tyr, or Phe that generate chymotrypsin cleavage sites, b) Lys or Arg that generate trypsin cleavage sites, c) deficient, or d) His, Leu, or Ser.

[0164] In certain embodiments, the human uroganine peptide or analogue comprises, consists of, or essentially comprises the amino acid sequence (IV) shown below. Asn1Xaa2Xaa3Xaa4Glu5Leu6Xaa7Val8Asn9Xaa 10 Xaa 11 Xaa 12 Thr 13 Xaa 14 Xaa 15 Leu 16 (Sequence ID 670) [In the formula, Xaa2 is either Asp or Glu, Xaa3 is either Asp or Glu. Xaa4 is either Cys or Mpt (mercaptoproline) or Pen (penicillamine) or Dpr (diaminopropionic acid) or Asp or Glu. Xaa7 is either Cys or Mpt (mercaptoproline) or Pen (penicillamine) or Dpr (diaminopropionic acid) or Asp or Glu. Xaa 10 It is either Val or Pro, Xaa 11 It is Ala or Aib (α-aminoisobutyric acid), Xaa 12 These are Cys or Mpt (mercaptoproline) or Pen (penicillamine) or Dpr (diaminopropionic acid) or Asp or Glu, Xaa 14 It is Gly or Ala, Xaa 15 [These are Cys or Mpt (mercaptoproline) or Pen (penicillamine) or Dpr (diaminopropionic acid) or Asp or Glu].

[0165] In a particular embodiment of formula IV, Xaa 15 Xaa7 is either a non-Cys amino acid or is missing, and Xaa7 is a Ser or a non-Cys amino acid.

[0166] In a particular embodiment, Xaa1, Xaa2, Xaa3, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, Xaa 10 Xaa 11 Xaa 13 Xaa 14 , and Xaa 16 One, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of these are any amino acids other than Cys. In some embodiments, Xaa9 is any amino acid other than Gln. In embodiments where Xaa2 and Xaa3 are Glu, Xaa9 is any amino acid other than Gln. In certain embodiments, Xaa1 and Xaa2 are missing, Xaa3 is Thr, Xaa5 is Glu, Xaa6 is Ile or Leu, Xaa8 is Ala, Val, or Ile, Xaa9 is Phe or Tyr, and Xaa 10 is Ala or Val, Xaa 11 is Ala and Xaa 13 is Ala or Thr, Xaa 14 Gly and Xaa 16 It is either Trp, Tyr, Phe, Lys, or Arg, or it is missing. Specific examples of human uroganiline analogs are provided in Table A7 below. [Table 7] TIFF0007894693000020.tif217156TIFF0007894693000021.tif139154

[0167] This also includes variants of the GC-C agonist peptides described herein. Examples include variant peptides that contain approximately, at least approximately, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or fewer amino acid substitutions, insertions, and / or deletions of any of the sequences of formula I, II, III, or IV, or sequence numbers 1, 5, 46-50, 650, and 670, or any of the sequences in Tables A1-A7. Substitutions may be conserved or non-conserved. An example of a conserved amino acid substitution is when an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-loading side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative substitutions can replace spontaneously occurring amino acids with non-spontaneous amino acids or amino acid analogs. Insertions and / or deletions may be located in the N-terminus, C-terminus, and / or internal region of the peptide (e.g., insertion or deletion of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the C-terminus, N-terminus, and / or N-terminus and / or C-terminus). In some cases, it may be desirable to use mutant peptides that bind to and stimulate GC-C receptors in the intestinal tract, but with lower activity than the non-mutant form of the peptide. This reduction in activity may result from reduced affinity to the receptor, reduced ability to activate the receptor upon binding, or reduced peptide stability.

[0168] GC-C agonist peptides can be cyclic or linear peptides. Furthermore, multiple copies of the same peptide can be incorporated into a single cyclic or linear peptide. Cyclic peptides can be prepared by methods known in the art. For example, macrocyclization is often achieved by forming an amide bond between the N-terminus and C-terminus of a peptide, between a side chain and the N-terminus or C-terminus [e.g., using K3Fe(CN)6 at approximately pH 8.5] (Samson et al., Endocrinology, 137:5182-5185, 1996), or between two amino acid side chains such as cysteine ​​(DeGrado, Adv Protein Chem, 39:51-124, 1988).

[0169] The peptides may contain amino acid sequences of peptides that occur spontaneously in vertebrate (e.g., mammalian) species or bacterial species. Furthermore, the peptides may be partially or entirely non-spontaneous peptides.

[0170] This specification also includes peptide analogues corresponding to the GC-C agonist peptides described herein. Peptide analogues are commonly used in the pharmaceutical industry as non-peptide drugs that have properties similar to those of the template peptide. These types of non-peptide compounds are called "peptide mimes" or "peptidomimetic compounds" (Luthman, et al., A Textbook of Drug Design and Development, 14:386-406, 2nd Ed., Harwood Academic Publishers, 1996; Joachim Grante, Angew. Chem. Int. Ed. Engl., 33:1699-1720, 1994; Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber and Freidinger TINS, p.392 (1985); and Evans et al., J. Med. Chem. 30:229, 1987). Peptidomimetic molecules are molecules that mimic the biological activity of peptides but whose chemical properties are no longer peptidic. Peptidomimetic compounds are known in the art and are described, for example, in U.S. Patent No. 6,245,886.

[0171] The present invention also includes peptoids. Peptoid derivatives of peptides are equivalent to other forms of modified peptides that retain important structural determinants for biological activity but remove peptide bonds, thereby conferring resistance to proteolysis (see, for example, Simon et al., PNAS USA. 89:9367-9371, 1992). Peptoids are oligomers of N-substituted glycine. Several N-alkyl groups corresponding to the side chains of native amino acids have been described. The peptoids of the present invention include compounds in which at least one amino acid, several amino acids, or all amino acid residues are substituted with the corresponding N-substituted glycine. A peptoid library is described, for example, in U.S. Patent No. 5,811,387.

[0172] In some embodiments, the GC-C agonist peptide contains or consists of approximately, at least approximately, or approximately 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 8, 7, 6, or fewer than 5 amino acids. In some embodiments, the peptide contains 5 or fewer amino acids that are the N-terminal side of Cys6 (of formula I or II). In some embodiments, the peptide contains Cys (of formula I or II) 18 It contains 20, 15, 10, or 5 or fewer amino acids at the C-terminus.

[0173] In some embodiments, the peptides are purified. The purified peptides are separated from other proteins, lipids, and nucleic acids, or from the compounds from which the peptides are synthesized or prepared. The purified peptides may constitute at least about 50, 60, 70, 80, 85, 90, 95, 96, 97, or 98% by dry weight of the purified preparation.

[0174] As described above, certain peptides described herein may contain one or more or all of the unnatural amino acids or amino acid analogs. In addition to those described elsewhere in this specification (e.g. above), examples include unnatural analogs of tyrosine; unnatural analogs of glutamine; unnatural analogs of phenylalanine; unnatural analogs of serine; unnatural analogs of threonine; alkyl, aryl, acyl, azide, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkyn, ether, thiol, sulfonyl, seleno, ester, thio acid, borate, boronic acid, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino-substituted amino acids, or similar. Any combination of these; amino acids having photoactivatable crosslinking agents; spin-labeled amino acids; fluorescent amino acids; amino acids with novel functional groups; amino acids that interact covalently or acovalently with other molecules; metal-binding amino acids; metal-containing amino acids; radioactive amino acids; photocage and / or photoisomerizable amino acids; biotin or biotin analog-containing amino acids; glycosylated or carbohydrate-modified amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyether; heavy atom-substituted amino acids (e.g., deuterium, tritium, 13 C, 15 N, or 18O-containing amino acids; chemically cleavable or photocleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar-substituted amino acids, e.g., sugar-substituted serine; carbon-bonded sugar-containing amino acids; redox-active amino acids; α-hydroxy-containing acids; aminothio acid-containing amino acids; α,α-disubstituted amino acids; β-amino acids; cyclic amino acids other than proline; O-methyl-L-tyrosine; L-3-(2-naphthyl)alanine; 3-methylphenylalanine; p-acetyl-L -Phenylalanine; 0-4-allyl-L-tyrosine; 4-propyl-L-tyrosine; tri-O-acetyl-GlcNAcβ-serine; L-dopa; fluorinated phenylalanine; isopropyl-L-phenylalanine; p-azide-L-phenylalanine; p-acyl-L-phenylalanine; p-benzoyl-L-phenylalanine; L-phosphoserine; phosphonoserine; phosphonotyrosine; p-iodophenylalanine; 4-fluorophenylglycine; p-bromophenylalanine n; p-amino-L-phenylalanine; isopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine; amino, isopropyl, or O-allyl-containing phenylalanine analogs; dopa, O-methyl-L-tyrosine; glycosylated amino acids; p-(propargyloxy)phenylalanine; dimethyl-lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine; 3-nitrotyrosine; norleucine; pyro-glutamic acid; Z (carbobenzoxyl); Examples include ε-acetyl-lysine; β-alanine; aminobenzoyl derivatives; aminobutyric acid (Abu); citrulline; aminohexanoic acid; aminoisobutyric acid; cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitro-arginine; nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindole carboxylates; ornithine; penicillamine; tetrahydroisoquinoline; acetamidomethyl-protected amino acids; and PEGylated amino acids. Further examples of unnatural amino acids and amino acid analogs can be found in U.S. Patent Applications 2003 / 0108885 and 2003 / 0082575, and the references cited herein.

[0175] In some embodiments, the amino acid may be substituted with a naturally occurring non-essential amino acid, such as taurine.

[0176] In some embodiments, one, two, three, four, five, or six cysteine ​​residues are deleted or substituted with a different amino acid. In certain embodiments, one or more cysteine ​​residues at the N-terminal and / or C-terminal end are deleted or substituted with a different amino acid. In certain embodiments, the different amino acid is alanine or serine.

[0177] Peptides can be polymers of L-amino acids, D-amino acids, or combinations thereof. For example, in certain embodiments, the peptide is a D-retro-inverso peptide. The term “retro-inverso isomer” refers to an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is reversed. See, for example, Jameson et al., Nature. 368:744-746, 1994 and Brady et al., Nature. 368:692-693, 1994. The net result of D-enantiomer bonding and inversion synthesis is that the positions of the carbonyl and amino groups within each amide bond are exchanged, while the positions of the side chain groups at each α-carbon are preserved. Unless specifically stated otherwise, any given L-amino acid sequence of the present invention can be converted to a D-retro-inverso peptide by synthesizing the inverse of its sequence relative to the corresponding native L-amino acid sequence.

[0178] Methods for producing peptides containing non-natural amino acids can be found, for example, in U.S. Patent Applications No. 2003 / 0108885 and No. 2003 / 0082575, Deiters et al., J Am Chem. Soc. 125:11782-3, 2003, Chin et al., Science. 301:964-7, 2003, and the references cited therein.

[0179] In some embodiments, the GC-C agonist peptide may have one or more conventional peptide bonds that are substituted with alternative bonds. Such substitutions may increase the stability of the peptide. For example, Cys (of formula I or II) by alternative bonds. 18 and Xaa 19 Substitution of peptide bonds between these compounds can reduce cleavage by carboxypeptidases and increase the half-life in the gastrointestinal tract. Examples of bonds that can substitute peptide bonds include, but are not limited to, retro-inverso bonds (C(O)-NH instead of NH-C(O); reducing amide bonds (NH-CH2); thiomethylene bonds (S-CH2 or CH2-S); oxomethylene bonds (O-CH2 or CH2-O); ethylene bonds (CH2-CH2); thioamide bonds (C(S)-NH); trans-olefin bonds (CH=CH); fluorosubstituted trans-olefin bonds (CF=CH); ketomethylene bonds (C(O)-CHR or CHR-C(O) (wherein R is H or CH3); and fluoro-ketomethylene bonds (C(O)-CFR or CFR-C(O) (wherein R is H or F or CH3)).

[0180] In some GC-C agonist peptides, one or both members of one or more pairs of Cys residues that normally form a disulfide bond are substituted with homocysteine, penicillamine, 3-mercaptoproline (see, e.g., Kolodziej et al., Int J Pept Protein Res. 48:274, 1996), β,β-dimethylcysteine ​​(see, e.g., Hunt et al., Int J Pept Protein Res. 42:249, 1993), or diaminopropionic acid (see, e.g., Smith et al., J Med Chem. 21:117, 1978) to form alternative internal crosslinks at the location of the normal disulfide bond.

[0181] In some embodiments, one or more disulfide bonds may be replaced by alternative covalent crosslinks, such as amide bonds (-CH2CH(O)NHCH2- or -CH2NHCH(O)CH2-), ester bonds, thioester bonds, lactam crosslinks, carbamoyl bonds, urea bonds, thiourea bonds, phosphonic acid ester bonds, alkyl bonds (-CH2CH2CH2CH2-), alkenyl bonds (-CH2CH=CHCH2-), ether bonds (-CH2CH2OCH2- or -CH2OCH2CH2-), thioether bonds (-CH2CH2SCH2- or -CH2SCH2CH2-), amine bonds (-CH2CH2NHCH2- or -CH2NHCH2CH2-), or thioamide bonds (-CH2CH(S)HNHCH2- or -CH2NHCH(S)CH2-). For example, Ledu et al. (PNAS.100:11263-78, 2003) describe a method for preparing lactam and amide crosslinks. Schafmeister et al. (J.Am.Chem.Soc.122:5891, 2000) describe stable hydrocarbon crosslinks. Hydrocarbon crosslinks can be produced via double decomposition (or, in the case of saturated hydrocarbon crosslinks, double decomposition followed by hydrogenation) using one or another of the Grubbs catalysts (available from Materia, Inc. and Sigma-Aldrich, and described, for example, in U.S. Patent Nos. 5,831,108 and 6,111,121). In some cases, the production of such alternative crosslinks requires the substitution of Cys residues with other residues such as Lys or Glu, or non-spontaneous amino acids. Furthermore, lactam, amide, and hydrocarbon crosslinks can be used to stabilize peptides even when they link amino acids at positions other than those occupied by Cys. Such crosslinking can occur, for example, between two amino acids separated by two amino acids, or between two amino acids separated by six amino acids (see, for example, Schafmeister et al., J.Am.Chem.Soc.122:5891, 2000).

[0182] GC-C agonist peptides can be modified using standard modifications. Modifications may occur at the amino (N) terminus, the carboxy (C) terminus, internally, or in any combination of the above. In some embodiments, more than one type of peptide modification may exist. Modifications include, but are not limited to, acetylation, amidation, biotinylation, cinnamoylation, farnesylation, formylation, myristoylation, palmitoylation, phosphate chlorination (Ser, Tyr, or Thr), stearoylation, succinylation, sulfurylation, and cyclization (via disulfide crosslinking or amide cyclization), as well as modifications with Cy3 or Cy5. The peptides of the present invention may also be modified with 2,4-dinitrophenyl (DNP), DNP-lysine, 7-amino-4-methyl-coumarin (AMC), fluorescein, NBD (7-nitrobenz-2-oxa-1,3-diazole), p-nitro-anilide, rhodamine B, EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl, dansyl, Texas Red, FMOC, and Tamra (tetramethylrhodamine). The peptides of the present invention can also be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 linear or branched alkyl groups); fatty acid radicals; combinations of PEG, alkyl groups, and fatty acid radicals (see U.S. Patent No. 6,309,633, Soltero et al., Innovations in Pharmaceutical Technology. 106-110, 2001); BSA and KLH (keyhole limpet hemocyanin). For example, in certain embodiments, the N-terminal amino acid, the C-terminal amino acid, or both are conjugated to the PEG molecule.

[0183] In certain embodiments, the GC-C agonist peptides described herein may be present with counterions. Exemplary counterions include acetate, benzenesulfonate, benzoate, calcium edetate, cansylate, carbonate, citrate, edetate (EDTA), edisylate, embonate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolyllarsanilate, hexylresorcinate, iodide, bromide, chloride, hydroxynaphthoate, isethionate, lactate, lactobionate, estolate, maleate, malate, mandelate, mesylate, mucate, napsylate, nitrate, pantothenate, phosphate, salicylate, stearate, succinate, and sulfuric acid. Examples include salts, tartrates, theoclates, acetamide benzoates, adipines, alginates, aminosalicylates, anhydromethylene citrates, ascorbic acid, aspartates, camphorates, caprinates, caproates, caprylates, cinnamates, cyclamates, dichloroacetates, formates, gentisinates, glucuronates, glycerophosphates, glycolates, hippurates, fluoride salts, malonates, napadisylates, nicotinates, oleates, orotates, oxalates, oxoglutarates, palmitates, pectinates, salts of pectinate polymers, phenylethyl barbiturates, picrinates, propions, pidolates, sebacinates, thiocyansides, tosylates, and tannates.

[0184] GC-C agonist peptides can be produced according to various techniques. For example, peptides may be produced in bacteria, including but not limited to Escherichia coli, or in other systems for peptide or protein production (e.g., Bacillus subtilis, baculovirus expression systems using Drosophila Sf9 cells, yeast or filamentous fungal expression systems, mammalian cell expression systems), or they may be chemically synthesized. When peptides or mutant peptides are produced in bacteria, such as Escherichia coli, the nucleic acid molecule encoding the peptide may optionally encode a leader sequence that enables the secretion of mature peptides from the cell. Thus, the sequence encoding the peptide may include, for example, the pre-sequence and pro-sequence of a spontaneously occurring bacterial ST peptide. The secreted mature peptides can be purified from the culture medium.

[0185] In some cases, the sequence encoding the peptide of the present invention is inserted into a vector capable of delivering and maintaining nucleic acid molecules within bacterial cells. The DNA molecule may be inserted into an autonomous replication vector (preferred vectors include, for example, pGEM3Z and pcDNA3, and their derivatives). The vector nucleic acid may be bacterial DNA, or bacteriophage DNA such as bacteriophage λ or M13, and their derivatives. Construction of a nucleic acid-containing vector described herein may be followed by transformation of a host cell, such as bacteria. Preferred bacterial hosts include, but are not limited to, Escherichia coli, Bacillus subtilis, Pseudomonas, and Salmonella. In addition to the encoding nucleic acid molecule, the gene construct may also include elements that enable expression, such as promoters and regulatory sequences. The expression vector may contain transcriptional regulatory sequences that control transcription initiation, such as promoters, enhancers, operators, and repressor sequences. Various transcriptional regulatory sequences are well known to those skilled in the art. The expression vector may also include translational control sequences (e.g., untranslated 5′ sequences, untranslated 3′ sequences, or intra-sequence ribosome entry sites). The vector may be capable of autonomous replication or can be incorporated into host DNA to ensure stability during peptide production. In some embodiments, vectors, expression systems, and methods described in U.S. Patent No. 5,395,490 may be used to produce the GC-C agonist peptides described herein.

[0186] The protein-coding sequence containing the peptide of the present invention may also be fused to a nucleic acid encoding a polypeptide affinity tag (e.g., glutathione S-transferase (GST), maltose E-binding protein, protein A, FLAG tag, hexa-histidine, myc tag, or influenza HA tag) to facilitate purification. The affinity tag or reporter fusion links the reading frame of the peptide of interest with the reading frame of the gene encoding the affinity tag so that translational fusion occurs. Expression of the fusion gene results in the translation of a single polypeptide containing both the peptide of interest and the affinity tag. In some cases where affinity tags are used, a DNA sequence encoding a protease recognition site is fused between the affinity tag and the reading frame of the peptide of interest.

[0187] Gene constructs and methods suitable for producing immature and mature peptides and mutants of the present invention in protein expression systems other than bacteria, and which are well known to those skilled in the art, can also be used to produce peptides in biological systems.

