SODIUM-HYDROGEN EXCHANGER 3 INHIBITOR COMPOUNDS

Abstract

The present invention relates to sodium-hydrogen exchanger 3 (NHE3) inhibitor compounds of the Formula:

##STR00001##

to pharmaceutical compositions comprising the compound and to the use of the compound for the treatment of certain diseases associated with elevated sodium and/or phosphate levels.

Claims

1. A compound of the formula: ##STR00019## wherein both R are CN or both R are C(O)NH.sub.2, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein both R are C(O)NH.sub.2, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein the compound is: ##STR00020## or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1, wherein the compound is: ##STR00021##

5. The compound of claim 1, wherein the compound is the dihydrochloride salt of: ##STR00022##

6. A compound which is: ##STR00023##

7. A compound which is the dihydrochloride salt of: ##STR00024##

8. A method of treating a disease selected from the group consisting of chronic kidney disease, hyperphosphatemia, secondary hyperparathyroidism, heart failure, hypertension and cardiovascular disease comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to claim 7, or a pharmaceutically acceptable salt thereof.

9. A method of treating a disease selected from the group consisting of chronic kidney disease, hyperphosphatemia, secondary hyperparathyroidism, heart failure, and cardiovascular disease comprising administering to a mammal in need thereof a therapeutically effective combination of a compound according to claim 7, or a pharmaceutically acceptable salt thereof, in combination with a compound of the formula: ##STR00025## or a pharmaceutically acceptable salt thereof.

10. The method of claim 8 wherein the mammal is a human.

11. The method of claim 8 wherein the mammal is a dog.

12. The method of claim 8 wherein the mammal is a cat.

13. A compound according to claim 7, or a pharmaceutically acceptable salt thereof, for use in therapy.

14. A compound according to claim 7, or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic kidney disease, hyperphosphatemia, secondary hyperparathyroidism, heart failure, hypertension or cardiovascular disease.

15. A compound according to claim 7, or a pharmaceutically acceptable salt thereof, for use in simultaneous, separate or sequential combination with a compound of the formula: ##STR00026## or a pharmaceutically acceptable salt thereof, in the treatment of chronic kidney disease, hyperphosphatemia, secondary hyperparathyroidism, heart failure or cardiovascular disease.

16. A compound of the formula: ##STR00027## or a pharmaceutically acceptable salt thereof, for use in simultaneous, separate or sequential combination with the compound according to claim 7, or a pharmaceutically acceptable salt thereof, in the treatment of chronic kidney disease, hyperphosphatemia, secondary hyperparathyroidism, heart failure or cardiovascular disease.

17. A pharmaceutical composition comprising a compound according to claim 7, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable excipient, carrier, or diluent.

18. The pharmaceutical composition according to claim 17 additionally comprising a compound of the formula: ##STR00028## or a pharmaceutically acceptable salt thereof.

Description

EXAMPLE 1

2-Cyano-1-[4-[[N′-cyano-N-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]carbamimidoyl]amino]butyl]-3-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]guanidine

[0057] ##STR00017##

[0058] To a solution of N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide (prepared according to Preparation 5; 740 mg, 1.47 mmol) in 1,4-dioxane (10 mL) and pyridine (10 mL) add diphenoxymethylenecyanamide (351 mg, 1.47 mmol) and stir at RT under nitrogen overnight. Add butane-1,4-diamine (65 mg, 0.74 mmol) and heat at 60° C. for 24 h, then heat at 90° C. overnight. Cool the reaction mixture to RT. Dilute the reaction mixture with EtOAc, wash with 5% aqueous potassium carbonate twice, dry over anhydrous magnesium sulfate, filter and concentrate in-vacuo to obtain an orange oil. Purify the crude mixture by silica gel column chromatography eluting with a gradient of 0 to 10% MeOH in DCM to obtain the title compound as a yellow foam (591 mg, 67%). ES/MS m/z (.sup.35Cl) 1193 [M+H].sup.+

Alternative Synthesis

[0059] To a solution of N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide (prepared according to Preparation 6; 90 wt % purity, 24.5 g, 44 mmol) in 1,4-dioxane (240 mL) add pyridine (17.7 mL, 219 mmol) and diphenoxymethylenecyanamide (10.5 g, 44 mmol) and stir at

