Compounds and methods for inhibiting phosphate transport

Abstract

Provided are non-NHE3-binding agents having activity as phosphate transport/uptake inhibitors in the gastrointestinal tract, including in the small intestine, methods for their use as therapeutic or prophylactic agents, and related methods of drug discovery.

1 . A method for inhibiting phosphate uptake in the gastrointestinal tract of a patient in need of phosphate lowering, comprising administering to the patient a guanylate cyclase C receptor (GC-C) agonist compound, where the GC-C agonist compound is substantially active in the gastrointestinal tract to inhibit transport of phosphate ions (Pi) therein upon administration to the patient in need thereof. 
     
     
         2 . A method for inhibiting phosphate uptake in the gastrointestinal tract of a patient in need of phosphate lowering, comprising administering to the patient a compound that does not bind NHE3, where the compound is substantially active in the gastrointestinal tract to inhibit transport of phosphate ions (Pi) therein upon administration to the patient in need thereof. 
     
     
         3 . The method of  claim 2 , where the compound decreases the cross-epithelial pH gradient (CEPG) in the small intestine, where the CEPG is defined as the difference in pH between (i) the cytoplasm of the epithelial cells of the surface of the small intestine, optionally at the subapical surface of the epithelial cell, and (ii) the unstirred layer at the apical surface of the small intestine, where the compound is substantially active in the gastrointestinal tract to inhibit transport of phosphate ions (Pi) therein upon administration to the patient in need thereof. 
     
     
         4 . The method of  claim 2 , where the compound decreases water absorption in the small intestine, optionally the jejunum, and where the compound is substantially active in the gastrointestinal tract to inhibit transport of phosphate ions (Pi) therein upon administration to the patient in need thereof. 
     
     
         5 . The method of  claim 2 , where the compound decreases the CEPG and decreases water absorption in the small intestine. 
     
     
         6 . The method of  claim 2 , where the compound decreases the CEPG in the small intestine without significantly decreasing water absorption in the small intestine. 
     
     
         7 . The method of  claim 2 , where the compound decreases water absorption in the small intestine without significantly decreasing the CEPG in the small intestine, optionally without significantly stimulating bicarbonate secretion and/or inhibiting acid secretion in the small intestine. 
     
     
         8 . The method of  claim 2 , where the method results in a method selected from one or more of:
 (a) a method for treating hyperphosphatemia, optionally postprandial hyperphosphatemia;   (b) a method for treating a renal disease, optionally chronic kidney disease (CKD) or end-stage renal disease (ESRD);   (c) a method for reducing serum creatinine levels;   (d) a method for treating proteinuria;   (e) a method for delaying time to renal replacement therapy (RRT), optionally dialysis;   (f) a method for reducing FGF23 levels;   (g) a method for reducing the hyperphosphatemic effect of active vitamin D;   (h) a method for attenuating hyperparathyroidism, optionally secondary hyperparathyroidism;   (i) a method for reducing serum parathyroid hormone (PTH) a method for improving endothelial dysfunction, optionally induced by postprandial serum phosphorus;   (k) a method for reducing vascular calcification, optionally intima-localized vascular calcification;   (l) a method for reducing urinary phosphorous;   (m) a method for normalizing serum phosphorus levels;   (n) a method for reducing phosphate burden in an elderly patient;   (o) a method for decreasing dietary phosphate uptake;   (p) a method for reducing renal hypertrophy; and   (q) a method for reducing heart hypertrophy.   
     
     
         9 . The method of  claim 2 , where the compound decreases the intracellular pH of the epithelial cells of the surface of the small intestine, optionally at the subapical surface of the epithelial cell. 
     
     
         10 . The method of  claim 2 , where the compound increases the pH of the unstirred layer at the apical surface of the small intestine. 
     
     
         11 . The method of  claim 2 , where the compound (a) stimulates bicarbonate secretion in the small intestine, or (b) inhibits acid secretion in the small intestine, or (c) stimulates bicarbonate secretion and inhibits acid secretion in the small intestine. 
     
     
         12 . The method of  claim 2 , where the compound increases one or more intracellular secondary messengers of epithelial cells of the surface of the small intestine. 
     
     
         13 . The method of  claim 12 , where the one or more intracellular secondary messengers are selected from Ca ++ , cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP). 
     
     
         14 . The method of  claim 2 , where the compound is substantially systemically non-bioavailable upon enteral administration to the patient. 
     
     
         15 . The method of  claim 14 , where the compound is substantially impermeable to the epithelium of the gastrointestinal tract. 
     
     
         16 . The method of  claim 14 , where the compound is substantially permeable to the epithelium of the gastrointestinal tract. 
     