[0188] Peptides and their variants can be synthesized by solid-phase chemical synthesis. For example, peptides can be synthesized on a Cyc(4-CH2Bxl)-OCH2-4-(oxymethyl)-phenylacetamidomethyl resin using a double bond program. Protecting groups must be used appropriately to generate the correct disulfide bond pattern. For example, the following protecting groups may be used: t-butyloxycarbonyl (α-amino group); acetamidomethyl (thiol groups of Cys residues B and E); 4-methylbenzyl (thiol groups of Cys residues C and F); benzyl (γ-carboxyl group of glutamic acid and hydroxyl group of threonine (if present)); and bromobenzyl (phenol group of tyrosine (if present)). Bonding is induced using symmetric anhydrides of t-butoxylcarbonyl amino acids or hydroxybenzotriazole esters (in the case of asparagine or glutamine residues), and the peptide is deprotected and cleaved from a solid support in hydrogen fluoride, dimethyl sulfide, anisole, and p-thiocresol at a ratio of 8 / 1 / 1 / 0.5 (v / v / v / w) for 60 minutes at 0°C. After removing anisole and p-thiocresol by extraction using hydrogen fluoride and dimethyl sulfide by reduced pressure, and then sequentially using ethyl ether and ethyl acetate, the crude peptide is extracted using a mixture of 0.5 M sodium phosphate buffer (pH 8.0) and N,N-dimethylformamide at a ratio of 1 / 1 (v / v). The disulfide bonds of Cys residues B and E were formed using dimethyl sulfoxide (Tam et al., J.Am.Chem.Soc.113:6657-62, 1991). The resulting peptide can be purified by reverse-phase chromatography. The disulfide bond between the Cys residues C and F is first formed by dissolving the peptide in 50% acetic acid in water. A saturated iodine solution is added to the glacial acetic acid (1 ml of iodine solution per 100 ml of solution). After incubation in a sealed glass container at room temperature for 2 days, the solution is diluted 5-fold with deionized water and extracted four times with ethyl ether to remove unreacted iodine. After removing the remaining ethyl ether by rotary evaporation, the crude product solution is freeze-dried and purified by continuous reverse-phase chromatography.

[0189] Peptides can also be synthesized by many other methods, including solid-phase synthesis using conventional FMOC protection (i.e., binding with DCC-HOBt and deprotection with piperidine in DMF). The Cys thiol group can be trityl protected. Treatment with TFA can be used for final deprotection of the peptide and release of the peptide from the solid resin. In many cases, air oxidation is sufficient to obtain appropriate disulfide bond formation.

[0190] The ability of peptides and other agents to bind to and / or stimulate GC-C receptors in the intestinal tract can be tested by assays such as the intestinal GC-C receptor binding assay. In one example, cells from the T84 human colon cancer cell line (American Type Culture Collection (Bethesda, Md.)) are grown in a 24-well culture plate until confluent using a 1:1 mixture of Ham's F12 medium supplemented with 5% fetal bovine serum and Dulbecco's modified Eagle medium (DMEM). The cells used in the assay are optionally passaged 54-60. Briefly, the T84 cell monolayer in the 24-well plate is washed twice with 1 ml of binding buffer (DMEM containing 0.05% bovine serum albumin and 25 mM HEPES, pH 7.2), and then incubated at 37°C for 30 minutes in the presence of mature radiolabeled E. coli ST peptides and test material at various concentrations. Next, the cells are washed four times with 1 ml of DMEM and solubilized with 0.5 ml / well of 1N NaOH. The level of radioactivity in the solubilized material is determined using standard methods.

[0191] Examples of additions of GC-C agonist peptides include, for example, U.S. Patent Nos. 7,041,786, 7,304,036, 7,371,727, 7,494,979, 7,704,947, 7,799,897, 7,745,409, 7,772,188, and 7,879,802. U.S. applications Nos. 7,910,546, 8,034,782, 8,080,526, 8,101,579, 8,114,831, 8,110,553, 8,357,775, and 8,367,800; U.S. applications Nos. 2013 / 0096071 and 2013 / 019023 No. 8, No. 2012 / 0040892, No. 2012 / 0040025, No. 2012 / 0213846, No. 2012 / 0289460, No. 2 011 / 0118184, 2010 / 0152118, 2010 / 0048489, 2010 / 0120694, 2010 / 02 These are described in publications 61877, 2009 / 0253634, 2009 / 0192083, and 2009 / 0305993; as well as the PCT publications International Publication 2006 / 086653 and International Publication 2002 / 098912, which are incorporated herein by reference in their entirety.

[0192] D. Soluble guanylate cyclase agonists In certain embodiments, the compound is a soluble guanylate cyclase (sGC) agonist. Guanine nucleotidyl (guanylyl; guanylate) cyclase (GC) is a widely distributed signaling enzyme that converts GTP to the secondary messenger cyclic GMP (cGMP) in response to various cellular stimuli. Both membrane-bound and soluble guanylate cyclases exist, and both can increase the intracellular concentration of cGMP.

[0193] In intestinal cells of the gut, increased cGMP production inhibits Na+ / H+ exchange activity in the gut, leading to alkalization of the intestinal mucosa. See, for example, Fawcus et al., Comp Biochem. Physiol A Physiol. 118:291-295, 1997. Although not limited to one mechanism, in certain aspects, soluble guanylate cyclase activators inhibit or reduce phosphate uptake in the gastrointestinal tract, increasing cGMP production and thereby enhancing alkalization of the intestinal mucosa.

[0194] Common examples of sGC agonists include heme-dependent and heme-independent activators. See, for example, Evgenov et al., Nat. Rev. Drug Discov. 5:755-768, 2006. According to one non-limiting theory, these and other sGC activators can be used to selectively activate sGC in the intestinal tract, as described herein, to increase cGMP concentration and thereby inhibit phosphate uptake.

[0195] Although not limited to any one mechanism, in some embodiments, sGC agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0196] Non-limiting examples of sGC agonists include Bay 41-2271, Bay 58-2667, and the compounds shown in Figures 9A–9L. Additional structures of exemplary sGC agonists, along with methods for their synthesis, are disclosed in U.S. Patent No. 7,087,644 and the PCT publication International Publication No. 2013 / 101830, which are incorporated herein by reference in their entirety.

[0197] E. Adenylyl cyclase agonists In certain embodiments, the compound is an adenylyl cyclase agonist, optionally a selective agonist. Adenylyl cyclase (or adenylyl cyclase) refers to a type of enzyme that catalyzes the conversion of ATP to 3',5'-cyclic AMP (cAMP) and pyrophosphate. Divalent cations (e.g., Mg) are often involved in this enzymatic activity. cAMP produced by adenylyl cyclase functions as a regulatory signal mediated by specific cAMP-binding proteins, including transcription factors or other enzymes (e.g., cAMP-dependent kinases).

[0198] Adenylyl cyclase is an effector molecule in one of the most widely used signaling pathways. Its product, cAMP, regulates cell growth and differentiation from bacteria to higher eukaryotic cells in organisms. In animals, transmembrane adenylyl cyclase (tmAC) and soluble adenylyl cyclase (sAC) exist. See, for example, Tresguerres et al., Kidney Int. 79:1277-1288, 2011. Unlike tmAC, sAC is not a transmembrane protein and is found distributed throughout the cytoplasm and within specific organelles that are thought to be the source of secondary messengers mediating the intracellular function of cAMP. See, for example, Buck and Levin, Sensors. (Basel) 11:2112-2128, 2011. Therefore, tmAC is directly regulated by G proteins that translate extracellular signals into intracellular cAMP changes. In contrast, sAC isoforms are regulated by intracellular signaling involving bicarbonate, calcium, and ATP.

[0199] The transmembrane regulatory factor (CFTR) for cystic fibrosis is a chloride and bicarbonate ion channel that functions in the epithelium of multiple tissues. This channel is responsible for the bicarbonate that determines the pH on the mucosal surface in the small intestine. 3-It has been shown to be responsible for secretion. See, for example, Kunzelmann and Mall, Physiol Rev. 82:245-289, 2002. CFTR is regulated by cAMP, and phosphate chloride of the CFTR regulatory domain by cAMP-dependent protein kinase A (PKA) increases its activity. Therefore, selective activation of this ion channel leads to alkalization of the luminal membrane, thereby reducing or decreasing CEPG. Thus, according to one non-limiting theory, selective stimulation of tmAC in the intestinal tract should increase intracellular cAMP, stimulate PKA, increase CFTR activity, and thereby inhibit Pi uptake via CEPG action. In certain embodiments, this compound selectively activates tmAC compared to, for example, sAC.

[0200] Adenylyl cyclase agonists, such as forskolin, have been shown to optionally increase cAMP-mediated duodenal bicarbonate secretion (without increasing gastric bicarbonate secretion) via CFTR signaling. See, for example, Takeuchi et al., Am.J.Physiol.272(3 Pt 1):G646-53, 1997. While not limited to any single mechanism, in certain aspects, adenylyl cyclase agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine.

[0201] Although not limited to any one mechanism, in some embodiments, adenylyl cyclase agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0202] In certain embodiments, the compound is an adenylyl cyclase III (AC-III) agonist, optionally one or more agonists of the AC-III isoforms ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and / or ADCY10.

[0203] Specific examples of adenylyl cyclase agonists include labdane diterpenes such as forskolin and its analogs / derivatives, including water-soluble forskolin analogs such as colforcin (NKH477). Forskolin is a diterpene compound isolated from plants that activates all mammalian tmACs except tmAC IX (mammalian sACs are insensitive to forskolin). See, for example, Kamenetsky et al., J.Mol.Biol.362:623-639, 2006. Forskolin stimulation can result in potent and prolonged cAMP changes. See, for example, Tresguerres et al., Kidney Int.79:1277-1288, 2011. The structures of forskolin and some forskolin analogs are illustrated in Figure 10. Water-soluble derivatives of forskolin include those acylated at C-6 or C-7 with polar aliphatic amines. These derivatives typically exhibit less off-target activity and are more selective for AC. See, for example, Hartzell and Budnitz, Molecular Pharmacology 41:880-888, 1992. Therefore, certain embodiments involve the use of soluble forskolin analogs that selectively activate adenylyl cyclase in cells lining the gastrointestinal tract.

[0204] Specific examples of forskolin analogs / derivatives include aminoalkylcarbamyl derivatives of forskolin, including 1-aminoalkylcarbamate, 9-aminoalkylcarbamate, 7-aminoalkylcarbamate, 6-aminoalkylcarbamate, 6,7-diaminoalkylcarbamate, 1,6-diaminoalkylcarbamate, 1,7-diaminoalkylcarbamate, and 1,6,7-triaminoalkylcarbamate, which can be used as intermediates in the synthesis of forskolin derivatives. See U.S. Patent No. 5,350,864. Additional examples of forskolin analogs / derivatives include 12-halogenated forskolin derivatives, including 12-chlorodesacetylforskolin, 12-chloroforskolin, 12-bromodesacetylforskolin, 12-bromodesacetylforskolin, 12-fluorodesacetylforskolin, and 12-fluoroforskolin. See U.S. Patent No. 4,871,764.

[0205] In some embodiments, the forskolin analog / derivative is 6-acetyl-7-deacetyl-forskolin, 7-deacetyl-forskolin, 7-deacetyl-6-(N-acetylglycyl)-forskolin, 7-deacetyl-7-β-hemisacnyl-forskolin, 7-deacetyl-7-(ON-methylpiperazino)-γ-butryl-dihydroclone-forskolin, 7-HPP-forskolin, 6-HPP-forskolin, or colforsin dalopate hydrochloride (NKH477). For example, see U.S. Patent Applications 2011 / 0171195, 2006 / 0004090, and 2011 / 0077292, Laurenza et al., Mol Pharmacol. 32:133-9, 1987, Lal et al., Bioorg Med Chem. 6:2075-83, 1998, and Mori et al., J. Cardiovasc. Pharmacol. 24:310-6, 2004. Also, see Levin, Tetrahedon Letters. 37:3079-3082, 1996 for exemplary methods of synthesizing forskolin analogs, and Lal et al., Indian J. Chemistry. 45B:232-246, 2006 for additional examples of water-soluble forskolin analogs and methods of synthesizing them. Additional structures of exemplary adenylyl cyclase agonists, along with methods for their synthesis, are disclosed in U.S. Patent No. 4,954,642 and Khandelwal et al., J Med Chem. 31:1872-9, 1988. For exemplary methods / assays for detecting adenylyl cyclase activity stimulated by agonists, see Cunliffe et al., Electrophoresis. 28:1913-20, 2007. These references are incorporated herein by reference in their entirety.

[0206] F. Imidazolin-1 receptor agonists In certain embodiments, the compound is an imidazoline-1 receptor agonist, optionally a selective agonist. Imidazolin receptors include a family of non-adrenergic high-affinity binding sites for clonidine, idazoxane, and other imidazoles. There are three types of imidazoline receptors: the I1 receptor, which lowers blood pressure by mediating the sympathetic inhibitory effect of imidazoline; the I2 receptor, which is an allosteric binding site for monoamine oxidase and is involved in pain regulation and neuroprotection; and the I3 receptor, which regulates insulin secretion from pancreatic β-cells. In some embodiments, the compound is, for example, an imidazoline-1 receptor agonist that is selective to imidazoline-2 and / or imidazoline-3 receptors.

[0207] Subclasses of the imidazoline-1 receptor partially mediate the central antihypertensive effects of clonidine-like drugs. According to one non-limiting theory, activated imidazoline-1 receptors induce hydrolysis of phosphatidylcholine to DAG, which then induces the synthesis of secondary messengers such as arachidonic acid and downstream eicosanoids such as PGE2. See, e.g., Ernsberger, Ann. NY Acad. Sci. 881:35-53 1999. PGE2 is an endogenous inducer of DBS. See, e.g., Takeuchi et al., Gastroenterology. 113:1553-1559, 1997. Although not bound to any one mechanism, in some aspects, imidazoline-1 receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by increasing DBS.

[0208] According to another non-restrictive theory, imidazoline-1 receptor agonists, such as moxonidine, have also been shown to reduce acid secretion in the gastrointestinal tract. See, for example, Glavin and Smyth, Br J Pharmacol. 114:751-4, 1995. Therefore, although not limited to one mechanism, in certain embodiments, imidazoline-1 receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by inhibiting or reducing acid secretion in the gastrointestinal tract, for example, in the small intestine. Although not limited to one mechanism, in certain embodiments, imidazoline-1 receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by increasing DBS and reducing acid secretion in the small intestine.

[0209] Although not limited to any one mechanism, in some embodiments, imidazoline-2 receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0210] Non-exclusive examples of imidazoline-1 receptor agonists include agmatine, apraclonidine, clonidine, efaloxane, moxonidine, rilmenidine, S-23515, S-23757, LNP-509, LNP-911, LNP-509, S-23515, PMS-812, PMS-847, BU-98008, and TVP1022 (the S-enantiomer of rasagiline). See also Head and Mayorov, Cardiovasc Hematol Agents Med Chem. 4:17-32, 2006, which is incorporated herein by reference in its entirety.

[0211] Exemplary structures of imidazoline receptor agonists are shown in Figure 11 and, together with methods for their synthesis, are further disclosed in U.S. Patents 4,323,570, 5,686,477, 3,988,464, 6,300,366, 5,492,912, 5,492,912, and International Publication 200141764, which is a PCT publication, each of which is incorporated herein by reference in its entirety.

[0212] Additional examples of imidazoline receptor agonists are described in U.S. Patent No. 7,309,706, U.S. Patent No. 5,686,477 (European Patent No. 710,658), U.S. Patent No. 5,925,665 (European Patent No. 846,688), International Publication No. 2001 / 41764, and International Publication No. 2000 / 02878. The 5-(aryloxymethyl)-oxazoline derivative described in U.S. Patent No. 5,686,477 is characterized by selective affinity for the imidazoline-1 receptor. The imidazoline derivative described in U.S. Patent No. 5,925,665 binds to the imidazoline receptor without significantly binding to the adrenergic receptor. International Publication No. 2001 / 41764 describes isoquinoline and quinoline derivatives that possess affinity for the imidazoline receptor. International Publication No. 2000 / 02878 describes exemplary β-carbolin derivatives as ligands for potential imidazoline receptors. These references are incorporated herein by reference in their entirety.

[0213] G. Cholinergic agents In certain embodiments, the compound is a cholinergic agent, optionally a selective cholinergic agent. Examples of cholinergic agents include indirect cholinergic agents that stimulate the production or release of acetylcholine (e.g., astethylcholinesterase inhibitors), and direct cholinergic agents that bind to and stimulate one or more acetylcholine receptors. Acetylcholine (2-acetoxy-N,N,N-trimethylethaneaminium), a neurotransmitter, is an ester of acetic acid and choline. Common examples of acetylcholine receptors include nicotinic acetylcholine receptors and muscarinic acetylcholine receptors. Nicotinic acetylcholine receptors are ligand-opening ion channels consisting of five protein subunits.

[0214] Muscarinic acetylcholine receptors (i.e., M1, M2, M3, M4, and M5) are G protein-bound receptors that activate other ionic channels via a secondary messenger cascade. These receptors are expressed in the gastrointestinal tract, including in the salivary glands and in smooth muscle and mucosal cells of the stomach and intestines. In certain embodiments, the compound is a pan-agonist of muscarinic receptor subtypes. The endogenous agonists of all five muscarinic receptor subtypes are acetylcholine, which exerts physiological regulation through both hormonal and neural mechanisms. See, for example, Eglen, Ann. NYAcad. Sci. 881:35-53, 2012. Several spontaneously occurring compounds also modulate muscarinic receptors (see Figure 12), including agonists such as muscarine (a toxin from the mushroom Aminita muscaria, from which this receptor family derives its name) and pilocarpine, as well as antagonists such as atropine or (-)-hyostine (from plants of the Solanaceae family). When administered in vivo, muscarinic agonists induce saliva production, while muscarinic antagonists cause dry mouth.

[0215] In some embodiments, the compound is a relative selective agonist of the M3 muscarinic receptor. The secretory response of M3 is physiologically stimulated by acetylcholine (ACh). Specifically, ACh binds to the G protein-bound M3 muscarinic ACh receptor, which causes phospholipase C to produce inositol 1,4,5-trisphosphate (InP3). InP3 binds to and opens the InP3 receptor on the endoplasmic reticulum, and according to one non-limiting theory, this opens Ca 2+ It releases [Ca 2+ ] i An increase in Cl- channels in the apical membrane and K+ channels in the basal plane activates Cl- channels into the acinar lumen. - The outward current causes Na+ to flow throughout the cell, and the resulting osmotic gradient induces fluid secretion. See, for example, Tobin et al., J. Physiol Pharmacol. 60:3-21, 2009. This fluid is rich in bicarbonates.

[0216] The regulation of bicarbonate secretion by muscarinic receptors has been repeatedly demonstrated, and muscarinic agonists administered intravascularly or subcutaneously increase the release of bicarbonate into the intestinal lumen, a response that is blocked by muscarinic antagonists. For example, according to one non-limiting theory, cholinergic agonists such as bethanechol (a muscarinic receptor selective agonist), carbachol (a muscarinic and nicotinic acetylcholine receptor agonist), and McN-A-343 (an M1 receptor selective agonist) have been shown to increase duodenal bicarbonate secretion. See, for example, Safsten et al., Am J Physiol. 267(1 Pt 1):G10-7, 1994. Although not limited to one mechanism, in certain aspects, cholinergic agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine.

[0217] While not limited to any one mechanism, in some embodiments, cholinergics inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0218] In some embodiments, muscarinic receptor agonists possess a structure that is sterically constrained with respect to the endogenous ligand acetylcholine, such as the cis-trimethyl-(2-methyl-[1,3]dioxolan-4-ylmethyl)ammonium iodide structure shown in Figure 12. See, for example, Piergentili et al., Bioorganic & Medicinal Chemistry 15:886-896, 2007. This structure contains a ketal, a significantly more stable bioequivalent that retains both hydrogen-bonding receptors of ACh instead of the unstable ester of acetylcholine. Similarly, the structures of carbechol and betanecol (also shown in Figure 12) are examples of agonists, as they substitute the unstable ester group of ACh with a non-hydrolyzable carbamic acid functional group.

[0219] Non-limiting examples of indirectly acting cholinergics include acetylcholinesterase inhibitors such as carbamates (e.g., physostigmine, neostigmine, pyridostigmine), piperidines (e.g., donepezil), edrophonium, huperzine A, ladostigil, ungeremine, lactucopicrin, tacrine, galantamine, trans-δ-9-tetrahydrocannabinol, and phosphates (e.g., isoflurofate, ecothiophate, parathion, malathion). Preferably, the methods provided herein use reversible acetylcholinesterase inhibitors.

[0220] Non-exclusive examples of direct-acting cholinergics include acetylcholine, nicotine, succinylcholine, metacholine (acetyl-β-methylcholine), McN-A-343, carbacol (carbamoylcholine), bethanecol (carbamoyl-β-meslicolin), muscarine, pilocarpine, oxotremoline, loberine, and dimethylphenylpipalazinium.