[0060] RT under nitrogen overnight. To the reaction mixture, add butane-1,4-diamine (1.93 g, 21.9 mmol), split the reaction into four equal portions, and heat each portion to 90° C. for 21 h. Add butane-1,4-diamine (102 mg, 1.16 mmol) and 1,4-dioxane (5 mL) to each portion of the reaction and continue heating at 90° C. for 23 h. Add butane-1,4-diamine (42 mg, 0.48 mmol) and 1,4-dioxane (5 mL) to portions of the reaction mixture showing greater than a 1:5 ratio of cyanoisourea intermediate : title compound as determined by LC-MS. Continue heating all portions of the reaction mixture at 90° C. overnight and then cool to RT. Combine the reaction portions. Combine the mixture with two other batches prepared in essentially the same way using N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide (90 wt % purity) (10.05 g, 18.00 mmol and 5.20 g, 9.32 mmol respectively). Remove the solvents under reduced pressure, dissolve the residue in DCM (250 mL), then wash the organics with 5% aqueous potassium carbonate (2×80 mL). Combine the aqueous washes and extract with DCM (2×40 mL). Combine all organic washes together and wash with saturated aqueous sodium chloride (80 mL). Dry the organics over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a yellowish brown oil. Purify the crude material by silica gel column chromatography eluting with a gradient of 8 to 16% EtOH in DCM. Then re-purify the material twice by silica gel chromatography eluting with gradients of 5 to 12% and 2 to 12% MeOH in DCM. Re-purify impure fractions by silica gel chromatography with a gradient of 2 to 12% MeOH in DCM and combine pure materials to obtain the title compound as an off-white foamy solid (15.3 g, 36%). ES/MS m/z (.sup.35Cl) 1193 [M+H].sup.+.

EXAMPLE 2

[[4-[[N′-Carbamoyl-N-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]carbamimidoyl]amino]butylamino]-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethylamino]methylene]urea dihydrochloride

[0061] ##STR00018##

[0062] To Example 1 (670 mg, 0.562 mmol) add trifluoroacetic acid (10 mL) and water (1 mL) and stir at RT overnight. Remove volatiles under reduced pressure. Dissolve residue in a minimum amount of MeOH and purify through a 10 g Isolute® SCX column to obtain a yellow foam (629 mg). Purify by preparative HPLC [column: Phenomenex® Kinetex® EVO C.sub.18 100×30 mm, 5 μm particle size, inline heater at 50° C.; eluent: 51 to 86% (5% MeOH in 10 mM NH.sub.4HCO.sub.3) in ACN] to obtain the title compound as a free base as an orange oil (253 mg, 37%). ES/MS m/z (.sup.35Cl) 1229 [M+H].sup.+. In a vial, dissolve the free base compound (223 mg, 0.181 mmol) in DCM (0.5 mL) and add hydrochloric acid (1M solution in diethyl ether, 1 mL) drop wise with shaking. White solids precipitate out of solution. Shake the mixture for 5 min and then concentrate in-vacuo to obtain the title compound as a fine white powder (236 mg, 100%). ES/MS m/z (.sup.35Cl) 1229 [free base M+H].sup.+, 1227 [free base M−H].sup.+

Alternative Synthesis

[0063] Prepare two mixtures of equal portions in the following manner: For each portion, mix the compound of Example 1 (5.1 g, 4.3 mmol), 4 M hydrochloric acid in dioxane (50 mL, 200 mmol) and water (25 mL). Heat each solution to 65° C. for 2 h. Cool the mixtures to RT and stir at RT overnight, then combine them together with another batch prepared in essentially the same manner starting with the compound of Example 1 (3.1 g, 2.6 mmol). Concentrate the mixture in-vacuo. Add water/EtOH (1:2, 100 mL) to the residue and concentrate in-vacuo. Repeat this operation three times. To the residue add EtOH and stir at 45° C. for 10 min, then concentrate in-vacuo. Repeat this operation three times. Dry the residue in-vacuo at RT for 22 h. To the residue, add another batch of product prepared in essentially the same manner as described above with Example 1 (1 g, 0.8 mmol). Dry in-vacuo at RT for 23 h, 50° C. 16 h, 55° C. 6 h, and at RT for 72 h to yield the title compound (14.7 g, 94%). ES/MS m/z (.sup.35Cl) 1229 [free base M+H].sup.+.