     
         17 . The method of  claim 2 , where administration to the patient in need thereof (a) reduces serum phosphorus concentrations or levels to about 150% or less of normal serum phosphorus levels, and/or (b) reduces uptake of dietary phosphorous by at least about 10% relative to an untreated state. 
     
     
         18 . The method of  claim 2 , where administration to the patient in need thereof increases phosphate levels in fecal excretion by at least about 10% relative to an untreated state. 
     
     
         19 . The method of  claim 2 , where administration to the patient in need thereof reduces urinary phosphate concentrations or levels by at least about 10% relative to an untreated state. 
     
     
         20 . The method of  claim 2 , where the patient in need thereof has ESRD, and where administration to the patient reduces serum phosphorus concentrations or levels by at least about 10% relative to an untreated state. 
     
     
         21 . The method of  claim 2 , where the patient in need thereof has CKD, and where administration to the patient reduces FGF23 levels and serum intact parathyroid hormone (iPTH) levels by at least about 10% relative to an untreated state. 
     
     
         22 . The method of  claim 2 , where the compound is selected from one or more of a guanylate cyclase C receptor (GC-C) agonist, a P2Y agonist, an adenosine A2b receptor agonist, a soluble guanylate cyclase agonist, an adenylate cyclase receptor agonist, an imidazoline-1 receptor agonist, a cholinergic agonist, a prostaglandin EP4 receptor agonist, a dopamine D1 agonist, a melatonin receptor agonist, a 5HT4 agonist, an atrial natriuretic peptide receptor agonist, a carbonic anhydrase inhibitor, a phosphodiesterase inhibitor, and a Down-Regulated in Adenoma (DRA or SLC26A3) agonist. 
     
     
         23 . The method of  claim 1  or  22 , where 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. 
     
     
         24 . The method of  claim 23 , where the GC-C agonist peptide comprises the amino acid sequence (I): Xaa 1  Xaa 2  Xaa 3  Xaa 4  Xaa 5  Cys 6  Cys 7  Xaa 8  Xaa 9  Cys 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) where: Xaa 1  Xaa 2  Xaa 3  Xaa 4  Xaa 5  is Asn Ser Ser Asn Tyr (SEQ ID NO:2) or is missing or Xaa 1  Xaa 2  Xaa 3  Xaa 4  is missing. 
     
     
         25 . The method of  claim 24 , where Xaa 5  is Asn, Trp, Tyr, Asp, or Phe. 
     
     
         26 . The method of  claim 24 , where Xaa 5  is Thr or Ile. 
     
     
         27 . The method of  claim 24 , where Xaa 5  is Tyr, Asp, or Trp. 
     
     
         28 . The method of  claim 24 , where Xaa 8  is Glu, Asp, Gln, Gly, or Pro. 
     
     
         29 . The method of  claim 24 , where Xaa 9  is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr, or Phe. 
     
     
         30 . The method of  claim 24 , where Xaa 9  is Leu, Ile, Val, Lys, Arg, Trp, Tyr, or Phe. 
     
     
         31 . The method of  claim 24 , where Xaa 12  is Asn, Tyr, Asp, or Ala. 
     
     
         32 . The method of  claim 24 , where Xaa 13  is Ala, Pro, or Gly. 
     
     
         33 . The method of  claim 24 , where Xaa 14  is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp. 
     
     
         34 . The method of  claim 24 , where Xaa 16  is Thr, Ala, Asn, Lys, Arg, or Trp. 
     
     
         35 . The method of  claim 24 , where Xaa 17  is Gly, Pro, or Ala. 
     
     
         36 . The method of  claim 24 , where Xaa 19  is Trp, Tyr, Phe, Asn, or Leu. 
     
     
         37 . The method of  claim 24 , where Xaa 19  is Lys or Arg. 
     
     
         38 . The method of  claim 24 , where Xaa 20  Xaa 21  is AspPhe or Xaa 20  is Asn or Glu and Xaa 21  is missing. 
     
     
         39 . The method of  claim 24 , where Xaa 19  Xaa 20  Xaa 21  is missing. 
     
     
         40 . The method of  claim 24 , where the GC-C agonist peptide comprises 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 a variant thereof having 1, 2, 3, 4, or 5 deletions, insertions, and/or substitutions. 
     
     
         41 . The method of  claim 24 , where the peptide comprises the amino acid sequence: Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:4), or a variant thereof having 1, 2, 3, 4, or 5 deletions, insertions, and/or substitutions. 
     