[0221] H. Prostaglandin EP4 receptor agonists In certain embodiments, the compound is an E-type prostanoid receptor 4 (EP4) agonist (or prostaglandin EP4 receptor agonist), optionally a selective agonist. The EP4 receptor is a prostaglandin that stimulates adenylyl cyclase and causes an increase in intracellular cAMP levels. αs Initially described as a protein-binding receptor, the EP4 receptor was named "EP2" when it was first cloned as a prostaglandin E2 (PGE2) receptor that stimulated cAMP formation. However, with the discovery of another cAMP-stimulated PGE2 receptor sensitive to porcine prost, the porcine prost-insensitive receptor that mediated vasodilation was renamed "EP4." This is one of four receptors identified for PGE2.

[0222] According to one non-restrictive theory, prostaglandin EP4 receptor agonists have been shown to stimulate duodenal bicarbonate secretion, for example, through a mechanism mediated by cAMP. See, for example, Aoi et al., Am J Physiol Gastrointest Liver Physiol. 287:G96-103, 2004; Lundgren, Acta Physiol Scand. 185:87, 2005; and Takeuchi et al., Gastroenterology. 113:1553-1559, 1997. Therefore, although not limited to any one mechanism, in certain aspects, prostaglandin EP4 receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine.

[0223] While not limited to any one mechanism, in some embodiments, EP4 agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0224] Non-exclusive examples of prostaglandin EP4 receptor agonists include PGE2, PGE2 analogues, AE1-329, AGN205203, APS-999 Na, Cay10598(19a), CP-044519-02, CJ-023,423, EP4RAG, ER-819762, L-902688, lubiprostone, ONO-4819CD, ONO AE1-329, ONO AE1-734, PGE1-OH, TCS2510, γ-lactam PGE analogue 3, 11-deoxy-PGE1, γ-lactam PGE analogue 2a, and γ-lactam PGE analogue 4. See, for example, Konya et al., Pharmacol Ther. 138:485-502, 2013.

[0225] Non-limiting examples of PGE2 analogs include 16,16-dimethylPGE2, 16-16-dimethylPGE2p-(p-acetamidobenzamide)phenyl ester, 11-deoxy-16,16-dimethylPGE2, 9-deoxy-9-methylene-16,16-dimethylPGE2, 9-deoxy-9-methylenePGE2, 9-ketofluprostenol, 5-transPGE2, 17-phenyl-ω-trinolePGE2, and PG. Examples include E2 selinolamide, PGE2 methyl ester, 16-phenyltetranol PGE2, 15(S)-15-methyl PGE2, 15(R)-15-methyl PGE2, 8-iso-15-keto PGE2, 8-iso PGE2 isopropyl ester, 20-hydroxy PGE2, 11-deoxy PGEi, nocloprost, sulprostone, buprost, 15-keto PGE2, and 19(R) hydroxyy PGE2. See, for example, U.S. Patent Application No. 2012 / 0202288.

[0226] Additional examples of prostaglandin EP4 receptor agonists include those described in U.S. applications 2001 / 0056060, 2002 / 0040149, 2005 / 0164949, and 2011 / 0098481. U.S. applications 4,219,479, 4,049,582, 4,423,067, 4,474,802, 4,692,464, 4,708,963, 5,010,065, 5,013,758, 6,747,037, and 7,776,Patent No. 896, European Patent No. 0084856, Canadian Patent No. 1248525, US applications No. 2004 / 0102499, 2005 / 049227, 2005 / 228185, 2006 / 106088, 2006 / 111430, 2007 / 0010495, 2007 / 0123568, 2007 / 0123569, 2005 / 0020686, 2008 / 0234337, 2010 / 0010222, 2010 / 0216689, and 2004 / 01987 No. 01, No. 2004 / 0204590, No. 2005 / 0227969, No. 2005 / 0239872, No. 2006 / No. 0154899, No. 2006 / 0167081, No. 2006 / 0258726, No. 2006 / 0270721, No. 2009 / 0105234, 2009 / 0105321, 2009 / 0247596, 2009 / 025891 No. 8, No. 2009 / 0270395, No. 2004 / 0087624, No. 2004 / 0102508, No. 2006 / 02 Publications 52799, 2009 / 0030061, 2009 / 0170931, 2010 / 0022650, 2009 / 0312388, 2009 / 0318523, 2010 / 0069457, 2010 / 0076048, 2007 / 0066618, 2004 / 0259921, 2005 / 0065133, and 2007 / 0191319, as well as PCT publications International Publications 2004 / 4071428, 2006 / 052630, and 2006 Prostaglandin EP4 receptor agonists described in Nos. / 047476, 2006 / 058080, 2004 / 065365, 2003 / 047513, 2004 / 085421, 2004 / 085430, 2005 / 116010, 2005 / 116010, 2007 / 014454, 2006 / 080323, and 2006 / 137472 are also included (along with the associated synthesis methods), and these are each incorporated herein by reference in their entirety.

[0227] Examples of specific EP4 receptor agonists are shown in Figure 13.

[0228] In certain embodiments, the EP4 receptor agonist is lubiprostone (similarly a calcium-activated chloride channel agonist). Lubiprostone is a bicyclic fatty acid derived from prostaglandin E1 that acts by specifically activating ClC-2 chloride channels on the apical lateral surfaces of gastrointestinal epithelial cells, resulting in the secretion of chloride-rich fluids. These secretions soften the stool, increase motility, and promote spontaneous bowel movements (SBM). Lubiprostone stimulates CFTR-dependent duodenal bicarbonate secretion without altering net Cl- secretion. See, for example, Muzimori et al., J Physiol. 573:827-842, 2006. Here, lubiprostone-induced duodenal bicarbonate secretion was abolished by co-perfusion with the potent EP4 receptor antagonist AH23848, but had no effect with the EP1 / EP2 receptor antagonist AH6809. These results suggest that lubiprostone may increase duodenal bicarbonate secretion by stimulating prostaglandin EP4 receptors. Therefore, in certain embodiments, lubiprostone inhibits or reduces phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine.

[0229] As described above, certain embodiments include prostaglandin EP4 receptor selective agonists. EP4 selective agonists affect IC in EP4 receptor subtypes. 50 IC in EP1, EP2, and / or EP3 receptor subtypes is at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times greater. 50 It contains compounds that have the following properties.

[0230] I. Dopamine D1 receptor agonists In certain embodiments, the compound is a dopamine D-1 receptor agonist, optionally a selective agonist. The dopamine D1 G protein-binding receptor is the most highly expressed dopamine receptor subtype in the dopamine receptor family. It stimulates adenylyl cyclase, which activates cyclic AMP-dependent protein kinase.

[0231] Based on one non-restrictive theory, dopamine D1 receptor agonists and peripheral catechol-O-methyl-transferase (COMT) inhibitors such as nitecapone (COMT inhibitors reduce histolytic degradation of catecholamines, including dopamine) have been shown to stimulate bicarbonate secretion in the intestines and increase the production of cyclic AMP in isolated duodenal intestinal cells. See, for example, Flemstrom and Safsten, Dig Dis Sci. 39:1839-42, 1994; Knutson et al., Gastroenterology. 104:1409-13, 1993; Iwatsuki et al., Eur J Pharmacol. 218:237-41, 1992; and Fraga et al., Cell Physiol Biochem. 18:347-60, 2006. While not limited to any single mechanism, in certain aspects, dopamine D1 receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine.

[0232] While not limited to any one mechanism, in some embodiments, dopamine D1 agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0233] Non-exclusive examples of dopamine D1 receptor agonists include dopamine (e.g., dopamine hydrochloride, NPEC-caged dopamine), dihydroexidine (e.g., dihydroexidine hydrochloride), benzazepine, and their analogs / derivatives. Specific examples of dihydroexidine derivatives include A86929, dinapsolin, dinoxyline, and doxanthrine, while specific examples of benzazepine derivatives include SKF81297, SKF82958, SKF38393, phenoldopam, and 6-Br-APB. The dopamine D1 receptor agonists shown in Figure 14 are also included.

[0234] Additional non-exclusive examples of dopamine D1 receptor agonists include A68930, A77636, (R)-(-)-apomorphine hydrochloride, CY208-243, SKF89145, SKF89626, 7,8-dihydroxy-5-phenyl-octahydrobenzo[h]isoquinoline, YM435, ABT-431, NNC01-0012, SCH23390, SKF7734, SKF81297, SKF38322, SKF83959, cabergoline, phenoldapam (e.g., phenoldapam hydrochloride), bromocriptine, ropinirole, pramipexole, entacapone, tolcapone, dihexadine, IPX-750, and pergolide. See also Zhang et al., Med Res Rev.29:272-94, 2009; Yvonne Connolly Martin, International Journal of Medicinal Chemistry, vol.2011, Article ID 424535, 8 pages, 2011. doi:10.1155 / 2011 / 424535; Salmi et al., CNS Drug Rev.10:230-42, 2004; and Bourne, CNS Drug Rev.7:399-414, 2001. Furthermore, D1 receptor agonists can be identified using standard screening methods known in the art. As a non-limiting example, a cell-based functional assay for high-throughput drug screening of dopamine D1 receptor agonists is described in Jiang et al., Acta Pharmacol Sin.26:1181-6, 2005. These references are incorporated herein by reference in their entirety.

[0235] As described above, certain embodiments include dopamine D1 receptor selective agonists. Dopamine D1 selective agonists affect IC in D1 receptor subtypes. 50 IC in D2, D3, D4, and / or D5 receptor subtypes is at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times greater. 50It contains compounds that have the following properties.

[0236] J. Melatonin receptor agonists In certain embodiments, the compound is a melatonin receptor agonist, optionally a selective agonist. Melatonin receptors refer to a family of high-affinity G protein-binding receptors that bind to melatonin, a pineal hormone. See Reppert, Biol Rhythms. 12:528-31, 1997.

[0237] Examples of melatonin receptors include MT1 and MT2 receptors. In some embodiments, melatonin receptor agonists bind to both MT1 and MT2 receptors. In some embodiments, melatonin receptor agonists selectively bind to either MT1 or MT2 receptors, for example, binding to MT2 but not significantly to MT1, or binding to MT1 but not significantly to MT2.

[0238] According to non-restrictive theories, melatonin receptor agonists, such as melatonin, have been shown to stimulate duodenal bicarbonate secretion, for example, through their action on intestinal cell MT2 receptors. See, for example, Sjoblom et al., J Clin Invest. 108:625-33, 2001 and Sjoblom and Flemstrom, J. Pineal Res. 34:288-293, 2003. Although not limited to any one mechanism, in certain aspects, melatonin receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine.

[0239] Although not limited to any one mechanism, in some embodiments, melatonin receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0240] Examples of melatonin receptor agonists include melatonin (N-acetyl-5-methoxytryptamine) and melatonin analogs that bind to and activate melatonin receptors. The general structure of melatonin contains an indole ring with a methoxy group (5-methoxy group) at position 5 and an acylaminoethyl side chain at position 3. These two side chains contribute to binding to and activation of melatonin receptors. The indole ring has been evaluated at all positions by substitution. See, for example, Rivara et al., Curr Top Med Chem. 8:954-68, 2008, and Sugen et al., Pigment Cell Research. 17:454-460, 2004.

[0241] Specific examples of melatonin receptor agonists include circadine, agomelatine, ramelteon, tasimelteon, β-methyl-6-chloromelatonin (TIK-301 or LY156735), TAK-375, VEC-162, GR196429, S20242, S23478, S24268, S25150, GW290569, BMS-214778, 8-methoxy-2-chloroacetamidotetraline, 8-methoxy-2-propionamide-tetraline, N-acetyltryptamine, 6-chloromelatonin, 2-iodomelatonin, 8-M-PDOT, and 2-phenylmelatonin, as well as 2-iodomelatonin, 6-chloromelatonin, 6,7-dichloro-2-methylmelatonin, and 8-hydroxymelatonin, all of which contain a 5-methoxyindole ring as part of their structure. See, for example, U.S. Patent Application No. 2005 / 0164987, which is incorporated in its entirety herein by reference. This includes exemplary melatonin receptor (MT2) agonists shown in Figure 15.

[0242] A method for screening melatonin receptor agonists is described, for example, in U.S. Patent Application No. 2003 / 0044909, which is incorporated in whole by reference herein.

[0243] K5HT4 receptor agonist In certain embodiments, the compound is a 5HT4 receptor agonist, optionally a selective agonist. 5-hydroxytryptamine receptor 4 (5HT4) is a G protein-bound serotonin receptor that stimulates cAMP production in response to serotonin (5-hydroxytryptamine, i.e., 5-HT) or other agonists.

[0244] Based on one non-restrictive theory, serotonin has been shown to increase protective duodenal bicarbonate secretion, for example, via enteric ganglia, cAMP-dependent and Ca2+-dependent signaling pathways, and 5HT4-dependent pathways. See, for example, Safsten et al., Scand J Gastroenterol. 41:1279-89, 2006 and Tuo et al., Am J Physiol Gastrointest Liver Physiol 286:G444-G451, 2004. Although not limited to any one mechanism, in certain aspects, 5HT4 receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine.

[0245] While not limited to any one mechanism, in some embodiments, 5HT4 agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0246] Non-exclusive examples of 5HT4 agonists include serotonin and its analogues, BIMU-8, cisapride, cleovoprid, CL033466, ML10302, mosapride, pulcalopride, lenzaprid, RS67506, RS67333, SL650155, tegaserod, zacoprid, nalonopride (ATI-7505), and bercetrag (TD-5108).

[0247] In some embodiments, the 5HT4 receptor agonist or partial agonist is a substituted benzamide such as cisapride, including each or a combination of cisapride enantiomers ((+)cisapride and (-)cisapride), mosapride, or lenzaprid. In some embodiments, the 5HT4 receptor agonist is a benzofuran derivative such as prukalopride, an indole such as tegaserod, or benzimidazolon. Other non-exclusive examples of 5HT4 receptor agonists or partial agonists include zacoprid (CAS RN 90182-92-6), SC-53116 (CAS RN 141196-99-8), and its racemic mixture SC-49518 (CAS RN 146388-57-0), BIMU1 (CAS RN 127595-43-1), TS-951 (CAS RN 174486-39-6), ML10302 (CAS RN 148868-55-7), metoclopramide, 5-methoxytryptamine, RS67506, 2-[1-(4-piperonyl)piperazinyl]benzothiazole, RS66331, BIMU8, SB Examples include 205149 (an n-butyl quaternary analog of lenzaprid) and indolecarbazimidamide, as described in Buchheit et al., J Med. Chem. 38:2331-8, 1995. Norcisapride (CAS RN 102671-04-5), a metabolite of cisapride; mosapride citrate; the maleate form of tegaserod (CAS RN 189188-57-6); zacoprid hydrochloride (CAS RN 99617-34-2); mezacoprid (CAS RN 89613-77-4); SK-951 ((+-)-4-amino-N-(2-(1-azabicyclo(3.3.0)octan-5-yl)ethyl)-5-chloro-2,3-dihydro-2-methylbenzo[b]furan-7-carboxamide hemifumarate); ATI-7505, a cisapride analog; and SDZ-216-454 (e.g., Markstein et al.), a selective 5HT4 receptor agonist that stimulates cAMP formation in a concentration-dependent manner. al., Naunyn-Schmiedebergs Arch Pharmacol.See 359:454-9, 1999); SC-54750, i.e., aminomethyl azaadamantane; Y-36912, i.e., 4-amino-N-[1-[3-(benzylsulfonyl)propyl]piperidine-4-ylmethyl]-5-chloro-2-methoxybenzamide (Sonda et al., Bioorg See Med.Chem.12:2737-47,2004; TKS159, i.e., 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2-hydroxymethyl-4-pyrrolidinyl]benzamide; RS67333, i.e., 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-n-butyl-4-piperidinyl)-1-propanone; KDR-5169, i.e., 4-amino-5-chloro-N-[1-(3-fluoro-4-methoxybenzyl)piperidine-4-yl]-2-(2-hydroxyethoxy)benzamide hydrochloride dihydrate (Tazawa, et al., Eur J See Pharmacol. 434:169-76, 2002; SL65.0155, i.e., 5-(8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-yl)-3-[1-(2-phenylethyl)-4-piperidinyl]-1,3,4-oxadiazole-2(3H)-one monohydrochloride; and Y-34959, i.e., 4-amino-5-chloro-2-methoxy-N-[1-[5-(1-methylindole-3-ylcarbonylamino)pentyl]piperidine-4-ylmethyl]benzamide.

[0248] Additional examples of 5HT4 receptor agonists and partial agonists include metoclopramide (CAS RN 364-62-5), 5-methoxytryptamine (CAS RN 608-07-1), RS67506 (CAS RN 168986-61-6), 2-[1-(4-piperonyl)piperazinyl]benzothiazole (CAS RN 155106-73-3), RS66331 (see Buccafusco et al., Pharmacology. 295:438-446, 2000), BIMU8 (endo-N-8-methyl-8-azabicyclo[3.2.1]octo-3-yl)-2,3-dehydro-2-oxo-3-(prop-2-yl)-1H-benzimidoazole-1-carboxamide), or SB This includes compounds related to metoclopramide, such as metoclopramide dihydrochloride (CAS RN 2576-84-3), metoclopramide dihydrochloride (CAS RN 5581-45-3), and metoclopramide hydrochloride (CAS RN 7232-21-5 or 54143-57-6). See, for example, U.S. Patent Application No. 2009 / 0325949, De Maeyer et al., Neurogastroenterology and Motility. 20:99-112, 2008, Manabe et al., Expert Opin Investig Drugs. 19:765-75, 2010, and Tack et al., Alimentary Pharmacology & Ther. 35:745-767, 2012. These references are incorporated herein by reference in their entirety.

[0249] L. Atrial natriuretic peptide receptor agonists In some embodiments, the compound is an atrial natriuretic peptide (NP) receptor agonist. The NP receptor is a single transmembrane catalytic receptor with intracellular guanylyl cyclase (GC) activity. There are three isoforms of the NP receptor: NPR1, NPR2, and NPR3. These receptors conserve the catalytic domain, regulatory domain, and branched ligand-binding domain.

[0250] Natriuretic peptide receptors (NP receptors) are found in the brain, vascular structures of the kidneys, and the gastrointestinal tract, and bind to α-atrial natriuretic peptides, brain natriuretic peptides, and C-type natriuretic peptides with varying affinities. The primary physiological role of NP receptors is homeostasis of body fluid volume. According to one non-restrictive theory, exogenous natriuretic peptides stimulate GC activity in the gastrointestinal tract. See, for example, Rambotti et al., Histochem. J. 29: 117-126, 1997.

[0251] While not limited to any single mechanism, in certain aspects, NP receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion in the small intestine and / or inhibiting acid secretion.

[0252] Although not limited to any one mechanism, in some embodiments, NP receptor agonists inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0253] The structures of exemplary peptide agonists of NP receptors are shown in Figure 16 and are described, for example, in their entirety by reference to von Geldern et al., J.Med.Chem. 35:808-816, 1992.

[0254] In certain embodiments, the NP receptor agonist comprises, consists of, or essentially consists of the atrial natriuretic peptide amino acid sequence Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr (SEQ ID NO: 7) (including active variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deletions, insertions, and / or substitutions). Specific examples of deletion mutants include those having the sequences Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys (SEQ ID NO: 8) and Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg (SEQ ID NO: 9). As described elsewhere in this specification, such peptides may consist of any combination of spontaneously occurring and non-spontaneously occurring amino acids.

[0255] M. Carbonic anhydrase inhibitors In some embodiments, the compound is a carbonic anhydrase inhibitor. Bicarbonate uptake into epithelial cells is performed by CO2 diffusion and subsequent HCO3 uptake by cellular carbonic anhydrase (CA). - and H + It is generated by the conversion to . Next, bicarbonate is secreted across the apical membrane by anion exchange. CA hydrates CO2 to form HCO3. - and H + This enzyme produces HCO3, which is present in most tissues, including the epithelial cells of the duodenum. See, for example, Kaunitz and Akiba, 2006. - It is an important source of transported bicarbonates.

[0256] Carbonic anhydrase has at least 15 isoforms. Carbonic anhydrase IV (CAIV) is a membrane-bound isoform, while CAII is cytoplasmic, ubiquitous, and highly active (metabolic turnover rate, approximately 10⁻¹⁰⁻¹ 6 s -1 See, for example, Shandro and Casey, 2007. Carbonic anhydrase II appears to functionally bind (directly and indirectly) to bicarbonate transport proteins such as CFTR, SLC26A6, and DRA. See, for example, Seidler and Sjoblom, 2012. In general, the COOH-terminal tails of all bicarbonate transport proteins except DRA possess a common carbonic anhydrase II-binding motif. See, for example, Dudeja and Ramaswamy, 2006.