Assays

In Vitro NHE3 Inhibition Activity

[0064] To create an NHE3 assay, cells selectively expressing NHE3 are generated by overexpressing human NHE3 in an endogenously NHE-deficient cell line. The NHE-deficient cell line is created by chemical-induced mutagenesis. After the mutagenesis, NHE-deficient cells are selected by subjecting lithium (Li) loaded cells to an acidic extracellular environment. Under these conditions, NHE exchanges intracellular Li.sup.+ for extracellular protons, thus only NHE-deficient cells can avoid a toxic intracellular acidosis and survive.

[0065] An NHE-deficient cell line, NHD C8, is generated by mutagenesis and selection from the cell line Dede (ATCC® CCL-39™). The mutagenesis is induced by treating Dede CCL-39 cells with EMS (Sigma-Aldrich) in growth media at a final concentration of 500 μg/ml for 20 h. NHE-deficient cells are selected by culturing in a LiCl solution (130 mM LiCl, 5 mM KCl, 1 mM MgSO.sub.4, 2 mM CaCl.sub.2, 5 mM glucose and 20 mM Hepes-Tris, pH 7.4) for 2 h followed by culturing in a choline chloride solution (130 mM choline chloride, 5 mM KCl, 1 mM MgSO.sub.4, 2 mM CaCl.sub.2, and 20 mM 2-(N-morpholino) ethanesulfonic acid-Tris, pH 5.5) for 30 min. The surviving cells after growing in standard culture media to 90% confluence are subjected to a second cycle of selection. Single-clone colonies are isolated, grown to full confluence, and are subjected to a third cycle of selection. Surviving clones are expanded and tested for the activity of sodium-hydrogen exchanger (see assay below).

[0066] The cDNA encoding human NHE3 with a myc-tag is subcloned into plasmid pcDNA3.1, and a stable cell line is generated in the above-mentioned NHE-deficient NHD C8 cells. Stably NHE3 over-expressing cells are maintained in McCoy's 5A medium (Hyclone-GE Healthcare Bio science) with 10% FBS, 400 mg/ml G418 (Gibco-ThermoFisher Scientific), and antibiotic/antimycotic solution.

[0067] To measure NHE activity, the intracellular pH of the cells is reduced by addition of NH.sub.4Cl. The sodium-dependent intracellular pH change is then measured by the intracellular pH-sensitive fluorescein dye, BCECF-AM (Molecular Probes-ThermoFisher Scientific). NHD8/NHE3 cells are dispersed with a multi-channel pipette into 96-well poly-D-lysine plates (Corning) at 30,000 per well. The cells are incubated at 37° C. plus 5% CO.sub.2 for 24 h. Cell culture media are aspirated, cells are washed with 100 μl of NaCl-HEPES solution (100 mM NaCl, 10 mM glucose, 5 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.1% BSA, 50 mM HEPES, pH 7) twice, and then cells are incubated with 100 μl of 5 μM BCECF-AM in NH.sub.4Cl/pluronic F-127/probenecid solution (130 mM NH.sub.4Cl, 5 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.1% BSA, 0.0625% pluronic F-127, 2 mM probenecid, 20 mM HEPES, pH 7) for 60 min at RT. Following the incubation, cells are washed with 100 μl of ammonium free, sodium free, HEPES/0.1% BSA solution (100 mM choline chloride, 10 mM glucose, 5 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.1% BSA, 50 mM HEPES, pH 7) twice. 86 μl of ammonium free, sodium free, HEPES/0.1% BSA containing 30 μM amiloride with compound or controls are then added to the appropriate wells. The NHE activity is initiated by the addition of 14 μl of 1M NaCl to achieve a final concentration of 140 mM. The plate is immediately read for fluorescent intensity at an excitation wavelength of 505 nm and emission wavelength of 550 nm. Percentage of inhibition at each concentration tested is calculated relative to NHE activity with 1% DMSO as 0% inhibition and NHE activity with a saturating concentration of a standard inhibitor as 100% inhibition. A 9-concentration response curve from 1000 nM to 0.152 nM plus no compound is fitted to a 4-parameter model using Prism to determine the IC.sub.50.

[0068] Example 2 inhibits human NHE3 in a concentration-dependent manner with an IC.sub.50 of 5.76 nM (geometric mean of individual IC.sub.50, n=7 with a standard error of geometric mean of 0.72). Example 2 is selective for NHE3, in that it was not a potent NPT2b (IC.sub.50=58.4 μM) or SGLT1 (IC.sub.50=6.6 μM) inhibitor. Example 1 inhibits human NHE3 in a concentration-dependent manner with an IC.sub.50 of 37.9 nM (geometric mean of individual IC.sub.50, n=2 with a standard error of geometric mean of 6.2).