     
         42 . The method of  claim 23 , where the GC-C agonist peptide comprises the amino acid sequence (III): Xaa 1  Xaa 2  Xaa 3  Cys 4  Xaa 5  Xaa 6  Xaa 7  Xaa 8  Xaa 9  Xaa 10  Xaa 11  Cys 12  Xaa 13  Xaa 14  Xaa 15  Xaa 16  (SEQ ID NO:5), where Xaa 1  is: Ser, Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is missing; Xaa 2  is His, Asp, Glu, Ala, Ser, Asn, Gly, or is missing; Xaa 3  is Thr, Asp, Ser, Glu, Pro, Val or Leu; Xaa 5  is Asp, Ile or Glu; Xaa 6  is Ile, Trp or Leu; Xaa 7  is Cys, Ser, or Tyr; Xaa 8  is Ala, Val, Thr, Ile, Met or is missing; Xaa 9  is Phe, Tyr, Asn, or Trp; Xaa 10  is Ala, Val, Met, Thr or Ile; Xaa 11  is Ala or Val; Xaa 13  is Thr or Ala; Xaa 14  is Gly, Ala or Ser; Xaa 15  is Cys, Tyr or is missing; and Xaa 16  is His, Leu or Ser. 
     
     
         43 . The method of  claim 42 , where the peptide comprises 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 a variant thereof having 1, 2, 3, 4, or 5 deletions, insertions, and/or substitutions. 
     
     
         44 . The method of  claim 22 , where the P2Y agonist is selected from a compound in  FIG. 4  or  FIGS. 5A-5C . 
     
     
         45 . The method of  claim 22 , where the adenosine A2b receptor agonist is selected from a compound in  FIGS. 6A-6C . 
     
     
         46 . The method of  claim 22 , where the soluble guanylate cyclase agonist is selected from a compound in  FIGS. 9A-9L . 
     
     
         47 . The method of  claim 22 , where the adenylate cyclase receptor agonist is selected from a compound in  FIG. 10 . 
     
     
         48 . The method of  claim 22 , where the imidazoline-1 receptor agonist is selected from moxonidine and a compound in  FIG. 11 . 
     
     
         49 . The method of  claim 22 , where the cholinergic agonist is selected from a compound in  FIG. 12 . 
     
     
         50 . The method of  claim 22 , where the prostaglandin EP4 receptor agonist is selected from PGE 2  or its analogs/derivatives and a compound in  FIG. 7  or  FIG. 13 . 
     
     
         51 . The method of  claim 22 , where the dopamine D1 agonist is selected from a compound in  FIG. 14 . 
     
     
         52 . The method of  claim 22 , where the melatonin receptor agonist is selected from melatonin and a compound in  FIG. 15 . 
     
     
         53 . The method of  claim 22 , where the 5HT4 agonist is selected from serotonin and its analogs, prucalopride, metoclopramide, cleobopride, mosapride, prucalopride, renzapride, tegaserod, zacopride, norcisapride, naronopride, and velusetrag. 
     
     
         54 . The method of  claim 22 , where 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. 
     
     
         55 . The method of  claim 22 , where the carbonic anhydrase inhibitor is selected from a compound in  FIG. 17 . 
     
     
         56 . The method of  claim 22 , where the phosphodiesterase inhibitor is selected from a compound in  FIG. 18 . 
     
     
         57 . The method of  claim 22 , where the DRA agonist is selected from  FIGS. 21A-B . 
     
     
         58 . The method of  claim 2 , where the compound is substantially systemically non-bioavailable upon enteral administration to the patient and has (i) a tPSA of at least about 200 Å 2 . 
     
     
         59 . The method of  claim 58 , where the compound has a tPSA of at least about 250 Å 2 . 
     
     
         60 . The method of  claim 58 , where the compound has a tPSA of at least about 270 Å 2 . 
     
     
         61 . The method of  claim 58 , where the compound has a tPSA of at least about 300 Å 2 . 
     
     
         62 . The method of  claim 58 , where the compound has a tPSA of at least about 350 Å 2 . 
     
     
         63 . The method of  claim 58 , where the compound has a tPSA of at least about 400 Å 2 . 
     
     
         64 . The method of  claim 58 , where the compound has a tPSA of at least about 500 Å 2 . 
     
     
         65 . The method of  claim 58 , where the compound has a molecular weight of at least about 500 Da. 
     
     
         66 . The method of  claim 58 , where the compound has a molecular weight of at least about 1000 Da. 
     
     
         67 . The method of  claim 58 , where the compound has a molecular weight of at least about 2500 Da. 
     
     
         68 . The method of  claim 58 , where the compound has a molecular weight of at least about 5000 Da. 
     
     
         69 . The method of  claim 58 , where the compound has (i) a total number of NH and/or OH and/or other potential hydrogen bond donor moieties greater than about 5; (ii) a total number of O atoms and/or N atoms and/or other potential hydrogen bond acceptors greater than about 10; and/or (iii) a Moriguchi partition coefficient greater than about 10 5  or less than about 10. 
     