[0257] Carbonic anhydrase is involved in several physiological processes, including pH homeostasis. Conventional carbonic anhydrase inhibitors, such as acetazolamide and benzoramide, have been shown to inhibit multiple CA isoforms, including CAII and CAIV. See, for example, Scozzafava et al., J.Med.Chem. 45:1466-1476, 2002. According to one non-limiting theory, inhibition of carbonic anhydrase affects the subapical intracellular pH. i It is expected to reduce [something]. Although not tied to any one mechanism, selective inhibition of CA in duodenal intestinal cells may thereby reduce CEPG and lead to a decrease in phosphate transport.

[0258] Although not limited to any one mechanism, in some embodiments, carbonic anhydrase inhibitors inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0259] Figure 17 shows the structures of exemplary carbonic anhydrase inhibitors, including, in particular, dorzolamide and brinzolamide. In certain embodiments, carbonic anhydrase inhibitors may be used in combination with compounds of a type that can increase cAMP, cGMP, calcium, or other secondary messengers in the mucosal cells on the apical side of the gastrointestinal tract.

[0260] N. Phosphodiesterase inhibitors In some embodiments, the compound is a phosphodiesterase inhibitor. Phosphodiesterases (PDEs) are a family of related phosphohydrolyases that selectively catalyze the hydrolysis of the 3'-cyclic phosphate bond in adenosine and / or the 3',5'-cyclic monophosphate (cAMP and / or cGMP) of guanine. They control their cellular level, localization, and duration of action by controlling the rate of degradation of their secondary messengers.

[0261] There are 11 subtypes of PDE, named PDE1-11. PDE4, 7, and 8 selectively degrade cAMP; PDE5, 6, and 9 selectively degrade cGMP; and PDE1, 2, 3, 10, and 11 degrade both cyclic nucleotides. PDEs are ubiquitously expressed, and each subtype has a specific tissue distribution. Figure 18 shows the structures of exemplary phosphodiesterase inhibitors with various subtype specificities, including theophylline, cilostazol, vinpocetine, amrinone, EHNA, trequinine, dorotaverine, roflumilast, and sildenafil.

[0262] According to one non-limiting theory, phosphodiesterase inhibitors can modulate duodenal bicarbonate secretion (DBS) alone and in combination with agents that increase cytoplasmic cAMP and cGMP by maintaining levels of these secondary messengers in intestinal cells. PDE1 and PDE3 inhibitors are particularly involved in the modulation of DBS. See, for example, Hayashi, Biochem.Pharmacol. 74:1507-1513, 2007. Although not limited to one mechanism, in certain embodiments, phosphodiesterase inhibitors inhibit or reduce phosphate uptake in the gastrointestinal tract by stimulating bicarbonate secretion into the small intestine, or DBS.

[0263] Although not limited to any one mechanism, in some embodiments, phosphodiesterase inhibitors inhibit or reduce phosphate uptake in the gastrointestinal tract by decreasing water absorption in the small intestine.

[0264] In certain embodiments, PDE inhibitors can slow the degradation of cyclic AMP (cAMP) and / or cyclic GMP (cGMP), which can then lead to a relative increase in the intracellular concentration of cAMP and / or cGMP. Common examples include PDE1 inhibitors, PDE3 inhibitors, PDE4 inhibitors, PDE5 inhibitors, PDE3 / 4 inhibitors, and PDE3 / 4 / 5 inhibitors. Although this is merely a non-exclusive example, PDE inhibitors are included in the following patent applications and patents: German Nos. 1470341, 2108438, 2123328, 2305339, 2305575, 2315801, 2402908, 2413935, 2451417, 2459090, 2646469, 2727481, 2825048, 2837161, 2845220, and 28 No. 47621, No. 2934747, No. 3021792, No. 3038166, No. 3044568, No. 3142982, No. 1116676, No. 2162096, European No. 000718, No. 0008408, No. 0010759, No. 0059948, No. 0075436, No. 0096517, No. 0112987, No. 0116948, No. 0150937, No. 0158380, No. 0161632, No. 0 No. 161918, No. 0167121, No. 0199127, No. 0220044, No. 0247725, No. 0258191, No. 0272910, No. 0272914, No. 0294647, No. 03007 No. 26, No. 0335386, No. 0357788, No. 0389282, No. 0406958, No. 0426180, No. 0428302, No. 0435811, No. 0470805, No. 0482208, No. No. 0490823, No. 0506194, No. 0511865, No. 0527117, No. 0626939, No. 0664289, No. 0671389, No. 0685474, No. 0685475, No. 0685 No. 479, No. 0293063, No. 0463756, No. 0482208, No. 0579496, No. 0667345, No. 0163965, No. 0393500, No. 0510562, No. 0553174,Japanese Patent Nos. 92234389, 94329652, 95010875; U.S. Patents Nos. 4,963,561, 5,141,931, and 6,331,543; and International Publications of Patent Applications Nos. 9117991, 9200968, 9212961, 9307146, 9315044, 9315045, 9318024, 9319068, and 93197 No. 20, No. 9319747, No. 9319749, No. 9319751, No. 9325517, No. 9402465, No. 9406423, No. 9412461, No. 9420455, No. 9 No. 422852, No. 9425437, No. 9427947, No. 9500516, No. 9501980, No. 9503794, No. 9504045, No. 9504046, No. 9505386, No. 9508534, No. 9509623, No. 9509624, No. 9509627, No. 9509836, No. 9514667, No. 9514680, No. 9514681, No. 95173 No. 92, No. 9517399, No. 9519362, No. 9522520, No. 9524381, No. 9527692, No. 9528926, No. 9535281, No. 9535282, No. 9 This may include the disclosures in U.S. applications 600218, 9601825, 9602541, 9611917, 9307124, 9501338, and 9603399, as well as those disclosed in U.S. application 2005 / 0004222 (including formulas I-XIII and those disclosed in paragraphs 37-39, 85-0545, and 557-577), each of which is incorporated herein by reference in its entirety.

[0265] Examples of PDE5 inhibitors include RX-RA-69, SCH-51866, KT-734, Vesnarinone, Zaprinast, SKF-96231, ER-21355, BF / GP-385, NM-702, and sildenafil (Viagra®). Examples of PDE4 inhibitors include RO-20-1724, MEM 1414 (R1533 / R1500, Pharmacia). Roche), Denbuphylline, Lolipram, Oxagrelate, Nitraquazone, Y-590, DH-6471, SKF-94120, Motapizone, Lixazinone, Indolidan, Olprinone, Atizolam, KS-506-G, Dipamphylline, BMY-43351, Atizolam, Allophylline, Filaminaster, PDB-093, UCB-29646, CDP-840, SKF-107806, Examples include picramirast, RS-17597, RS-25344-000, SB-207499, tibenelast, SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, mopidamol, anagrelide, ibudilast, amrinone, pimobendan, cilostazol, quazinone, and N-(3,5-dichloropyrido-4-yl)-3-cyclopropylmethoxy-4-difluoromethoxybenzamide. Examples of PDE3 inhibitors include sulmazole, ampizone, cilostamide, carbazelan, pyroximon, imazodan, CI-930, ciguazodan, azibendan, saterinone, SKF-95654, SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, EMD-57033, NSP-306, NSP-307, revidinone, NM-702, WIN-62582 and WIN-63291, enoximon, and milrinone. Examples of PDE3 / 4 inhibitors include benafentrin, trequinin, ORG-30029, zardaverine, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, and trafentrin.Other examples of PDE inhibitors include siromilast, pentoxifylline, roflumilast, tadalafil (Cialis®), theophylline, vardenafil (Levitra®), and zaprinast (PDE5 specific).

[0266] In certain embodiments, phosphodiesterase inhibitors may be used in combination with compounds of a type that can increase cAMP, cGMP, calcium, or other secondary messengers in the mucosal cells on the apical side of the gastrointestinal tract.

[0267] O.DRA agonist (SLC26A3) In certain embodiments, the compound is an agonist of SLC26A3, a chloride / bicarbonate exchange transporter, also known as a downregulated exchange transporter (DRA) in adenomas. One non-limiting function of intestinal DRA is to absorb chloride ions from the lumen and secrete bicarbonate ions. Pharmacological stimulation of DRA is expected to result in a phosphate-lowering effect, for example, by increasing the pH of the UWL, as described herein.

[0268] Examples of DRA agonists include lysophosphatic acid (LPA) and structurally related compounds. These types of compounds are thought to act on DRA activity by stimulating signaling of LPA receptors (e.g., LPA2) via the Pi3K / AKT pathway, not only by activating DRA gene transcription but also by increasing DRA surface accumulation (Singla et al. Am.J. Physiol Gastrointest. Liver Physiol. 298:G182-G189, 2010; Singla et al. Am.J. Physiol Gastrointest. Liver Physiol. 302:G618-G627, 2012). Examples of LPA-related compounds with potential roles in DRA stimulation include Jiang et al.,Bioorg.Med.Chem.Lett.23:1865-1869,2013, Kiss et al.,Molecular Pharmacology 82:1162-1173,2012, Kozian et al. al.,Bioorg.Med.Chem.Lett.22:5239-5243,2012, Parrill,Expert.Opin.Ther.Pat.21:281-286,2011, Gupte et al.,Bioorg.Med.Chem.Lett.20:7525-7528,2010, Liliom et al. al., Biochim. Biophys. Acta 1761:1506-1514, 2006, and Durgam et al., Journal of Medicinal This is described in Chemistry 48:4919-4930, 2005.

[0269] According to one non-restrictive theory, protein kinase C inhibitors can increase DRA activity and also generate transepithelial pH gradients. For example, phorbol 12-myristate 13-acetate (PMA), an in vitro PKC agonist, increases apical membrane Cl - / HCO3 -It has been shown to directly inhibit activity (Gill et al., Physiology of the Gastrointestinal Tract, Chapter 67, 2012). Although not limited to one mechanism, inhibition of the appropriate PKC isoform is shown to be effective in Cl - / HCO3 - Conversely, this may increase activity and thereby inhibit phosphate uptake via the mechanism described herein.

[0270] Figures 21A-B (Mochly-Rosen et al., Nature Reviews Drug Discovery 11, 937-957, 2012) show that among other potential mechanisms of action, Cl - / HCO3 - Representative examples of subtype-selective PKC inhibitors that may increase activity are shown. Other possible DRA agonists include all-trans retinoic acid (ATRA) and related compounds, and more broadly, compounds that activate retinoic acid receptors (RAR) α, β, and γ, preferably RAR-β. RAR-β agonists are thought to induce DRA at the transcriptional level (total trans retinoic acid increases SLC26A3 (DRA) expression via HNF-1 (Priyamvada et al., DDW 2013, Orlando)). Another exemplary compound is S20787, which has been shown to stimulate the activity of human DRA expressed in oocytes (Chernova et al., J. Physiol., 549, 1, 3-19, 2003). Agonists of neuropeptide Y1 and Y2 receptors stimulate DRA activity within the Caco2 monolayer. NPY-stimulated DRA has been found to be membrane transport-independent and associated with the localization of DRA to lipid rafts (Saksena et al. Am. J. Physiol. Gastrointest Liver). Physiol. 299: G1334-G1343, 2010). Representative examples of NPY1 and NPY2 agonists include NPY, [Leu31,Pro34]-NPY, NPY 13-36, peptide YY(3-36), and GR 231118.

[0271] II. Compounds that are substantially unbiogenic A. Physical properties and performance characteristics of compounds that can be localized in the gastrointestinal tract Certain compounds described herein are designed to be substantially active or localized within the gastrointestinal lumen of a human or animal subject. The term “gastrointestinal lumen” is used herein interchangeably with the term “lumen” and refers to the space or cavity within the gastrointestinal tract (gastrointestinal (GI) tract, also referred to as the intestine) separated by the apical membrane of the gastrointestinal epithelial cells of the subject. In some embodiments, the compounds are not absorbed through the layer of epithelial cells of the gastrointestinal tract (also known as gastrointestinal (GI) epithelium). “Gastrointestinal mucosa” refers to the layer(s) of cells that separate the gastrointestinal lumen from the rest of the body and includes the mucosa of the stomach and intestines, such as the mucosa of the small intestine. As used herein, “gastrointestinal epithelial cells” or “intestinal epithelial cells” refers to any epithelial cells on the surface of the gastrointestinal mucosa facing the lumen of the gastrointestinal tract, including, for example, gastric epithelial cells, intestinal epithelial cells, colonic epithelial cells, etc.

[0272] Where used herein, “substantially bioavailable throughout the body” and / or “substantially impermeable” (and variations thereof) generally refer to situations in which a statistically significant amount of the compound, and in some embodiments essentially the entirety, remains in the gastrointestinal lumen. For example, according to one or more embodiments of this disclosure, preferably at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 99.5% of the compound remains in the gastrointestinal lumen. In such cases, localization to the gastrointestinal lumen means reducing the net movement of the compound across the layers of gastrointestinal epithelial cells, for example, by both transcellular and paracellular transport, and by active and / or passive transport. In such embodiments, the compound is prevented from net permeation through the layers of gastrointestinal epithelial cells, for example, through the apical membrane of the epithelial cells of the small intestine, in transcellular transport. In these embodiments, the compound is also prevented from net permeation through "tight junctions" during paracellular transport between gastrointestinal epithelial cells lining the lumen.

[0273] In this regard, it should be noted that in certain embodiments, the compound is essentially not absorbed at all by the gastrointestinal tract or gastrointestinal lumen. As used herein, the terms “substantially impermeable” or “substantially systemically unbiogenic” include embodiments in which no absorption, permeation, or systemic exposure of the compound is detected in amounts detectable by means of means commonly known in the art.

[0274] However, in this regard, in alternative embodiments, “substantially impermeable” or “substantially unbiodable throughout the body” may refer to a compound that results in or causes some limited absorption in the gastrointestinal tract, more specifically in the intestinal epithelium (e.g., some detectable amount of absorption, e.g., at least about 0.1%, 0.5%, 1% or more, and about 30%, 20%, 10%, or less than 5%, and the range of absorption is, for example, about 1% to 30%, or 5% to 20%); in other words, “substantially impermeable” or “substantially unbiodable throughout the body” may refer to a compound that exhibits some detectable permeability of less than about 20% of the administered compound to the epithelial layer of cells in the gastrointestinal tract (e.g., about 15%, about 10%, or even about 5%, 4%, 3%, or less than 2%, and e.g., about 0.5% or 1%), but is eliminated by the liver (i.e., hepatic excretion) and / or kidneys (i.e., renal excretion).

[0275] In this regard, it should be further noted that in certain embodiments, due to the substantial impermeability and / or substantial systemic non-biogenicity of the compounds of the present invention, about 50%, 60%, 70%, 80%, 90%, or more than 95% of the compounds of the present invention may be recoverable from feces over a period of 24, 36, 48, 60, 72, 84, or 96 hours after administration to a subject requiring it. In this regard, it should be understood that the recovered compounds may include the parent compound and its metabolites derived from the parent compound by any other modification, such as hydrolysis, conjugation, reduction, oxidation, N-alkylation, glucuronidation, acetylation, methylation, sulfation, phosphate chlorination, or any other modification that adds or removes atoms from the parent compound (metabolites are produced by the action of any enzyme or exposure to any physiological environment, including pH, temperature, pressure, or interaction with food when food is present in the digestive environment).

[0276] The fecal recovery rates of compounds and metabolites can be measured using standard methodologies. For example, the compound may be administered orally at a suitable dose (e.g., 10 mg / kg), and feces may be collected at predetermined times after administration (e.g., 24, 36, 48, 60, 72, 96 hours). The parent compound and metabolites may be extracted with organic solvents and quantitatively analyzed using mass spectrometry. To determine the recovery percentage in feces, a mass balance analysis of the parent compound and metabolites (including parent = M, metabolite 1 [M+16], and metabolite 2 [M+32]) may be used.

[0277] (i) Transparency In this regard, it should be noted that in various embodiments, the ability of the compound to be substantially unbiogenically unavailable throughout the body depends on the compound's charge, size, and / or other physicochemical parameters (e.g., polar surface area, the number of hydrogen bond donors and / or acceptors therein, the number of freely rotatable bonds, etc.). More specifically, it should be noted that the absorption characteristics of a compound can be selected by applying pharmacokinetic principles, for example, Lipinski's Law, also known as the "Rule of Five." While not a law but rather a set of guidelines, Lipinski shows that small molecule drugs having (i) molecular weight, (ii) several hydrogen bond donors, (iii) several hydrogen bond acceptors, and / or (iv) a water / octanol partition coefficient (Moriguchi log P) above a certain threshold generally do not exhibit significant systemic concentrations (i.e., are generally not absorbed to any significant degree). (See, for example, Lipinski et al., Advanced Drug Delivery Reviews, 46:3-26, 2001, which is incorporated herein by reference.) Thus, compounds that are substantially bioavailable throughout the body may be designed to have a molecular structure that exceeds one or more of Lipinski's thresholds. (See also Lipinski et al., Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings, Adv. Drug Delivery Reviews, 46:3-26, 2001, which is incorporated entirely herein by reference, and Lipinski, Drug-like Properties and the Causes of Poor Solubility and Poor Permeability, J. Pharm. & Toxicol. Methods, 44:235-249, 2000.

[0278] In some embodiments, for example, the substantially impermeable or substantially whole-body unbiogenic compounds of the Disclosure have the following properties: (i) (in the non-salt form of the Compound) about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1000 Da, about 1200 Da, about 1300 Da, about 1400 Da, about 1500 Da, about 1600 Da, about 1800 Da, about 2000 Da, about 2500 Da, about 3000 Da, about 4000 Da, about 5000 Da, about 7500 Da, about 1 (ii) a total of more than 0,000 Da MW; (ii) a total of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 NH and / or other possible hydrogen bond donors; (iii) a total of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 O atoms and / or N atoms and / or other possible hydrogen bond acceptors; (iv) about 10 5 It may be configured to feature one or more of the following: (i.e., logarithmic P greater than approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, etc.), or alternatively less than approximately 10 (i.e., logarithmic P less than 1, or even 0); and / or (v) the total number of rotatable bonds greater than approximately 5, approximately 10, or approximately 15, or greater. In certain embodiments, the compound has a logarithm P that is not 14, i.e., less than about 14, for example, a logarithm P in the range of about 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 8-9, 8-10, 8-11, 8-12, 8-13, 9-10, 9-11, 9-12, 9-13, 10-11, 10-12, 10-13, 11-12, 11-13, or 12-13.