Effect of Example 2 on Blood Pressure

[0069] Male spontaneously hypertensive (SHR) rats weighing 250-300 g with age of 7 weeks are acclimated to a reversed light cycle (8:00 am lights off and 8:00 pm lights on) and fed regular chow and water ad libitum. Implantable telemetry devices (model TA11PA-C40, Data Sciences International) are implanted into the abdominal aorta. After recovering from surgery, rats are placed in individual cages in a quiet telemetry facility room for measuring blood pressure (mean arterial pressure, MAP) and heart rate (HR). Digitized pressure signals are acquired for 20 second every 5 min using DSI Dataquest IV 4.0 software.

[0070] The 31 SHR rats are randomized by MAP into two groups and treated twice daily for 14 days with vehicle (n=15) (10% acacia, 0.05% antifoam in purified water) or 0.15 mg/kg/day Example 2. The first dose is given between 6:00 and 6:30 am (2 h prior to lights off) and the second dose is given between 4:00 and 4:30 pm. The daily food consumption and body weight gain are also monitored.

[0071] MAP, HR, and body weight (BW) data are analyzed by repeated measures analysis of covariance (ANCOVA) with baseline as the covariate. Food consumption (FC) data are analyzed by repeated measures analysis of variance (ANOVA). The mean blood pressure across the time course of the study (days 1-14) for the group treated with 0.15 mg/kg/day of Example 2 is 4.83 mm Hg less than that of the control group with a p-value of 0.0007 (Table 1). No significant effect is observed on HR, BW, or FC.

TABLE-US-00001 TABLE 1 Effect of Example 2 on mean arterial pressure (MAP) in male spontaneously hypertensive rats (SHR) Treatment Mean blood pressure Difference group over days 1-14 (mm Hg) SEM (mm Hg) p-value Vehicle 158.16 0.85 4.83 0.0007 Example 2 153.33 0.97 0.15 mg/kg/day

Effect of Example 1 on Sodium Absorption

[0072] Urinary sodium excretion following an oral bolus dose of sodium chloride is an indirect measure of sodium absorption in the intestine.

[0073] Male Sprague-Dawley with body weights ranging from 200 to 250 g are randomized into groups based on equal mean body weights. Henceforth the following formulation is referred to as “1% HEC”: 1% hydroxyethyl cellulose (HEC) and 0.25% Tween® 80 with antifoam vehicle in water. Example 1 at 0.1 mg/mL (final dose 1 mg/kg dosed in 10 mL/kg volume) is formulated with 1% HEC in a glass vial containing a pre-weighed amount of compound, and then probe sonicating until it appears as a uniform suspension. To ensure that the compound does not adhere to the sides or bottom of the vial, a stir bar is added to the bottle and the suspension is stirred throughout the formulation and dosing process. The subsequent dosing solutions of Example 1 are prepared by serial dilutions with 1% HEC. NaCl at 10 mg/ml (final dose 200 mg/kg dosed in 20 ml/kg volume) is formulated by adding sterile water.

[0074] On the day before the study, the animals are placed into clean cages without food but with access to water for an overnight fast. On the day of the study, rats are orally dosed with vehicle or varying doses of Example 1. 30 min later, animals are dosed with NaCl, then immediately transferred to metabolic cages. Urine samples are collected for 2 h. Net urine volume is recorded. Urinary sodium, and creatinine are assessed using a clinical biochemistry analyzer.

[0075] Values expressed as the ratio of urinary sodium to creatinine (mM/mM) are calculated and presented as mean±SEM. The curves are fitted with 4-parameter logistic curve fitting tool GraphPad Prism 6 to calculate the ED50. For the purpose of curve fitting, the dose of Example 1 is artificially set to 0.001 mg/kg in the software for the vehicle-alone group.

[0076] Following oral administration of Example 1 and a NaCl bolus, urinary sodium excretion decreases in a dose dependent fashion (Table 2). The ED.sub.50 of Example 1 on urinary sodium excretion is 0.058 mg/kg. The dose-dependent decrease in urinary sodium excretion is consistent with a dose-dependent inhibition of intestinal sodium absorption with Example 1.