     
         70 . The method of  claim 58 , where the compound has a permeability coefficient, P app , of less than about 100×10 −6  cm/s, or less than about 10×10 −6  cm/s, or less than about 1×10 −6  cm/s, or less than about 0.1×10 −6  cm/s. 
     
     
         71 . The method of  claim 2 , further comprising administering one or more additional biologically active agents. 
     
     
         72 . The method of  claim 71 , where the compound and the one or more additional biologically active agents are administered as part of a single pharmaceutical composition. 
     
     
         73 . The method of  claim 71 , where the compound and the one or more additional biologically active agents are administered as individual pharmaceutical compositions. 
     
     
         74 . The method of  claim 73 , where the individual pharmaceutical compositions are administered sequentially. 
     
     
         75 . The method of  claim 73 , where the individual pharmaceutical compositions are administered simultaneously. 
     
     
         76 . The method of  claim 71 , where the additional biologically active agent is selected from vitamin D 2  (ergocalciferol), vitamin D 3  (cholecalciferol), active vitamin D (calcitriol) and active vitamin D analogs (e.g. doxercalciferol, paricalcitol). 
     
     
         77 . The method of  claim 71 , where the additional biologically active agent is a phosphate binder. 
     
     
         78 . The method of  claim 77 , where the phosphate binder is selected from the group consisting of sevelamer (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 hydroxycarbonate (e.g., Fermagate™), aluminum hydroxide (e.g., Alucaps®, Basaljel®), APS1585, SBR-759, and PA-21. 
     
     
         79 . The method of  claim 71 , where the additional biologically active agent is a NaPi2b inhibitor. 
     
     
         80 . The method of  claim 71 , where the additional biologically active agent is niacin or nicotinamide. 
     
     
         81 . The method of  claim 71 , where the subject has CKD and the additionally active biological agent is selected from one or more of ACE inhibitors, antiogensin II receptor blockers, beta-blockers, calcium channel blockers, direct renin inhibitors, diuretics, vasodilators, erythropoietin therapy, iron replacement therapy, inhibitors of advanced glycation end products, vitamin D, and statins. 
     
     
         82 . The method of  claim 2 , where the compound is administered orally. 
     
     
         83 . The method of  claim 82 , where the compound is administered orally once-a-day. 
     
     
         84 . A method of screening for an inhibitor of phosphate uptake, comprising (a) culturing intestinal cells, (b) contacting the cultured intestinal cells with a test compound, and (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 an inhibitor of phosphate uptake where the pH from (c)(i) increases relative to a control, the intracellular pH from (c)(ii) decreases relative to a control, and/or phosphate uptake from (c)(iii) decreases relative to a control. 
     
     
         85 . The method of  claim 84 , where step (a) comprise culturing intestinal cells to monolayers. 
     
     
         86 . The method of  claim 84 , where step (a) comprises isolating the cells from intestinal crypts and culturing under conditions sufficient to form enteroids. 
     
     
         87 . The method of  claim 84 , where step (a) comprises culturing isolated embryonic stem cells, endoderm cells, or pluripotent stem cells under conditions sufficient to form organoids. 
     
     
         88 . The method of  claim 84 , where step (a) comprises culturing intestinal section(s) in a Ussing chamber. 
     
     
         89 . The method of  claim 84 , where step (c)(i) comprises contacting the cells with a pH-sensitive fluorescent dye and measuring fluorescence of the dye. 
     
     
         90 . The method of  claim 84 , where step (c)(ii) comprises contacting the cells with  33 P-labeled phosphate ions and measuring uptake of the labeled phosphate ions. 
     
     
         91 . The method of  claim 84 , where the increase and/or decrease of (d) is statistically significant. 
     
     
         92 . The method of  claim 84 , where the test compound is a small molecule or peptide that is known or suspected to stimulate bicarbonate secretion and/or inhibit acid secretion in the small intestine. 
     
     
         93 . The method of  claim 92 , where the test compound is selected from one or more of a P2Y agonist, an adenosine A2b receptor agonist, a guanylate cyclase C receptor agonist, a soluble guanylate cyclase agonist, an adenylate cyclase receptor agonist, an imidazoline-1 receptor agonist, a cholinergic agonist, a prostaglandin EP4 receptor agonist, a dopamine D1 agonist, a melatonin receptor agonist, a 5HT4 agonist, an atrial natriuretic peptide receptor agonist, a carbonic anhydrase inhibitor, a phosphodiesterase inhibitor, and a Down-Regulated in Adenoma (DRA or SLC26A3) agonist.