[0279] In addition to the parameters mentioned above, the polar surface area of ​​a molecule (i.e., "PSA"), which can be characterized as a surface belonging to a polar atom, has also been shown to correlate deeply with passive transport across membranes and is therefore a descriptor that enables prediction of drug transport properties. This has been successfully applied to the prediction of intestinal absorption and Caco2 cell monolayer permeability. For details of exemplary Caco2 cell monolayer permeability tests, see, for example, the description of the Caco2 model provided in U.S. Patent No. 6,737,423, incorporated herein by reference, in particular the text describing the Caco2 model that may be applied, for example, to the evaluation or testing of the compounds of the present invention. PSA is Å 2 Expressed in square angstroms, it is calculated from a three-dimensional representation of the molecule. Faster calculation methods are also available using desktop computers and commercially available chemical graphics tool packages such as ChemDraw (see, for example, Ertl et al., Journal of Medicinal Chem. 43:3714-3717, 2000, whose entire content is incorporated herein by reference for all purposes of relevance and consistency). The term "topological PSA" (tPSA) has been coined for this faster calculation method. tPSA correlates well with human absorption data for common drugs (see Table 1 in Ertl et al., J.Med.Chem. 43:3714-3717, 2000). [Table 8] TIFF0007894693000023.tif37110

[0280] Therefore, in some embodiments, the compounds of the Disclosure are made substantially impermeable (e.g., cellular impermeable) or substantially unbiogenic (as defined elsewhere herein) at a distance of about 100 Å. 2 , about 116Å 2 Approximately 120 Å 2 , approximately 130 Å 2 , or approximately 140 Å 2 In some cases, it is extremely high, and in some instances, approximately 150 Å. 2, about 160Å 2 , about 170Å 2 , about 180Å 2 Approximately 190 Å 2 , about 200Å 2 , about 225Å 2 , about 250Å 2 , about 270Å 2 , about 300Å 2 , about 350Å 2 Approximately 400 Å 2 Approximately 450 Å 2 , about 500Å 2 , approximately 750 Å 2 , or even approximately 1000 Å 2 , or approximately 100-120 Å 2 , 100~130 Å 2 , 100~140 Å 2 , 100~150 Å 2 , 100~160 Å 2 , 100~170 Å 2 , 100~170 Å 2 , 100~190 Å 2 , 100~200 Å 2 , 100~225Å 2 , 100~250Å 2 , 100~300Å 2 , 100~400Å 2 , 100~500 Å 2 , 100~750 Å 2 , 100~1000 Å 2 , 116~120 Å 2 , 116~130 Å 2 , 116~140 Å 2 , 116~150 Å 2 , 116~160 Å 2 , 116~170 Å 2 , 116~170 Å 2 , 116~190 Å 2 , 116~200Å 2 , 116~225Å 2 , 116~250Å 2 , 116~300Å 2 , 116~400Å 2 , 116~500Å 2 , 116~750 Å 2 , 116~1000 Å2 、120~130Å 2 、120~140Å 2 、120~150Å 2 、120~160Å 2 、120~170Å 2 、120~170Å 2 、120~190Å 2 、120~200Å 2 、120~225Å 2 、120~250Å 2 、120~300Å 2 、120~400Å 2 、120~500Å 2 、120~750Å 2 、120~1000Å 2 、130~140Å 2 、130~150Å 2 、130~160Å 2 、130~170Å 2 、130~170Å 2 、130~190Å 2 、130~200Å 2 、130~225Å 2 、130~250Å 2 、130~300Å 2 、130~400Å 2 、130~500Å 2 、130~750Å 2 、130~1000Å 2 、140~150Å 2 、140~160Å 2 、140~170Å 2 、140~170Å 2 、140~190Å 2 、140~200Å 2 、140~225Å 2 、140~250Å 2 、140~300Å 2 、140~400Å 2 、140~500Å 2 、140~750Å 2 、140~1000Å 2 ,150~160Å 2 、150~170Å 2 、150~170Å2 , 150~190 Å 2 , 150~200Å 2 , 150~225Å 2 , or 150-250 Å 2 , 150~300Å 2 , 150~400Å 2 , 150~500 Å 2 , 150~750Å 2 , 150~1000 Å 2 , 200~250 Å 2 , 200~300Å 2 , 200~400Å 2 , 200~500Å 2 , 200~750Å 2 , 200~1000Å 2 , 250~250Å 2 , 250~300Å 2 , 250~400Å 2 , 20~500Å 2 , 250~750Å 2 , or 250~1000Å 2 It can be configured to show tPSA values ​​within a certain range.

[0281] Because Lipinski's "law" or the tPSA model has exceptions, the permeability properties of the compounds of this disclosure can be experimentally screened. Permeability coefficients can be determined by methods known to those skilled in the art, including, for example, by Caco-2 cell permeability assays and / or by using an artificial membrane as a model of gastrointestinal epithelial cells. To mimic the net permeability properties of the gastrointestinal mucosa, synthetic membranes impregnated with, for example, lecithin and / or dodecane can be used as a model of the gastrointestinal mucosa. Using this membrane, compartments containing the compounds of this disclosure can be separated from compartments where the rate of permeation is monitored. Similarly, parallel artificial membrane permeability assays (PAMPA) can be performed. Such in vitro measurements can reasonably demonstrate actual in vivo permeability (see Wohnsland et al., J.Med.Chem. 44:923-930, 2001, Schmidt et al., Millipore Corp. Application Note, 2002, n AN1725EN00, and n AN1728EN00, incorporated herein by reference).

[0282] Therefore, in some embodiments, the compounds used in the methods of the present disclosure, when measured using means known in the art (e.g., permeability experiments as described in Wohnsland et al., 2001 (above)), have a concentration of approximately 100 × 10⁻¹⁶. -6 Less than cm / s, or approximately 10 × 10 -6 Less than cm / s, or approximately 1 × 10⁻⁶ -6 Less than cm / s, or approximately 0.1 × 10⁻⁶ -6 Transmission coefficient P less than cm / s app It may have.

[0283] As previously described, in accordance with the Disclosure, compounds can be modified to prevent their net absorption through layers of intestinal epithelial cells so as to make them substantially systemically unbiogenic. In some specific embodiments, the compounds of the Disclosure include compounds linked, bound, or attached to non-absorbable portions which may be oligomeric, polymeric, hydrophobic, hydrophilic, and / or charged portions, thereby making the overall compound substantially impermeable or substantially systemically unbiogenic. In some preferred embodiments, the compounds are linked to multimer or polymeric portions or sites so that the resulting molecules are substantially impermeable or substantially systemically unbiogenic. The multimer or polymer portion or site may have a molecular weight of about 500 Daltons (Da), about 1000 Da, about 2500 Da, about 5000 Da, more than about 10,000 Da, or higher, and in particular may have a molecular weight in the range of about 1000 Daltons (Da) to about 500,000 Da, preferably in the range of about 5000 to about 200,000 Da, and more preferably may have a molecular weight high enough to essentially eliminate any net absorption of the compound through the layer of intestinal epithelial cells. In these or other specific embodiments, the compound is modified to substantially prevent its net absorption through the layer of intestinal epithelial cells.

[0284] (ii)C max and IC 50 or EC 50 In some embodiments, the substantially systemically unbiogenic compounds detailed herein, when administered (e.g., enterally) to a subject requiring them, either alone or in combination with one or more additional pharmaceutically active compounds or agents, reach an IC2 concentration that inhibits phosphate ion (Pi) transport or uptake of the compound. 50 C is approximately the same as or less than C max This indicates the maximum concentration detectable in serum, defined as C. max This is to inhibit Pi transport or acquisition, IC 50It is approximately or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower. In some embodiments, C max This is to inhibit Pi transport or acquisition, IC 50 These are approximately 0.01, 0.02, 0.03, 0.04, 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, and 0.9 times, respectively.

[0285] In certain embodiments, one or more of the substantially systemically unbiogenic compounds detailed herein, when administered (e.g., enterally) to a subject requiring it, (in a manner that inhibits Pi transport or renewal) C max and IC 50 These are expressed in the same units, approximately 0.01, 0.02, 0.03, 0.04, 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, 0.9, or 1.0, or less, or approximately 0.01 to 1.0, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0. C in the range of 0.7, 0.01~0.6, 0.01~0.5, 0.01~0.4, 0.01~0.3, 0.01~0.2, or 0.01~0.1, or approximately 0.1~1.0, 0.1~0.9, 0.1~0.8, 0.1~0.7, 0.1~0.6, 0.1~0.5, 0.1~0.4, 0.1~0.3, or 0.1~0.2 max :I C 50 It may have a ratio of .

[0286] In some embodiments, the substantially systemically unbiogenic compounds detailed herein, when administered (e.g., enterally) to a subject requiring them, either alone or in combination with one or more additional pharmaceutically active compounds or agents, increase the EC of the compounds to increase the fecal excretion of phosphates. 50 C is approximately the same as or less than C maxIt represents the maximum concentration detectable in serum as defined, and the amount excreted in feces increases by approximately or at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. For example, in some embodiments, C max EC increases the amount of phosphate excreted in the feces. 50 It is approximately or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower. In some embodiments, C max EC increases the amount of phosphate excreted in the feces. 50 These are approximately 0.01, 0.02, 0.03, 0.04, 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, and 0.9 times, respectively.

[0287] In some embodiments, one or more of the substantially systemically unbiogenic compounds detailed herein, when administered (e.g., enterally) to a subject requiring it, or measured in animal models or cell-based assays, increases fecal excretion of phosphate in amounts of approximately 10 μM, 9 μM, 8 μM, 7 μM, 7.5 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2.5 μM, 2 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM, or less than or equal to 0.01 μM EC, either alone or in combination with one or more additional pharmaceutically active compounds or agents, to a subject requiring it, or as measured in animal models or cell-based assays. 50 It may have, IC 50 For example, this ranges from approximately 0.01 μM to approximately 10 μM, or approximately 0.01 μM to approximately 7.5 μM, or approximately 0.01 μM to approximately 5 μM, or approximately 0.01 μM to approximately 2.5 μM, or approximately 0.01 μM to approximately 1.0 μM, or approximately 0.1 μM to approximately 10 μM, or approximately 0.1 μM to approximately 7.5 μM, or approximately 0.1 μM to approximately 5 μM, or approximately 0.1 μM to approximately 2.5 μM, or approximately 0.1 μM to approximately 1.0 μM, or approximately 0.5 μM to approximately 10 μM, or approximately 0.5 μM to approximately 7.5 μM, or approximately 0.5 μM to approximately 5 μM, or approximately 0.5 μM to approximately 2.5 μM, or approximately 0.5 μM to approximately 1.0 μM.

[0288] In certain embodiments, the substantially systemically unbiogenic compounds detailed herein, when administered (e.g., enterally) to a subject requiring them, either alone or in combination with one or more additional pharmaceutically active compounds or agents, reduce the urinary excretion of phosphates, and the EC of the compounds is such that it reduces the urinary excretion of the compounds. 50 C is approximately the same as or less than C max It represents the maximum concentration detectable in serum as defined, and the urinary excretion is reduced by approximately or at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. For example, in some embodiments, C max To reduce the urinary excretion of phosphates, EC 50 It is approximately or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower. In some embodiments, C max To reduce the urinary excretion of phosphates, EC 50 These are approximately 0.01, 0.02, 0.03, 0.04, 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, and 0.9 times, respectively.

[0289] In some embodiments, one or more of the substantially systemically unbiogenic compounds detailed herein, when administered (e.g., enterally) to a subject requiring it, or when measured in an animal model or cell-based assay, at concentrations of approximately 10 μM, 9 μM, 8 μM, 7 μM, 7.5 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2.5 μM, 2 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM, or less than or equal to 0.01 μM EC, to reduce urinary phosphate excretion, either alone or in combination with one or more additional pharmaceutically active compounds or agents, or when measured in an animal model or cell-based assay. 50 It may have, IC 50For example, this ranges from approximately 0.01 μM to approximately 10 μM, or approximately 0.01 μM to approximately 7.5 μM, or approximately 0.01 μM to approximately 5 μM, or approximately 0.01 μM to approximately 2.5 μM, or approximately 0.01 μM to approximately 1.0 μM, or approximately 0.1 μM to approximately 10 μM, or approximately 0.1 μM to approximately 7.5 μM, or approximately 0.1 μM to approximately 5 μM, or approximately 0.1 μM to approximately 2.5 μM, or approximately 0.1 μM to approximately 1.0 μM, or approximately 0.5 μM to approximately 10 μM, or approximately 0.5 μM to approximately 7.5 μM, or approximately 0.5 μM to approximately 5 μM, or approximately 0.5 μM to approximately 2.5 μM, or approximately 0.5 μM to approximately 1.0 μM.

[0290] In certain embodiments, one or more of the substantially systemically unbiogenic compounds detailed herein, when administered (e.g., enterally) to a subject in need, may cause (e.g., to increase fecal excretion of phosphate, or to decrease urinary excretion of phosphate) C max and EC 50 These are expressed in the same units, approximately 0.01, 0.02, 0.03, 0.04, 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, 0.9, or 1.0, or less, or approximately 0.01 to 1.0, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0. C in the range of 0.7, 0.01~0.6, 0.01~0.5, 0.01~0.4, 0.01~0.3, 0.01~0.2, or 0.01~0.1, or approximately 0.1~1.0, 0.1~0.9, 0.1~0.8, 0.1~0.7, 0.1~0.6, 0.1~0.5, 0.1~0.4, 0.1~0.3, or 0.1~0.2 max :EC 50 It may have a ratio of .

[0291] Furthermore, or alternatively, one or more of the substantially systemically unbiogenic compounds detailed herein, when administered (e.g., enterally) to a subject in need, either alone or in combination with one or more additional pharmaceutically active compounds or agents, may result in a C content of approximately 10 ng / ml, approximately 7.5 ng / ml, approximately 5 ng / ml, approximately 2.5 ng / ml, approximately 1 ng / ml, or approximately 0.5 ng / ml or less. max It may have C max For example, this ranges from approximately 1 ng / ml to approximately 10 ng / ml, or from approximately 2.5 ng / ml to approximately 7.5 ng / ml.

[0292] III. Pharmaceutical Compositions and Methods of Treatment For the purpose of administration, the compounds of the present invention may be administered to a patient or subject as raw chemical substances, or they may be formulated as pharmaceutical compositions. Pharmaceutical compositions of the present invention generally comprise the compounds of the present invention and pharmaceutically acceptable carriers, diluents, or excipients. The compounds are present in the composition in amounts effective for treating a particular disease or condition of interest, as described herein, and preferably have toxicity acceptable to the subject. The activity of the compound(s) may be determined by those skilled in the art, for example, as described herein and in the following examples. Appropriate concentrations and dosages may be readily determined by those skilled in the art.

[0293] The compounds or compositions of the present invention may be used in methods to treat essentially any disease or other condition of a subject who would benefit from inhibition of phosphate uptake in the gastrointestinal tract.

[0294] For example, and this is an explanation rather than an exemplification, renal impairment reduces the production and activity of 1-α-hydroxylase in the kidneys, leading to a decrease in 1,25-dihydroxyvitamin D. Decreased vitamin D levels limit gastrointestinal calcium absorption, leading to a decrease in serum calcium levels. The combination of lower 1,25-dihydroxyvitamin D and lower serum calcium levels synergistically stimulates parathyroid tissue to produce and secrete PTH. Nephron loss also impairs Pi excretion, but serum P levels are actively defended by the action of PTH and FGF-23, and by higher serum P levels, which significantly improve urinary PO4 excretion. However, the tubular action of PTH and FGF-23 cannot maintain serum P levels in the face of continuous nephron loss. When renal failure progresses to a loss of approximately 40-50% of renal function, the reduction in the amount of functioning renal tissue prevents the excretion of the entire amount of ingested phosphate necessary to maintain homeostasis. As a result, hyperphosphatemia develops. Furthermore, elevated serum phosphorus levels interfere with renal 1-α-hydroxylase activity, further suppressing activated vitamin D levels, which in turn stimulates PTH and leads to secondary hyperparathyroidism (sHPTH).

[0295] However, phosphorus imbalance does not necessarily correspond to hyperphosphatemia. Rather, the majority of CKD patients who are not yet on dialysis are orthophosphoric, but their phosphorus balance is positive as excess phosphorus is discarded into the vascular structure in the form of ectopic calcification, such as intimal localized vascular calcification. Clinically, patients with CKD have elevated levels of FGF-23 which are significantly associated with worsening renal function and decreased calcitriol levels, and it is hypothesized that FGF-23 synthesis is induced by the presence of excess phosphorus in the body, leading to renal failure.

[0296] Furthermore, an unrecognized effect on cardiovascular disease is postprandial phosphatemia, i.e., serum phosphorus deviation secondary to food intake. Moreover, the acute effects of phosphorus loading on endothelial function in vitro and in vivo have been investigated. Exposure of bovine aortic endothelial cells to phosphorus loading increased the production of reactive oxygen species and decreased nitric oxide, a known vasodilator. In the aforementioned acute phosphorus loading studies in healthy volunteers, blood flow-mediated dilation was found to be inversely correlated with postprandial serum phosphorus (see, for example, Shuto et al., J.Am.Soc.Nephrol.20:1504-12, 2009).

[0297] Therefore, in certain embodiments, the compounds or compositions of the present invention can be used in the following ways: a method for treating hyperphosphatemia, optionally postprandial hyperphosphatemia; a method for treating renal diseases (e.g., chronic kidney disease (CKD), end-stage renal disease (ESRD)); a method for reducing serum creatinine levels; a method for treating proteinuria; a method for delaying the time to renal replacement therapy (RRT), such as dialysis; a method for reducing FGF23 levels; a method for reducing the hyperphosphatemic effect of active vitamin D; a method for alleviating hyperparathyroidism, such as secondary hyperparathyroidism; and blood The method may be used in a manner selected from one or more of the following: a method for reducing parathyroid hormone (PTH or iPTH); a method for improving endothelial dysfunction optionally induced by postprandial serum phosphorus; a method for reducing vascular calcification or reducing intimal localized vascular calcification; a method for reducing urinary phosphorus; a method for normalizing serum phosphorus levels; a method for reducing phosphorus load in elderly patients; a method for reducing dietary phosphate uptake; a method for reducing postprandial calcium absorption; a method for reducing renal hypertrophy; and a method for reducing cardiac hypertrophy. In certain embodiments, subjects requiring phosphate reduction have one or more of the aforementioned conditions. In some embodiments, the method includes selecting or identifying such subjects before treatment, optionally based on one or more clinical or diagnostic parameters described herein.

[0298] Hyperphosphatemia refers to a condition in which elevated levels of phosphate are present in the blood. The average serum phosphorus level in human adults is typically in the range of approximately 2.5–4.5 mg / dL (approximately 0.81–1.45 mmol / L). Levels are often about 50% higher in infants and about 30% higher in children, often due to the effects of growth hormone. Therefore, certain methods involve treating adult human patients with hyperphosphatemia, who have a serum phosphorus level of approximately or at least about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5 mg / dL. In some embodiments, the treatment reduces the serum phosphorus concentration or level of a subject with hyperphosphatemia to approximately 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, or 100% (normalization) of normal serum phosphorus levels (e.g., 2.5–4.5 mg / dL or 0.81–1.45 mmol / L for adults). In some embodiments, the treatment regimen results in and / or includes monitoring phosphate levels so that they remain within the range of approximately 2.5–4.5 mg / dL (approximately 0.81–1.45 mmol / L). In some embodiments, the treatment shifts the external phosphorus balance toward net excretion by increasing net phosphite excretion by about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or more, compared to the untreated state, with or without reduction of serum phosphorus concentration or levels.

[0299] Methods for treating pediatric or adolescent human patients having a serum phosphorus mass of approximately or at least approximately 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 mg / dL. As described herein, in these and related embodiments, administration of the compounds or compositions described herein may reduce the serum phosphorus mass of the subject by approximately or at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more.

[0300] A particular embodiment relates to a method for treating chronic kidney disease (CKD), a condition characterized by progressive loss of renal function. Common causes of CKD include diabetes mellitus, hypertension, and glomerulonephritis. Thus, a particular method involves treating a subject having CKD, who optionally also has one or more of the aforementioned conditions.

[0301] In some aspects, subjects, regardless of whether they exhibited similar renal impairment, consumed 60 mL / min / 1.73 m³ for approximately 3 months. 2 If a glomerular filtration rate (GFR) is less than 10, the patient is classified as having chronic kidney disease (CKD). Therefore, certain methods are used at approximately 60, 55, 50, 45, 40, 30, 35, 20, 25, 20, 15, or 10 mL / min / 1.73 m 2 This includes treating subjects having a GFR of about or less (e.g., initial GFR before treatment). In certain embodiments, administration of the compounds or compositions described herein may result in an increase in GFR of about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more.

[0302] CKD is most often characterized according to the disease stage: Stage 1, Stage 2, Stage 3, Stage 4, and Stage 5. Stage 1 CKD is characterized by renal impairment and a blood flow rate of approximately 90 mL / min / 1.73 m³.2 This includes subjects with a normal or relatively high GFR, or exceeding that. Stage 2 CKD is characterized by renal impairment and approximately 60-89 mL / min / 1.73m² 2 This includes subjects with GFR and [specific characteristics]. Stage 3 CKD is characterized by renal impairment and approximately 30-59 mL / min / 1.73m 2 This includes subjects with GFR and [specific characteristics]. Stage 4 CKD is characterized by renal impairment and approximately 15-29 mL / min / 1.73m 2 This includes subjects with GFR and [specific characteristics]. Stage 5 CKD is defined as confirmed renal failure and approximately 15 mL / min / 1.73 m 2 This includes subjects having a GFR of less than 500. Stage 5 CKD is also referred to as end-stage renal disease (ESRD). Thus, in certain ways, subjects have one or more of the following clinical characteristics (e.g., defined GFR, renal impairment): stage 1, 2, 3, 4, or 5 CKD. In some embodiments, subjects have one or more of the following clinical characteristics: ESRD and one or more of the following clinical characteristics, as described herein and known in the art.