TABLE-US-00002 TABLE 2 Effect of Example 1 on Urinary Sodium in Rats Urinary Sodium to Creatinine Ratio Treatment (mM/mM, Means ± SEM) Vehicle (n = 8) 1.43 ± 0.16 Example 1, 0.03 mg/kg (n = 5) 1.19 ± 0.19 Example 1, 0.1 mg/kg (n = 5) 0.80 ± 0.23 Example 1, 0.3 mg/kg (n = 5) 0.37 ± 0.06 Example 1, 1.0 mg/kg (n = 5) 0.63 ± 0.23

Effect of Example 2 on Sodium and Phosphate Absorption

[0077] Urinary sodium and phosphate excretion following an oral bolus dose of sodium phosphate is an indirect measure of sodium and phosphate absorption in the intestine.

[0078] Male Sprague-Dawley rats at an age of about 7 weeks with body weights ranging from 195 to 221 g are randomized into groups based on equal mean body weights. Example 2 at 0.3 mg/mL (final dose 3 mg/kg dosed in 10 mL/kg volume) is formulated by adding 1% HEC to a glass vial containing a pre-weighed amount of compound, and then probe sonicating until it appears as a uniform suspension. To ensure that the compound does not adhere to the sides or bottom of the vial, a stir bar is added to the bottle and the suspension is stirred throughout the formulation and dosing process. The subsequent dosing solutions of Example 2 are prepared by serial dilutions with 1% HEC. NaH.sub.2PO.sub.4 at 34.5 mg/ml (final dose 690 mg/kg dosed in 20 ml/kg volume) is formulated by adding sterile water.

[0079] On the day of the study, the animals are placed into clean cages without food but with access to water for a 4 h fast before the study. Rats are orally dosed with vehicle or varying doses of Example 2. 15 min later, animals are dosed with sterile water or NaH.sub.2PO.sub.4, then immediately transferred to metabolic cages. Urine samples are collected for 4 h. Net urine volume is recorded. Urinary sodium, creatinine, and phosphate are assessed using a clinical biochemistry analyzer. Values expressed as the ratio of urinary-to-dietary phosphorus or sodium are calculated and presented as mean±SEM. Curves are fitted with a 4-parameter logistic curve fitting tool GraphPod Prism 6 to calculate the ED50. For the purpose of curve fitting, the dose of Example 2 is artificially set to 0.00001 mg/kg in the software for the vehicle-alone group.

[0080] Following oral administration of Example 2 and a phosphate bolus, urinary sodium and phosphate excretion decrease in a dose dependent fashion (Table 3). The ED.sub.50 of Example 2 on urinary phosphorus excretion is 0.041 mg/kg and ED.sub.50 of Example 2 on sodium excretion is 0.058 mg/kg. The dose-dependent decrease in urinary sodium and phosphate excretion is consistent with a dose-dependent inhibition of intestinal sodium and phosphate absorption with Example 2.

TABLE-US-00003 TABLE 3 Effect of Example 2 on Urinary Phosphorus and Sodium in Rats Ratio of Urinary-to- Ratio of Urinary-to- Dietary Phosphorus Dietary Sodium Treatment (Means ± SEM) (Means ± SEM) Vehicle 0.297 ± 0.039 0.271 ± 0.031 Example 2, 0.001 mg/kg 0.337 ± 0.017 0.326 ± 0.042 Example 2, 0.003 mg/kg 0.350 ± 0.023 0.413 ± 0.055 Example 2, 0.01 mg/kg 0.320 ± 0.042 0.290 ± 0.06  Example 2, 0.03 mg/kg 0.268 ± 0.04  0.276 ± 0.06  Example 2, 0.1 mg/kg 0.197 ± 0.045 0.105 ± 0.042 Example 2, 0.3 mg/kg 0.168 ± 0.027 0.039 ± 0.010 Example 2, 1 mg/kg 0.191 ± 0.039 0.051 ± 0.027 Example 2, 3 mg/kg 0.144 ± 0.009 0.022 ± 0.009
Effect of Example 2 in Combination with an NPT2b Inhibitor (Compound A) on Phosphate Absorption in Rats

[0081] NPT2b inhibitor dosing solutions: Compound A and poly-1-vinylpyrrolidone-co-vinyl acetate (PVP-VA, Sigma-Aldrich) are weighed into a bottle at a ratio of 30% Compound A and 70% PVP-VA by weight. A clear yellow solution is prepared by diluting the mixture in MeOH followed by the addition of 2 mol NaOH per mol Compound A using 5 N NaOH. The solution is spray dried by a stream of hot nitrogen and the solid dispersion powder is collected and then further dried in a vacuum oven. Doses for the sprayed dried solid dispersion (SDD) are expressed as active pharmaceutical ingredient (API) throughout.