[0303] CKD can be characterized according to the affected area of ​​the kidney. For example, in certain embodiments, CKD includes vascular CKDs, including large vascular diseases such as bilateral renal artery stenosis, as well as small vascular diseases such as ischemic nephropathy, hemolytic uremic syndrome, and vasculitis. In certain embodiments, CKD includes glomerular CKDs, including primary glomerular diseases such as focal segmental glomerulosclerosis and IgA nephritis, as well as secondary glomerular diseases such as diabetic nephropathy and lupus nephritis. Tubulointerstitial CKDs are also included, including polycystic kidney disease, drug and toxin-induced chronic tubulointerstitial nephritis, and reflux nephropathy. Thus, certain subjects undergoing treatment for CKD may have one or more of the aforementioned CKD-related characteristics.

[0304] Certain embodiments relate to methods for treating subjects with renal impairment or one or more symptoms / clinical signs of renal impairment. Examples of renal impairment (e.g., renal impairment associated with chronic kidney disease) and its associated symptoms include pathological abnormalities and markers of damage, including abnormalities identified by blood tests (e.g., high blood or serum creatinine levels, creatinine clearance), abnormalities identified by urinalysis (e.g., proteinuria), and / or abnormalities identified by imaging studies.

[0305] Creatinine is a breakdown product of creatine phosphate in muscles and provides an easily measurable and useful indicator of kidney health. The normal range for healthy humans is approximately 0.5–1.0 mg / dL (approximately 45–90 μmol / L) for women and approximately 0.7–1.2 mg / dL (approximately 60–110 μmol / L) for men. Therefore, certain subjects for treatment according to the methods described herein may have blood or serum creatine levels (e.g., initially, before treatment) of approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / dL or greater. In these and related embodiments, administration of the compounds or compositions described herein may reduce the overall blood or serum creatinine level of a subject by about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more.

[0306] Creatinine clearance rate (C CrCreatinine clearance (or CrCl) refers to the volume of plasma per unit time from which creatinine is cleared, and is measured by comparing the level of creatinine in the blood to that in the urine over a period of time (e.g., 24 hours). Creatine clearance is often measured as milliliters / minute (ml / min) or as a function of body weight (ml / min / kg). Depending on the test performed, normal values ​​range from approximately 97 to 137 ml / min for men and approximately 88 to 128 ml / min for women. Reduced creatinine clearance provides a useful sign of renal impairment. Therefore, certain male subjects for treatment according to the methods described herein are approximately 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, or less than or equal to C Cr Certain female subjects for treatment according to the methods described herein may have (for example, initially, before treatment). Cr It may have (for example, initially, before treatment). In some embodiments, administration of the compound or composition described herein is to target C Cr It can be maintained or increased by approximately or at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more.

[0307] Proteinuria refers to a condition characterized by an excess of protein in the urine. This is associated with various pathological conditions, including kidney damage. Proteinuria is often characterized by a urinary protein / creatinine ratio greater than approximately 45 mg / millimole, or, in certain tests, an albumin / creatine ratio greater than approximately 30 mg / millimole. Certain subjects for treatment according to the methods provided herein, including subjects having a urinary protein / creatinine ratio of approximately 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 mg / millimole or greater, and / or a urinary albumin / creatinine ratio of approximately 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 mg / millimole or greater, have proteinuria (for example, before treatment) alone or in combination with CKD or other renal impairment. In these and related embodiments, administration of the compounds or compositions described herein can treat proteinuria, for example, by reducing the urinary protein / creatinine ratio and / or urinary albumin / creatinine ratio by about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more.

[0308] CKD is associated with a variety of clinical symptoms. Examples include hypertension, urinary retention, hyperkalemia, anemia, hyperphosphatemia, hypocalcemia, metabolic acidosis, and atherosclerosis. Therefore, in certain ways, subjects with CKD may have or be at risk of having one or more of the aforementioned clinical symptoms. In certain embodiments, subjects with CKD may have or be at risk of having hyperphosphatemia, as described herein.

[0309] Renal replacement therapy (RRT) relates to a range of life-sustaining treatments for renal failure, including those initiated in the later stages of chronic kidney disease (CKD) and advanced renal disease (ESRD). Examples of RRT include dialysis, hemodialysis, hemofiltration, and kidney transplantation. In certain embodiments, subjects for treatment according to the methods provided herein are either about to receive, are receiving, or have received one or more types of RRT. In some embodiments, the subject has not yet received RRT, and administration of the compounds described herein delays the time to the initiation of RRT (for example, compared to an untreated state) by about or at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or about or at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or about or at least about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, or longer.

[0310] Fibroblast growth factor 23 (FGF23) regulates the metabolism of phosphorus and vitamin D. It also promotes phosphateuria and reduces calcitriol production. Elevated FGF23 levels are associated with mortality, left ventricular hypertrophy (or left ventricular myocardial mass index), myocardial performance, endothelial damage, and progression of chronic kidney disease (CKD). In fact, FGF23 levels progressively increase in early CKD, possibly as a physiological adaptation to maintain normal serum phosphorus levels or a normal phosphorus balance. FGF23 levels may also directly contribute to tissue damage of the heart, blood vessels, and kidneys. Therefore, certain embodiments relate to the treatment of subjects with elevated FGF23 levels in their blood or serum, including subjects with CKD and subjects undergoing dialysis / hemodialysis (see, e.g., Kirkpantur et al., Nephrol Dial Transplant. 26:1346-54, 2011). In some embodiments, administration of the compounds or compositions described herein reduces the logarithm of FGF23 levels in blood or serum by about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more.

[0311] Vitamin D, in particular, stimulates the absorption of phosphate ions in the small intestine. Therefore, excessive levels or activity of vitamin D can lead to elevated phosphate levels and hyperphosphatemia. Accordingly, certain embodiments relate to methods for reducing the hyperphosphatemic effects of active vitamin D in subjects having elevated levels or activity of vitamin D. In some embodiments, the subjects have vitamin D toxicity resulting from excessive intake of vitamin D.

[0312] Hyperparathyroidism is a disorder in which the parathyroid glands produce excessive amounts of parathyroid hormone (PTH). Secondary hyperparathyroidism is characterized by excessive secretion of PTH in response to hypocalcemia and associated enlargement of the parathyroid glands. Generally, chronic kidney disease (CKD) is the most common cause of secondary hyperparathyroidism because the kidneys are unable to convert sufficient vitamin D to its active form and excrete sufficient phosphate. Insoluble calcium phosphate is formed in the body, and thus calcium is removed from circulation, causing hypocalcemia. The parathyroid glands then further increase PTH secretion in an attempt to raise serum calcium levels. Therefore, certain subjects for treatment according to the methods provided herein may present (e.g., initially, before treatment) with hyperparathyroidism and / or elevated PTH levels, optionally in combination with CKD, hyperphosphatemia, hypocalcemia, or other conditions or symptoms described herein. In some embodiments, administration of the compounds or compositions described herein may reduce hyperparathyroidism, including secondary hyperparathyroidism, in subjects requiring it. In some embodiments, administration of the compounds or compositions described herein may reduce PTH levels by about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more, for example, by reducing serum phosphorus levels and associated insoluble calcium phosphate formation, increasing available calcium, and thereby reducing PTH production that leads to hypocalcemia.

[0313] In certain embodiments, administration of the compounds described herein may produce multiple therapeutic effects in subjects with CKD. In some cases, administration of the compounds reduces FGF23 levels and serum parathyroid hormone (PTH) levels by about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more, compared to the untreated state; reduces blood pressure; and reduces proteinuria by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more, compared to the untreated state.

[0314] In certain embodiments, administration of the compounds described herein may produce multiple therapeutic effects in subjects having ESRD (or stage 5 CKD). In certain cases, administration of the compounds reduces serum phosphorus concentration or level by about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more, compared to the untreated state.

[0315] Hyperphosphatemia can cause endothelial damage independently of vascular calcification in both healthy and renal disease-affected subjects (see, e.g., Di Marco et al., Kidney International. 83:213-222, 2013). Restriction of dietary phosphates or management of serum phosphate levels with phosphate binders may prevent the development of cardiovascular disease in such subjects. Studies have also shown that restriction of dietary phosphates may improve aortic endothelial damage (e.g., in CKD with hyperphosphatemia) by increasing endothelial nitric oxide synthase and Akt activator phosphate chloride (see, e.g., Van et al., J Clin Biochem Nutr. 51:27-32, 2012). Certain subjects for treatment according to the methods provided herein may optionally have or be at risk of having endothelial damage in combination with hyperphosphatemia, renal disease, or any other condition described herein. By reducing postprandial or dietary phosphate uptake, either alone or in combination with dietary phosphate restriction, administration of the compounds or compositions described herein may reduce the risk of developing endothelial damage or improve existing endothelial damage, including postprandial serum phosphate-induced endothelial damage.

[0316] Hyperphosphatemia is a major inducer of vascular calcification (see Giachelli, Kidney Int. 75:890-897, 2009). Calcium phosphate deposition, primarily in the form of apatite, is characteristic of vascular calcification and can occur in blood vessels, myocardium, and heart valves. Along with the passive accumulation of calcium phosphate in extraskeletal tissues, inorganic phosphate can also induce arterial calcification directly through the "ossification" of the media within vascular structures. Furthermore, vascular smooth muscle cells respond to elevated phosphate levels by undergoing osteochondrogenic phenotypic changes and mineralizing their extracellular matrix through mechanisms requiring sodium-dependent phosphate cotransporters.

[0317] Intimal calcification is typically found in atherosclerotic lesions. Medial calcification is commonly observed in age-related atherosclerosis and diabetes mellitus, and is the primary form of calcification observed in ESRD. Indeed, extensive calcification of arterial walls and soft tissues is a common feature in patients with CKD, including those with ESRD. In the valves, calcification is a distinct feature of aortic stenosis and occurs in both the lobules and annulus, primarily at sites of inflammation and mechanical load. These mechanical changes are associated with increased arterial pulse wave velocity and pulse pressure, leading to impaired arterial distensibility, increased afterload supporting left ventricular hypertrophy, and coronary perfusion failure (see Guerin et al., Circulation. 103:987-992, 2001). Thus, both intimal and medial calcification can contribute to cardiovascular disease-related morbidity and mortality, and are likely to be the primary contributors to the significant increase in cardiovascular mortality observed in patients with CKD and ESRD. Therefore, controlling serum phosphorus can reduce the formation of calcium / phosphate products, thereby reducing vascular calcification. Accordingly, certain subjects for treatment according to the methods provided herein may have, or be at risk of having, vascular calcification, including intimal and / or internal calcification, optionally combined with any of the following: hyperphosphatemia, CKD, and ESRD. In some embodiments, administration of the compounds or compositions described herein reduces the risk of developing vascular calcification in subjects requiring it, or reduces its formation or level. In certain embodiments, administration of the compounds or compositions described herein can reduce vascular calcification by, for example, about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%, or more, compared to, for example, an untreated state.

[0318] Elderly patients may be particularly susceptible to the effects of increased phosphate levels. For example, dietary and genetic engineering studies provide in vivo evidence that phosphate toxicity accelerates the aging process, suggesting a novel role of phosphate in mammalian aging (see, e.g., Ohnishi and Razzaque, FASEB J.24:3562-71, 2010). These studies show that excess phosphate is associated with many signs of premature aging, including kyphosis, noncoordinated movement, hypogonadism, infertility, skeletal muscle wasting, emphysema, and osteopenia, as well as systemic atrophy of the skin, intestines, thymus, and spleen. Certain embodiments relate to reducing the phosphate load in elderly patients, for example, by administering the compounds described herein to elderly patients to reduce any one or more signs of premature aging. In some cases, the elderly patients are approximately or at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or older.

[0319] Hypertrophy refers to an increase in the volume of an organ or tissue resulting from the enlargement of its constituent cells. Hyperphosphatemia is associated with myocardial hypertrophy, including left ventricular hypertrophy (see Neves et al., Kidney Int. 66:2237-44, 2004, and Achinger and Ayus, Am Soc Nephrol. 17(12 Suppl 3):S255-61, 2006), and compensatory renal hypertrophy, including glomerular hypertrophy, the latter of which is commonly observed in CKD. Certain subjects for treatment according to the methods provided herein may have myocardial hypertrophy, renal hypertrophy, or both, alone or in combination with CKD or renal impairment (e.g., initially, before treatment). In some embodiments, administration of the compounds described herein may reduce myocardial hypertrophy and / or renal hypertrophy by about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more compared to the untreated state.

[0320] The administration of the compounds of the present invention or pharmaceutically acceptable salts thereof, in their pure form or in suitable pharmaceutical compositions, may be carried out by any acceptable mode of administration of a drug providing similar utility. Pharmaceutical compositions of the present invention may be prepared by combining the compounds of the present invention with a pharmaceutically acceptable carrier, diluent, or excipient, and may be formulated into preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, spheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and nasal administration. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, substernal, or infusion techniques. Pharmaceutical compositions of the present invention are formulated so that the active ingredients contained therein are bioavailable upon administration to a patient. The composition administered to the subject or patient may take the form of one or more drug dose units; for example, a tablet may be a single drug dose unit, and a container of the compound of the present invention in aerosol form may hold multiple drug dose units. Practical methods for preparing such dosage forms are known or will become apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In any event, the administered composition contains a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof for the treatment of the target disease or condition according to the teachings of the present invention.

[0321] The pharmaceutical composition of the present invention may be in solid or liquid form. In one embodiment, the carrier(s) are fine particles so that the composition may be in the form of, for example, tablets or powder. The carrier(s) may be liquid, and the composition may be, for example, an oral syrup, an injection solution, or an aerosol useful for, for example, inhalation administration.

[0322] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included within the range of forms considered as solid or liquid in this specification.

[0323] As solid compositions for oral administration, pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, or similar forms. Such solid compositions typically contain one or more inert diluents or food carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavoring; and colorants.

[0324] If the pharmaceutical composition is in the form of a capsule, for example a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol or oil in addition to the materials of the types described above.

[0325] Pharmaceutical compositions may be in the form of liquids, such as elixirs, syrups, solutions, emulsions, or suspensions. Liquids may, as two examples, be for oral administration or for delivery by infusion. For oral administration, preferred compositions contain, in addition to the compound, one or more of the following: sweeteners, preservatives, dyes / colorants, and flavor enhancers. Compositions intended for infusion may contain one or more of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0326] The liquid pharmaceutical compositions of the present invention, whether they are solutions, suspensions, or other similar forms, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic saline, fixing oils, such as synthetic mono or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as a solvent or suspension medium; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting osmotic pressure, such as sodium chloride or dextrose. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Physiological saline is a preferred adjuvant. The injectable pharmaceutical compositions are preferably sterile.

[0327] A liquid pharmaceutical composition of the present invention intended for either parenteral or oral administration should contain an amount of the compound of the present invention such that a suitable drug dose is obtained.

[0328] The pharmaceutical compositions of the present invention may be intended for topical administration, in which case the carrier may preferably comprise a solution, emulsion, ointment, or gel base. The base may comprise, for example, one or more of the following: diluents such as mineral oil, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, as well as emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. When intended for transdermal administration, the composition may comprise a transdermal patch or an ion electrophoresis apparatus.

[0329] The pharmaceutical compositions of the present invention may be intended for rectal administration, for example, in the form of suppositories that dissolve in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Examples of such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0330] The pharmaceutical compositions of the present invention may include various materials that modify the physical form of a solid or liquid drug dose unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and can be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0331] The pharmaceutical compositions of the present invention, in solid or liquid form, may contain agents that bind to the compounds of the present invention and thereby assist in the delivery of the compounds. Suitable agents that can act with this ability include monoclonal or polyclonal antibodies, proteins, or liposomes.

[0332] The pharmaceutical compositions of the present invention may consist of drug dose units that can be administered as aerosols. The term "aerosol" is used to mean a variety of systems, ranging from colloidal systems to systems consisting of pressurized packages. Delivery may be by liquefaction or compressed gas, or by a suitable pump system for dispensing the active ingredient. Aerosols of the compounds of the present invention may be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient(s). Aerosol delivery may include necessary containers, activators, valves, subcontainers, etc., which together form a kit. Those skilled in the art can determine a preferred aerosol without excessive experimentation.

[0333] The pharmaceutical compositions of the present invention can be prepared by methodologies well known in the pharmaceutical art. For example, a pharmaceutical composition intended for administration by infusion can be prepared by combining the compounds of the present invention with sterile distilled water to form a solution. A surfactant may be added to promote the formation of a homogeneous solution or suspension. The surfactant is a compound that interacts non-covalently with the compounds of the present invention to promote the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0334] The compounds of the present invention or their pharmaceutically acceptable salts are administered in therapeutically effective doses, which vary depending on a variety of factors, including the activity of the particular compound used; the metabolic stability and duration of action of the compound; the patient's age, weight, overall health, sex, and diet; the mode and timing of administration; the rate of excretion; drug combinations; the severity of a particular disorder or condition; and the subject receiving treatment.

[0335] In certain embodiments, typical drug doses of a substantially impermeable or substantially systemically unbiogenic compound may be about 0.2 mg to about 2 g per day, or about 1 mg to about 1 g per day, or about 5 mg to about 500 mg per day, or about 10 mg to about 250 mg per day, which are administered to subjects in need of treatment.

[0336] The frequency of administration of the compounds and compositions described herein may vary, such as once daily (QD), twice daily (BID), or three times daily (TID), and the precise frequency of administration will vary depending on, for example, the patient's condition and the dosage.

[0337] The compounds of the present invention or their pharmaceutically acceptable derivatives may be administered simultaneously with, before, or after the administration of one or more other therapeutic agents or bioactive agents, dietary supplements, or any combination thereof. Such combination therapy includes the administration of a single pharmaceutically active formulation containing the compounds of the present invention and one or more additional activators, as well as the administration of the compounds of the present invention and each activator in its own separate pharmaceutically active formulation. For example, the compounds of the present invention and other activators may be administered together to a patient in a single oral administration composition such as a tablet or capsule, or each agent may be administered in a separate oral administration formulation. When separate administration formulations are used, the compounds of the present invention and one or more additional activators may be administered essentially simultaneously, i.e., concurrently, or separately at staggered times, i.e., sequentially, and combination therapy is understood to include all of these regimens.

[0338] For example, in certain embodiments, additional bioactive agents included in the pharmaceutical composition (or method) of the present invention are selected from, for example, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), active vitamin D (calcitriol), and active vitamin D analogs (e.g., doxelcalciferol, paricalcitol).

[0339] In other specific embodiments, additional bioactive agents included in the pharmaceutical composition (or method) of the present invention are sevelamers (e.g., Renvela® (sevelamer carbonate), Renagel® (sevelamer hydrochloride)), lanthanum carbonate (e.g., Fosrenol®), calcium carbonate (e.g., Calcichew®, Titralac®), calcium acetate (e.g., PhosLo®, Phosex®), calcium acetate / magnesium carbonate (e.g., Renepho®, OsvaRen®), MCI-196, ferric citrate (e.g., Zerenex®), magnesium iron carbonate hydroxide (e.g., Fermagate®), aluminum hydroxide (e.g., Alucaps®, Basaljel®), phosphate binders such as APS1585, SBR-759, and PA-21.

[0340] In some embodiments, additional bioactive agents are inhibitors of the enteric sodium-dependent phosphate transporter (NaPi2b inhibitors). Examples of NaPi2b inhibitors can be found, for example, in International Patent Applications PCT / US2011 / 043267, PCT / US2011 / 043261, PCT / US2011 / 043232, PCT / US2011 / 043266, and PCT / US2011 / 043263, and U.S. Patent No. 8,134,015, each incorporated herein by reference in its entirety.

[0341] In certain embodiments, the additional bioactive agent is niacin or nicotinamide.

[0342] In some embodiments, the subjects have CKD or are undergoing treatment for it, and the additional bioactive agents are compounds used in the treatment or management of CKD. Examples of such compounds include antihypertensive agents, e.g., ACE inhibitors, antiogensin II receptor blockers, beta-blockers, calcium channel blockers, direct renin inhibitors, diuretics, and vasodilators; drugs for treating symptoms and complications of CKD, e.g., erythropoietin therapy and / or iron supplementation therapy for anemia, electrolytes for electrolyte imbalance, diuretics, ACE inhibitors, and antiogensin II receptor blockers, advanced glycation end product inhibitors (e.g., aminoguanidine, pyridoxamine), and vitamin D; lipid-lowering agents, e.g., HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase inhibitors or statins (e.g., atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), etc.

[0343] In this specification, it is understood that combinations of substituents and / or variables in expressed formulas are acceptable only if such contributions result in a stable or reasonably stable compound.

[0344] Those skilled in the art will understand that in the processes described herein, the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, and benzyl. Suitable protecting groups for amino, amidino, and guanidino groups include t-butoxycarbonyl and benzyloxycarbonyl. Suitable protecting groups for mercapto groups include -C(O)-R” (where R is alkyl, aryl, or arylalkyl), p-methoxybenzyl, and trityl. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups are known to those skilled in the art and can be added or removed according to the standard techniques described herein. The use of protecting groups is described in detail in Green, TWand PGMWutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As those skilled in the art will understand, the protecting group may be a polymer resin such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

[0345] While such protected derivatives of the compounds of the present invention do not possess such pharmacological activity, it will be understood by those skilled in the art that they may be administered to mammals and subsequently metabolized in the body to form pharmacologically active compounds of the present invention. Therefore, such derivatives may be described as "prodrugs." All prodrugs of the compounds of the present invention fall within the scope of the present invention.

[0346] Furthermore, all compounds of the present invention, existing in free base or acid form, can be converted to pharmaceutically acceptable salts thereof by treatment with appropriate inorganic or organic bases or acids using methods known to those skilled in the art. Salts of the compounds of the present invention can be converted to their free base or acid form by standard techniques.

[0347] IV. Drug Discovery This also includes methods for drug discovery of compounds that can inhibit phosphate uptake in the gastrointestinal tract. Specific embodiments include in vitro drug screening methods using cell cultures, such as intestinal cell cultures, or cell lines, including mammalian cell lines.

[0348] Therefore, a particular embodiment relates to a method for screening phosphate uptake inhibitors, comprising: culturing cells; contacting the cultured cells with a test compound; and measuring one or more of the following: pH at the apical surface of the cells, intracellular pH of the cells, bicarbonate secretion by the cells, acid secretion by the cells, water absorption, and / or phosphate uptake by the cells.

[0349] The following steps also include identifying a test compound as a phosphate uptake inhibitor if one or more of the following occur: the pH at the apical surface of the cell increases compared to the control; the intracellular pH of the cell decreases compared to the control; bicarbonate secretion by the cell increases compared to the control; acid secretion by the cell decreases compared to the control; water absorption decreases compared to the control; and / or phosphate uptake by the cell decreases compared to the control. In some embodiments, the increase or decrease is statistically significant. The terms “increase” and “decrease” and “statistically significant” are described elsewhere in this specification. The control may include a compound that does not contain the compound (e.g., vehicle only) or a compound known not to possess any of the above activities. The control may also include a predetermined reference value.

[0350] In certain embodiments, the cells are intestinal cells. Non-limiting examples of intestinal cell cultures include intestinal cell monolayers, enteroids, and intestinal cell organoids. Intestinal cell monolayers can be prepared according to routine techniques in the art. Non-limiting examples of intestinal cell monolayers include cell lines such as Caco-2, HCT-8, and T84 cell lines (see, e.g., Watson et al., Am J Physiol Cell Physiol. 281:C388-9, 2001, Shah et al., Biotechnol Prog. 22:186-9, 2006), as well as neonatal pig jejunal IPEC-J2 cell monolayers (see, e.g., Chapman et al., Pediatr Res. 72:576-82, 2012).

[0351] The term “enteroid” includes intestinal cell cultures obtained from intestinal crypts of fragments of intestinal tissue that optionally maintain structural integrity (e.g., three-dimensional structure of intestinal epithelium) and cell type of intestinal tissue, and replicate the genotype and phenotypic profile of primary intestinal tissue. Enteroid cell cultures can be prepared according to techniques known in the art. (See, for example, U.S. Patent Application No. 2010 / 0047853, International Publication No. 2010 / 090513, U.S. Patent Application No. 2012 / 0196312, and International Publication No. 2012 / 168930).

[0352] The terms “organoid” or “intestinal organoid” refer to intestinal cell cultures primarily made from precursor cells such as isolated embryonic stem cells, endodermal cells, or other pluripotent stem cells. Organoids can be prepared, for example, by stepwise differentiation (see, e.g., International Publication 2011 / 140441) into complex three-dimensional intestinal tissue (including intestinal tissue that may contain polarized columnar epithelium surrounded by mesenchyme containing a smooth muscle-like layer). In some embodiments, the epithelium is patterned into crypt-like proliferative zones and villous structures, which contain most, if not all, of the major functional cell types of the intestine. In some embodiments, the precursor cells are first selected or enriched for the expression of markers such as LGR5 and / or LGR6.

[0353] Cultures containing whole-thickness intestinal preparations (see, e.g., Binder et al., Am J Physiol. 225:1232-1239, 1973), as well as those prepared by pharmacological treatment and "seromuscular stripping" to minimize the influence of the intrinsic neuromuscular system (see, e.g., Clarke, Am. J. Physiol. Gastrointestin. Liver Physiol. 296:G1151-66, 2009). Seromuscular stripping removes the serosal (visceral peritoneum) and longitudinal / circular muscle layers of the intestinal wall, leaving only the underlying submucosal elements, muscle remnants, and epithelium. These cultures can be particularly useful when using a Ussing chamber.

[0354] In certain embodiments, a Ussing chamber may be used. The Ussing chamber provides a physiological system for measuring the transport of ions, nutrients, and drugs across various epithelial tissues, such as intestinal tissue (see, e.g., Clarke et al., above). For example, some methods may use pH stat techniques for measuring transepithelial bicarbonate secretion and / or isotope flux methods for measuring net secretion or absorption of substrates. In certain embodiments, the Ussing chamber is adapted for use with mouse or rat intestines, including whole-thickness intestinal preparations and those prepared by stripping of muscular serosa tissue (see, e.g., Clarke et al., above).

[0355] Certain screening methods may utilize various non-intestinal cell lines, including mammalian cell lines. Exemplary mammalian cell lines include human fetal kidney cell lines (e.g., HEK 293 cells), SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); TR1 cells; MRC Examples include 5 cells; FS4 cells; and human hepatocellular carcinoma cell lines (Hep G2). Other useful mammalian cell lines include Chinese hamster ovary (CHO) cells, which include DHFR-CHO cells and myeloma cell lines such as NSO and Sp2 / 0.

[0356] Techniques for measuring changes in pH, bicarbonate secretion, acid secretion, water absorption, and phosphate uptake are known in the art. For example, changes in intracellular pH can be measured by contacting cells or tissues with a pH-sensitive fluorescent dye or probe and measuring the fluorescence of the dye or probe. Examples of pH-sensitive dyes include 2",7"-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein 4 (BCECF), 2",7"-bis-(2-carboxypropyl)-5-(and-6-)-carboxyfluorescein (BCPCF 11), 5-(and 6)-carboxynaphthofluorescein, and others (see, for example, Figures 8A and 8B, Han and Burgess, Chem Rev. 110: 2709-28, 2010). Techniques for measuring bicarbonate transport (in vitro) through a single ion channel, individual cells, and intact epithelial layer are described, for example, in Hug et al., Methods Mol Biol. 741:489-509, 2011 and Feldman et al., Am.J.Physiol. 254:C383-90, 1988. As described above, changes in pH, bicarbonate secretion, and / or acid secretion can also be measured in a Ussing chamber, for example, using a pH stat or isotope flux method. Phosphate uptake can be measured, for example, in cells or tissues. 33 This can be measured by contacting the substance with a phosphorus-labeled phosphate ion and measuring the uptake of the labeled phosphate ion (see Example, Matsuo et al., Eur.J.Pharmacol. 517:111-19, 2005). Other techniques for measuring pH, bicarbonate secretion, acid secretion, and phosphate uptake will become apparent to those skilled in the art.

[0357] In certain embodiments, the test compound is a small molecule or peptide known or suspected to stimulate bicarbonate secretion (e.g., DBS) in the gastrointestinal tract, including the small intestine, inhibit acid secretion, and / or reduce water absorption. Examples of such compounds include, but are not limited to, P2Y agonists, adenosine A2b receptor agonists, guanylate cyclase C receptor agonists (e.g., peptide agonists), soluble guanylate cyclase agonists, adenylyl cyclase receptor agonists, imidazoline-1 receptor agonists, cholinergic agonists, prostaglandin EP4 receptor agonists, dopamine D1 agonists, melatonin receptor agonists, 5HT4 agonists, atrial natriuretic peptide receptor agonists, carbonic anhydrase inhibitors, and phosphodiesterase inhibitors. Non-limiting examples of such compounds are described elsewhere in this specification. In some embodiments, the compound is a derivative or analog of one or more such compounds. Such derivatives or analogues may include modifications, for example, to increase the systemic non-bioavailability of the compound, as described herein.

[0358] This includes any of the methods described above, or other screening methods known in the art, adapted for high-throughput screening (HTS). HTS typically uses automation to perform screening of assays against a library of candidate drugs (e.g., assays to measure increased or decreased binding and / or activity, as described herein).

[0359] Any of the screening methods provided herein may utilize small molecule libraries or libraries produced by combination chemistry. For example, such libraries may be used to screen for small molecules that bind to or interact with target molecules or induce a desired physiological response (e.g., decrease intracellular pH in intestinal cells, inhibit phosphate uptake). Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art. Examples of methods for synthesizing molecular libraries can be found in (Carell et al., 1994a, Carell et al., 1994b, Cho et al., 1993, DeWitt et al., 1993, Gallop et al., 1994, Zuckermann et al., 1994).

[0360] The drug library may be provided in solution (Houghten et al., 1992), on beads (Lam et al., 1991), on chips (Fodor et al., 1993), on bacteria, on spores (Ladner et al., U.S. Patent No. 5,223,409, 1993), on plasmids (Cull et al., 1992), or on phages (Cwirla et al., 1990, Devlin et al., 1990, Felici et al., 1991, Ladner et al., U.S. Patent No. 5,223,409, 1993, Scott and Smith, 1990). Libraries useful for the purposes of the present invention include, but are not limited to, (1) chemical libraries, (2) natural product libraries, and (3) combination libraries consisting of random peptides, oligonucleotides, and / or organic molecules.

[0361] Chemical libraries consist of known drugs or structural analogs of drugs identified as "hits" or "leads" by natural product screening. Natural product libraries are derived from a group of microorganisms, animals, plants, or marine organisms used to generate mixtures for screening by (1) fermentation and extraction of broths derived from soil, plants, or marine microorganisms, or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and their (non-spontaneously occurring) variants. See, for example, Cane et al., Science 282:63-68, 1998. Combination libraries may consist of a number of peptides or organic compounds as mixtures. They are prepared relatively easily by conventional automated synthesis methods, PCR, cloning, or specialized synthesis methods.

[0362] More specifically, a combinational chemical library is a diverse group of chemical agents produced by either chemical synthesis or biological synthesis by combining several chemical "components," such as reagents. For example, a linear combinational chemical library, such as a polypeptide library, is formed by combining a set of chemical components (amino acids) in all possible ways for a given compound length (i.e., the number of amino acids in the polypeptide). Millions of chemical agents can be synthesized by mixing such combinations of chemical components.

[0363] For an overview of combinatorial chemistry and the libraries produced therefrom, see, for example, Huc and Nguyen, (2001) Comb. Chem. High Throughput Screen. 4:53-74, Lepre, (2001) Drug Discov. Today 6:133-140, Peng, (2000) Biomed. Chromatogr. 14:430-441, Bohm, H. J. and Stahl, M. (2000) Curr. Opin. Chem. Biol. 4:283-286, Barnes and Balasubramanian, (2000) Curr. Opin. Chem. Biol. 4:346-350, Lepre et al., (2000) Mass Spectrom Rev. 19:139-161, Hall, (2000) Nat. Biotechnol. 18:262-262, Lazo and Wipf,(2000) J.Pharmacol.Exp.Ther.293:705-709, Houghten,(2000)Ann.Rev.Pharmacol.Toxicol.40:273-282, Kobayashi(2000)Curr.Opin.Chem.Biol.(2000)4:338-345, Kopylov Spiridonova, (2000) Mol. Biol. (Mosk) 34:1097-1113, Weber, (2000) Curr. Opin. Chem. Biol. 4: 295-302, Dolle, (2000) J. Comb. Chem. 2: 383-433, Floyd et al. al.,(1999)Prog.Med.Chem.36:91-168, Kundu et al. al.,(1999)Prog.Drug Res.53:89-156, Cabilly,(1999)Mol.Biotechnol.12:143-148, Lowe,(1999)Nat.Prod.Rep.16:641-651, Dolle and Nelson, (1999) J.Comb.Chem.1:235-282, Czarnick and Keene, (1998) Curr.Biol.8:R705-R707, Dolle, (1998) Mol.Divers.4:233-256, Myers, (1997) Curr.Opin.Biotechnol.See 8:701–707 and Pluckthun and Cortese, (1997) Biol. Chem. 378:443.

[0364] Instruments for preparing combination libraries are commercially available (see, for example, the 357 MPS and 390 MPS from Advanced Chem Tech (Louisville, Ky.), the Symphony from Rainin (Woburn, Mass.), the 433A from Applied Biosystems (Foster City, Calif.), and the 9050 Plus from Millipore (Bedford, Mass.)). Furthermore, many combination libraries themselves are commercially available (see, for example, ComGenex from Princeton, NJ (Asinex, Moscow, Ru.), Tripos, Inc. (St. Louis, Mo.), ChemStar, Ltd. (Moscow, Ru.), 3D Pharmaceuticals (Exton, Pa.), Martek Biosciences (Columbia, Md.), etc.).

[0365] Definitions and technical terms "Amino" refers to the -NH2 radical.

[0366] "Aminocarbonyl" refers to the -C(=O)NH2 radical.

[0367] "Carboxylate" refers to the -CO2H radical. "Carboxylate salt" refers to its salt or ester.

[0368] "Cyano" refers to the -CN radical.

[0369] "Hydroxy" or "hydroxyl" refers to the -OH radical.

[0370] "Imino" refers to the NH radical.

[0371] "Nitro" refers to the -NO2 radical.

[0372] "Oxo" or "carbonyl" refers to an =O radical.

[0373] "Thioxo" refers to the S radical.

[0374] "Guanidinyl" (or "guanidine") refers to the -NHC (=NH)NH2 radical.

[0375] "Amidinyl" (or "amidine") refers to the -C(=NH)NH2 radical.

[0376] "Phosphate" refers to the -OP(=O)(OH)2 radical.

[0377] "Phosphonate" refers to the -P(=O)(OH)2 radical.

[0378] "Phosphine salts" are each R a However, independently, it refers to the -PH(=O)OH radical, which is an alkyl group as defined herein.

[0379] "Sulfate" refers to the -OS(=O)2OH radical.

[0380] "Sulfonate" or "hydroxysulfonyl" refers to the -S(=O)2OH radical.

[0381] "Sulfinate" refers to the -S(=O)OH radical.

[0382] "Sulfonyl" refers to the part containing the -SO2- group. For example, "alkylsulfonyl" or "alkylsulfone" is R a However, the alkyl group as defined herein is -SO2-R a It refers to the base.

[0383] "Alkyl" is saturated or unsaturated (i.e., contains one or more double and / or triple bonds) and has 1 to 12 carbon atoms (C1- 12A alkyl group is a linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, preferably having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), and bonded to the remainder of the molecule by a single bond. Examples include methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, buto-1-enyl, pento-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless otherwise specified herein, alkyl groups may be optionally substituted.

[0384] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that is saturated or unsaturated (i.e., contains one or more double and / or triple bonds), has 1 to 12 carbon atoms, consists only of carbon and hydrogen, and the remainder of the molecule is bonded to a radical group, such as methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is bonded to the remainder of the molecule by single or double bonds and to the radical group by single or double bonds. The points of bond between the alkylene chain and the remainder of the molecule and the radical group may be through one carbon or any two carbons in the chain. Unless otherwise specifically stated herein, alkylene chains may be optionally substituted.

[0385] "alkoxy" is the formula -OR a This refers to the radical of the expression, where R is located in the formula. a This is an alkyl radical containing 1 to 12 carbon atoms as defined above. Unless otherwise specifically stated herein, the alkoxy group may be optionally substituted.

[0386] "Alkylamino" is formula -NHR a or -NR a R a This refers to the radical, and in the formula, each Ra This is an alkyl radical containing 1 to 12 carbon atoms as defined above. Unless otherwise specifically stated herein, the alkylamino group may be optionally substituted.

[0387] "Thioalkyl" is represented by formula -SR a This refers to the radical of the expression, where R is located in the formula. a This is an alkyl radical containing 1 to 12 carbon atoms as defined above. Unless otherwise specifically stated herein, the thioalkyl group may be optionally substituted.

[0388] "Aryl" refers to a hydrocarbon cyclic radical comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl radicals can be monocyclic, bicyclic, tricyclic, or tetracyclic systems, which may include condensed or bridging cyclic systems. Examples of aryl radicals include, but are not limited to, aryl radicals derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluorantene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specifically stated herein, the term "aryl" or the prefix "ar-" (as in "aralkyl") is intended to include optionally substituted aryl radicals.

[0389] "Aralkill" is formula -R b -R c This refers to the radical of the expression, where R is located in the formula. b R is the alkylene chain defined above, c This is one or more defined aryl radicals, such as benzyl or diphenylmethyl. Unless otherwise specifically stated herein, the aralkyl group may be optionally substituted.

[0390] A "cycloalkyl" or "carbocyclic ring" refers to a stable, non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms, having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, being saturated or unsaturated, and bonded to the remainder of the molecule by single bonds. This may include condensed or crosslinked ring systems. Examples of monocyclic radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic radicals include adamantyl, norbornyl, decalinyl, and 7,7-dimethylbicyclo[2.2.1]heptanyl. Unless otherwise specified herein, cycloalkyl groups may be optionally substituted.

[0391] "Cycloalkylalkyl" is defined by formula -R b R d This refers to the radical of the expression, where R is located in the formula. d R is the alkylene chain defined above, g is a cycloalkyl radical as defined above. Unless otherwise specified herein, cycloalkylalkyl groups may be optionally substituted.

[0392] "Condensation" refers to any ring structure described herein that is condensed onto an existing ring structure in the compound of the present invention. If the condensed ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure that becomes part of the condensed heterocyclyl ring or condensed heteroaryl ring may be substituted with a nitrogen atom.

[0393] "Halo" or "halogen" refers to bromo, chloro, fluoro, or iodine.

[0394] "Haloalkyl" refers to an alkyl radical as defined above, which is substituted with one or more halo radicals as defined above, such as trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc. Unless otherwise specifically stated herein, haloalkyls may be optionally substituted.

[0395] A "heterocyclyl" or "heterocyclic ring" refers to a stable 3- to 18-membered non-aromatic ring radical consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specifically stated herein, heterocyclyl radicals can be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which may include condensed or bridging ring systems, the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical may be optionally oxidized, the nitrogen atom may be optionally quaternized, and the heterocyclyl radical may be partially or completely saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanil, thienyl[1,3]dithianil, decahydroisoquinolyl, imidazolinil, imidazolidinil, isothiazolidinil, isoxazolidinil, morpholinil, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinil, 2-oxopiperidinil, 2-oxopyrrolidinil, oxazolidinil, piperidinil, piperazinil, 4-piperidonil, pyrrolidinil, pyrazolidinil, quinuclidinil, thiazolidinil, tetrahydrofuryl, trithianil, tetrahydropyranil, thiomorpholinil, thiamorpholinil, 1-oxo-thiomorpholinil, and 1,1-dioxo-thiomorpholinil. Unless otherwise specified herein, heterocyclyl groups may be optionally substituted.

[0396] "N-heterocyclyl" refers to the heterocyclyl radical as defined above, which contains at least one nitrogen atom, and the point of attachment of the heterocyclyl radical to the rest of the molecule is through the nitrogen atom within the heterocyclyl radical. Unless otherwise specifically stated herein, the N-heterocyclyl group may be optionally substituted.

[0397] "Heterocyclylalkyl" is a formula of -R b R e This refers to the radical of the expression, where R is located in the formula. b R is the alkylene chain defined above, e A is a heterocyclyl radical as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl can be bonded to an alkyl radical at the nitrogen atom. Unless otherwise specifically stated herein, heterocyclylalkyl groups may be optionally substituted.

[0398] "Heteroaryl" refers to a 5-14 membered cyclic radical comprising a hydrogen atom, 1-13 carbon atoms, 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For the purposes of the present invention, heteroaryl radicals can be monocyclic, bicyclic, tricyclic, or tetracyclic systems, which may include condensed or bridging cyclic systems, and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized, and the nitrogen atom may be optionally quaternized. Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranil, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanil, benzonaphthofuranil, benzoxazolyl, benzodioxolyl, benzodioxynil, benzopyranil, benzopyranonil, benzofuranil, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranil, dibenzothiophenyl, furanil, furanonil, isothiazolyl, imidazolyl, indazolyl, indazolyl, indazolyl, isoindo Examples include, but are not limited to, lyl, indolinyl, isoindolinyl, isoquinolyl, indolidinyl, isoxazolyl, naphthilidinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxyranil, 1-oxidepyridinyl, 1-oxidepyrimidinyl, 1-oxidepyradinyl, 1-oxidepyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxadinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridadinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless otherwise specified herein, heteroaryl groups may be optionally substituted.

[0399] "N-heteroaryl" refers to a heteroaryl radical as defined above, containing at least one nitrogen atom, and the point of attachment of the heteroaryl radical to the rest of the molecule is through the nitrogen atom within the heteroaryl radical. Unless otherwise specifically stated herein, the N-heteroaryl group may be optionally substituted.

[0400] "Heteroarylalkyl" is a compound of the formula -R b R f This refers to the radical of the expression, where R is located in the formula. b R is the alkylene chain defined above, f is a heteroaryl radical as defined above. Unless otherwise specified herein, heteroarylalkyl groups may be optionally substituted.

[0401] As used herein, the term "substituted" means that at least one hydrogen atom is replaced by halogen atoms such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl groups, carboxyl groups, phosphate groups, sulfate groups, alkoxy groups, and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfinic acid groups, sulfone groups, sulfonyl groups, and sulfoxide groups; phosphorus atoms in groups such as phosphinic acid groups and phosphonic acid groups; and in groups such as guanidine groups, amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines. "Substituted" also means any of the above groups (i.e., alkyl, alkylene, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl) in which one or more hydrogen atoms are substituted by a bond to a non-hydrogen atom, such as oxygen in oxo, carbonyl, carboxyl, and ester groups, and nitrogen in imine, oxymin, hydrazone, and nitrile groups. For example, "substituted" means any of the above groups in which one or more hydrogen atoms are substituted by a higher-order bond (e.g., a double or triple bond) to a heteroatom, such as oxygen in oxo, carbonyl, carboxyl, and ester groups, and nitrogen in imine, oxymin, hydrazone, and nitrile groups. g R h , -NR g C(=O)R h , -NR g C(=O)NR g R h , -NR g C(=O)OR h , -NR g SO2R h -OC(=O)NR g R h , -OR g , -SR g-SOR g , -SO2R g , -OSO2R g , -SO2OR g ,=NSO2R g , and -SO2NR g R h It contains one of the above groups which is substituted by -C(=O)R. "Substituted" means that one or more hydrogen atoms are replaced by -C(=O)R. g , -C(=O)OR g -C(=O)NR g R h -CH2SO2R g -CH2SO2NR g R h -(CH2CH2O) 1-10 R g -(CH2CH2O) 2-10 R g ,-(OCH2CH2) 1-10 R g , and -(OCH2CH2) 2-10 R g It also means any of the above bases that are substituted by R. g and R h These groups are the same or different and independently of each other: hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" further means any of the above groups in which one or more hydrogen atoms are substituted by bonding to amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. The above nonhydrogen groups are generally referred to herein as "substituents" or "nonhydrogen substituents." Furthermore, each of the above substituents may optionally be substituted with one or more of the above substituents.

[0402] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0403] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, length, or other unit of reference as described herein, which varies by the same amount as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%.

[0404] The term "activate" refers to the application of physical, chemical, or biochemical conditions, substances, or processes that cause a receptor (e.g., a pore receptor) to undergo a structural change in a way that allows the passage of ions, molecules, or other substances.

[0405] The term "active state" refers to the state or condition of a receptor in a non-resting state.

[0406] "Outward current" refers to the movement of a flux of ions, molecules, or other substances from the intracellular space to the extracellular space.

[0407] Enteral or intestinal administration refers to administration via the gastrointestinal tract, including oral, sublingual, sublabial, buccal, and rectal administration, and includes administration via the nutrient duct of the stomach or duodenum.

[0408] The term "inactive state" refers to the state of the receptor in its primordial endogenous state, or resting state.

[0409] The term “modulate” typically includes “increasing” or “improving” a statistically significant or physiologically significant amount compared to a control, as well as “decreasing” or “reducing.” An “increased” or “improved” amount is typically a “statistically significant” amount, approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2. This can include increases of 0.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.3, 4.4, 4.6, 4.8, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, and 50 times or more (e.g., 100, 200, 500, 1000 times) (including all integers and decimals greater than 1 in between, e.g., 5.5, 5.6, 5.7, 5.8, etc.). The amount "decreased" or "reduced" is typically a "statistically significant" amount and may include a decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and the decimals and ranges between them) of the amount or activity produced by a control (e.g., the absence of the compound, or a smaller amount of the compound, a different compound, or a therapeutic treatment) or the amount at an earlier point in time (e.g., before treatment with the compound).

[0410] The term "prodrug" is intended to refer to a compound that can be converted to the bioactive compound of the present invention under physiological conditions or by solvolysis. Therefore, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of the compound of the present invention. A prodrug may be inactive when administered to a target requiring it, but is converted to the active compound of the present invention in vivo. Prodrugs are typically rapidly converted in vivo, for example, by hydrolysis in the blood, to yield the parent compound of the present invention. Prodrug compounds often offer advantages in solubility, histocompatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). Further discussion of prodrugs is provided in Higuchi, T., et al., ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0411] The term “prodrug” is also intended to include any covalent carrier that, upon administration to a mammalian subject, releases the active compound of the present invention in vivo. Prodrugs of the compounds of the present invention can be prepared by modifying a functional group present in the compound of the present invention in such a manner that the modification is cleaved either in a routine operation or in vivo. Prodrugs include compounds of the present invention in which a hydroxyl, amino, or mercapto group is bonded to any group that, upon administration to a mammalian subject with a prodrug of the compound of the present invention, is cleaved to form a free hydroxyl group, a free amino group, or a free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols, or amide derivatives of amine functional groups in the compounds of the present invention.

[0412] The inventions disclosed herein are intended to also encompass in vivo metabolites of the disclosed compounds. Such products may arise, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily due to enzymatic processes. Accordingly, the present invention includes compounds produced by a process comprising administering the compounds of the present invention to a mammal for a period of time sufficient to yield its metabolites. Such products are typically identified by administering a radiolabeled compound of the present invention to an animal such as a rat, mouse, guinea pig, monkey, or human in a detectable dose, allowing sufficient time for metabolism to occur, and then isolating the conversion product from urine, blood, or other biological samples.

[0413] "Mammals" include humans, as well as domesticated animals such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domesticated animals such as wild animals.

[0414] "Optional" or "optionally" means that the event or situation described below may or may not occur, and that the description includes both instances in which the event or situation occurs and instances in which it does not occur. For example, "optionally substituted aryl" means that the aryl radical may or may not be substituted, and that the description includes both substituted aryl radicals and aryl radicals without substitution.

[0415] "Pharmacologically acceptable carriers, diluents, or excipients" include, but are not limited to, any adjuvants, carriers, excipients, lubricants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are approved by the United States Food and Drug Administration as acceptable for use in humans or domesticated animals.

[0416] "Pharmacologically acceptable salts" include both acid addition salts and base addition salts.

[0417] "Pharmacologically acceptable acid addition salts" are inorganic acids that are not biologically or otherwise undesirable and include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, as well as acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonate, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2 This refers to salts formed using organic acids, including but not limited to oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucinic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid, that retain their biological efficacy and free base properties.

[0418] "Pharmacologically acceptable base addition salts" refer to salts that are not biologically or otherwise undesirable and that retain the biological efficacy and properties of the free acid. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Examples of salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including spontaneously occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0419] In many cases, crystallization produces solvated compounds of the compounds of the present invention. As used herein, the term “solvated compound” refers to an aggregate containing one or more molecules of the compounds of the present invention together with one or more molecules of a solvent. The solvent may be water, in which case the solvent compound may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as hydrates, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the corresponding solvated forms. While the compounds of the present invention may be true solvated compounds, in other cases, the compounds of the present invention may simply retain exogenous water or be a mixture of water and some exogenous solvent.

[0420] "Pharmaceutical composition" refers to a formulation of the compound of the present invention with a medium generally accepted in the art for the delivery of a bioactive compound to a mammal, such as a human. Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients for that purpose.

[0421] The compounds of the present invention or their pharmaceutically acceptable salts may contain one or more chiral centers, and thus may give rise to enantiomers, diastereomers, and other stereoisomers that can be defined as (R) or (S) in terms of absolute stereochemistry, or as (D) or (L) in the case of amino acids. The present invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R) and (S), or (D) and (L) isomers may be prepared using chiral synthons or chiral reagents, or separated using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or separation of racemates (or racemates of salts or derivatives) using, for example, chiral high-pressure liquid chromatography (HPLC). Where a compound described herein contains an olefin double bond or other geometrically asymmetric center, unless otherwise stated, the compound is intended to include both E and Z geometric isomers. Similarly, all tautomers are also intended to be included.

[0422] "Stable compound" and "stable structure" are intended to indicate a compound that is robust enough to withstand isolation from the reaction mixture to a useful purity and formulation into an effective therapeutic agent.

[0423] "Statistically significant" means that the result is unlikely to have occurred by chance. Statistical significance can be determined by methods known in the relevant art. A commonly used measure of significance is the p-value, which is the frequency or probability that the observed event will occur if the null hypothesis were true. If the resulting p-value is smaller than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0424] "Substantially" or "essentially" means "almost entirely" or "completely," for example, including 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of some given quantity.

[0425] The term "secondary" refers to a situation or condition that may occur in conjunction with, follow, or result from another condition, situation, or treatment. This term also refers to situations where the condition, situation, or treatment plays only a minor role in the patient's final pathological state, symptoms, or symptoms or responses in the condition.

[0426] Subjects or patients (these terms are used interchangeably herein) who require treatment with the compounds of this disclosure include, for example, subjects requiring "phosphate reduction," which may include subjects requiring "phosphate management," such as prophylactic management of phosphate or phosphorus levels. The diseases and / or conditions described herein include mammals that have, or are at risk of having, diseases and / or conditions that can be treated with the compounds of the present invention, with or without other activators, to achieve beneficial therapeutic and / or prophylactic outcomes. Beneficial outcomes include reduced severity of symptoms, delayed onset of symptoms, maintenance of orthophosphate, reduced risk of developing hyperphosphatemia, modulation of one or more of the signs described herein (e.g., reduced serum or blood phosphorus levels in patients with or at risk of hyperphosphatemia, increased fecal excretion of phosphate ions in patients with or at risk of hyperphosphatemia), extended lifespan, and / or rapid or more complete recovery from the disease or condition.

[0427] A "stereoisomer" refers to a compound that consists of the same atoms bonded together by the same bonds, but has different, non-interchangeable three-dimensional structures. This invention aims to introduce various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers that are mirror images of each other and whose molecules cannot be superimposed.

[0428] "Tautomerism" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This invention includes any tautomer of such compound.

[0429] A “therapeutic effective dose” or “effective dose” is a certain amount of the compound of the present invention that, when administered to a mammal, preferably a human, is sufficient to inhibit or reduce the transport of phosphate ions from the gastrointestinal lumen, increase fecal excretion of phosphate ions, reduce serum levels of phosphate ions, treat hyperphosphatemia in a mammal, preferably a human, and / or treat any one or more of the conditions described herein. The amount of the compound of the present invention constituting a “therapeutic effective dose” will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal being treated, but can be routinely determined by a person skilled in the art considering their own knowledge and the present disclosure.

[0430] As used herein, “to treat” or “to treat” encompasses the treatment of the disease or condition of the subject in a mammal, preferably a human, that has the disease or condition of the subject, and (i) in particular, when the mammal is susceptible to the disease or condition but has not yet been diagnosed as having it, (ii) inhibiting a disease or condition, that is, stopping its development (iii) Relieving the disease or condition, that is, causing regression of the disease or condition, (iv) Relieving symptoms resulting from a disease or condition, i.e., relieving pain without addressing the underlying disease or condition. Where used herein, the terms “disease” and “condition” may be interchangeable, or they may differ in that a particular illness or condition may not have a known causative agent (and therefore its etiology has not yet been determined), and is therefore recognized not as a disease, but only as an undesirable condition or syndrome characterized by a more or less set of specific symptoms identified by a clinician. Examples

[0431] Example 1 An increase in intracellular pH leads to a decrease in intracellular phosphate uptake. We conducted experiments to examine the relationship between changes in intracellular pH and changes in phosphate ion (Pi) uptake within human fetal kidney cells (HEK-293 cells).

[0432] HEK-293 cells were seeded at 25,000 cells / well in 96-well plates and cultured overnight. The cells were then transfected with either rat or human NaP2b cDNA, or pseudo-transfected (without DNA) using Lipofectamine 2000 (Invitrogen). During a second overnight incubation, the cells were brought close to confluence.

[0433] The intracellular pH was reduced from approximately 7.4 to approximately 6.8 using an ammonium pulse technique. The culture medium was aspirated from the wells, and the cells were washed twice with NaCl-HEPES buffer (100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), and then incubated at room temperature for 30 minutes with NH4Cl-HEPES buffer containing 5 μM BCECF-AM (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4). + Cells were washed twice with HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) without ammonium ion, and incubated in the same buffer at room temperature for 10 minutes to reduce intracellular pH. The reduction in intracellular pH to approximately pH 6.8 was confirmed by monitoring the pH-sensitive change of BCECF fluorescence (λex 505 nm, λem 538 nm) normalized to pH-insensitive BCECF fluorescence (λex 439 nm, λem 538 nm). A control was included in which the ammonium pulse technique was omitted, and BCECF was used to show a normal intracellular pH of 7.4.

[0434] Next, the cells are washed with a sodium-free uptake buffer (14 mM Tris, 137 mM choline chloride, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 100 μM KH2PO4, 1 mg / mL bovine serum albumin, pH 7.4), and then coated with a sodium-containing uptake buffer (14 mM Tris, 137 mM sodium chloride, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 100 μM KH2PO4, 1 mg / mL bovine serum albumin, pH 7.4), 33 P uptake was initiated. In rat or human NaP2b-transfected cell lines, sodium-dependent uptake was initiated. 33 Endogenous PiT activity was suppressed with a PiT expression inhibitor so that only P uptake was attributable to NaP2b. Since the PiT expression inhibitor was not used on pseudotransfected cells, sodium-dependent PiT activity was suppressed. 33 P is solely due to PiT.

[0435] In the presence and absence of 5 μM EIPA, a specific inhibitor of NHE1, 33 P uptake was measured. After 23 minutes at room temperature, the assay mixture was removed and the cells were washed twice with ice-cold, sodium-free uptake buffer. The cells were lysed by adding 20 μL of 0.1% Tween 80 followed by 100 μL of scintillation solution and counted using TopCount (Perkin Elmer).

[0436] As shown in Figures 22A-22C, intracellular acidification is either PiT-mediated (22A) or NaPi2b-mediated (22B-22C). 33 It caused a decrease of over 75% in P uptake. EIPA, which blocks NHE1-mediated proton efflux from the cytoplasm, also caused a small but significant decrease in Pi uptake in cells that were not pretreated to lower intracellular pH.

[0437] Example 2 Guanylate cyclase C (GC-C) receptor agonists reduce phosphate absorption. Experiments were conducted and found that guanylate cyclase C (GC-C) receptor agonists 33 We determined whether it could reduce phosphate absorption / uptake in the small intestine as measured by P uptake. As shown below, 33 P and linaclotide were administered simultaneously to rats. 1. Vehicle (N=5 / group) 2.0.1 mg / kg of linaclotide (N=6 / group) 3.0.3 mg / kg of linaclotide (N=4 / group)

[0438] 33 Blood samples were collected at 5, 15, 30, 45, and 60 minutes after administration of phosphate (P), and plasma scintillation was performed. The results are shown in Figures 1A and 1B. Figure 1A shows the results of a two-way repeated measures ANOVA followed by Dunnett's multiple comparison test, while Figure 1B shows the results of a one-way ANOVA followed by Dunnett's multiple comparison test. These results indicate that both doses of linaclotide reduced phosphate absorption in the gastrointestinal tract.

[0439] Example 3 I1 receptor agonists and adenylyl cyclase agonists reduce phosphate absorption. Experiments were conducted, and other types of drugs were found to be... 33 We determined whether it could reduce phosphate absorption / uptake in the small intestine as measured by P uptake. As shown below, 33 Rats were simultaneously administered either P and an imidazoline subtype 1 (I1) receptor agonist (moxonidine) or an adenylyl cyclase agonist (NKH477, a water-soluble forskolin analog). 1. Vehicle 2.2 mg / kg of moxonidine 3.6 mg / kg of moxonidine NKH477 at 4.1 mg / kg 5.3 mg / kg of NKH477

[0440] 33Blood samples were collected at 5, 15, 30, 45, and 60 minutes after administration of P, and plasma scintillation counts were performed. The results are shown in Figures 2A and 2B. Figure 2A shows the results of two-way ANOVA with repeated measures, followed by Dunnett's multiple comparison test, and Figure 2B shows the results of one-way ANOVA, followed by Dunnett's multiple comparison test. These results show that all test compounds showed the same reaction at 15 minutes. 33 This indicates a significant decrease in phosphorus uptake / absorption.

[0441] Example 4 A2B and P2Y2 agonists reduce phosphate absorption. We conducted experiments to determine the mechanisms of intracellular calcium (Ca) ++ The increase in ) 33 We also determined whether it could reduce phosphate absorption in the small intestine as measured by P uptake. As shown below, 33 P and the test compound were administered simultaneously to rats. 1. Vehicle, n=6 2.10 mg / kg of BAY 60-6583 (adenosine A2B agonist) 3.15 mg / kg of Up4U (P2Y2 receptor agonist)

[0442] 33 Blood samples were collected at 5, 15, 30, 45, and 60 minutes after P administration, and plasma scintillation counts were performed. Figure 3 shows that Up4U (15 mg / kg), a P2Y2 receptor agonist, 33 This indicates a significant decrease in phosphorus uptake / absorption.

[0443] Example 5 Pharmacodynamic effects on acute phosphate uptake in rats The compounds were tested for their ability to reduce the appearance of circulating radiolabeled phosphate after administration to the gastrointestinal tract of rats. The rate of accumulation of radiolabeled phosphate tracers in the rat blood was measured as a surrogate for the rate of intestinal absorption of phosphate diets from the gastrointestinal tract. For this purpose, circulating radiolabeled phosphate was monitored after intragastric co-administration of a phosphate tracer diet combined with the compounds of the examples to rats. However, some of the...

Claims

1. A composition for treating hyperphosphatemia in patients requiring treatment for hyperphosphatemia, comprising a compound that is substantially active in inhibiting the transport of phosphate ions (Pi) within the gastrointestinal tract and is a guanylate cyclase C receptor (GC-C) agonist, The composition wherein the GC-C agonist is precanatide, bacterial thermostable enterotoxin, guaniline, proguaniline, uroguaniline, prouroguaniline, lymphoguaniline, or a peptide having the amino acid sequence Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 3), or Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu (SEQ ID NO: 6).

2. The composition according to claim 1, wherein the compound does not bind to NHE3.

3. The composition according to claim 1 or 2, wherein hyperphosphatemia is associated with kidney disease.

4. The composition according to claim 1, wherein the compound is precanatide.

5. The composition according to any one of claims 1 to 4, used in combination with one or more additional bioactive agents.

6. The composition according to claim 5, wherein the compound and the one or more additional bioactive agents are administered as part of a single pharmaceutical composition, as separate pharmaceutical compositions, sequentially, or simultaneously.

7. The aforementioned additional bioactive agent is vitamin D 2 (Ergocalciferol), Vitamin D 3 The composition according to claim 5 or 6, comprising one or more of the following: (cholecalciferol), active vitamin D (calcitriol), active vitamin D analog (e.g., doxelcalciferol, paricalcitol), phosphate binder, NaPi2b inhibitor, niacin, nicotinamide, ACE inhibitor, antiogensin II receptor blocker, β-blocker, calcium channel blocker, direct renin inhibitor, diuretic, vasodilator, erythropoietin therapy, iron supplementation therapy, advanced glycation end product inhibitor, vitamin D, and statin.

Images