[0082] To make the Compound A dosing solution, an appropriate amount of Compound A SDD is weighed in a vial and dissolved in water (10 mL/kg dosing volume). To ensure that the compound does not adhere to the sides or bottom of the vial, a stir bar is added to the bottle and the suspension is stirred throughout the formulation and dosing processes. The subsequent solutions for lower doses are prepared by serial dilutions with water. PVP-VA at 7 mg/mL is used as a vehicle control.

[0083] NHE3 inhibitor dosing solutions: To make the Example 2 dosing solution, an appropriate amount of the compound is weighed in a vial and dissolved in 1% HEC (10 mL/kg dosing volume). To ensure that the compound does not adhere to the sides or bottom of the vial, a stir bar is added to the bottle and the suspension is stirred throughout the formulation and dosing processes. The subsequent solutions for lower doses are prepared by serial dilutions with HEC.

[0084] To make the radiolabeled phosphate dosing solution, a 16.25 mM Na.sub.2HPO.sub.4, 0.9% saline, pH 7.4 solution is prepared and filtered using a sterile, 0.22 μm, polyethersulfone, Millex-GP Syringe Filter Unit (EMD Millipore). Radioactive phosphate (H.sub.3.sup.33PO.sub.4, Perkin Elmer) is added at about 2.5 μCi per mL of the solution and filtered again.

[0085] Three separate studies are performed. In these studies, male Sprague Dawley rats, are randomized into groups with approximately equal mean body weights. Following an overnight fast, all the animals are dosed at 10 mL/kg with either vehicle, varying doses of Compound A (Study 1), varying doses of Example 2 (Study 2) or 1.2 mg/kg Compound A and varying doses of Example 2 (Study 3). 15 min later, radiolabeled phosphate is orally dosed in a 2 mL volume. 15 min later, blood is collected and plasma is prepared. Radioactivity (dpm) in 50 μL plasma is measured by scintillation counting and is used to calculate phosphate absorption. The results are normalized with the vehicle controls representing 100% absorption. The curves are fitted with nonlinear regression with variable slope using GraphPad Prism 6 to calculate the ED.sub.50 and E.sub.max given in Table 7. For the purposes of curve fitting in the ED.sub.50 calculation, the dose of Example 2 is artificially set to 0.00001 mg/kg in the software for the vehicle-alone group in Study 2, and also for the vehicle+1.2 mg/kg Compound A group in Study 3. No curve fit was performed for Compound A alone in Study 1.

[0086] The results of the three separate studies performed in rats, a dose response study for Compound A (Study 1), a dose response study for Example 2 (Study 2) and a dose response study for Example 2 in the presence of 1.2 mg/kg Compound A (Study 3) are summarized in Tables 4-7 below. Example 2 and Compound A both inhibit phosphate absorption in a dose dependent fashion with a percent inhibition at the highest dose tested of 37% and 18%, respectively. However, when given in combination, an inhibition of 70% is achieved at the highest dose of Example 2 in combination with 1.2 mg/kg Compound A. Thus, the two compounds are more effective when dosed together than when either compound is dosed alone, consistent with each compound inhibiting distinct pathways contributing to intestinal phosphate absorption in rats.

TABLE-US-00004 TABLE 4 Effect of Compound A on Phosphate Absorption in Rats (Study 1) Absorption (vs. vehicle Treatment control, %, means ± SEM) Vehicle .sup. 100 ± 7.23 Compound A, 0.01 mg/kg 94.39 ± 7.88 Compound A, 0.1 mg/kg 87.61 ± 6.08 Compound A, 1 mg/kg 87.64 ± 7.26 Compound A, 10 mg/kg 81.53 ± 5.50

TABLE-US-00005 TABLE 5 Effect of Example 2 on Phosphate Absorption in Rats (Study 2) Absorption (vs. vehicle Treatment control, %, means ± SEM) Vehicle 100.00 ± 14.22  Example 2, 0.001 mg/kg 97.16 ± 20.76 Example 2, 0.003 mg/kg 100.81 ± 20.74  Example 2, 0.01 mg/kg 75.15 ± 15.52 Example 2, 0.03 mg/kg 83.17 ± 17.99 Example 2, 0.1 mg/kg 57.29 ± 11.20 Example 2, 0.3 mg/kg 71.09 ± 13.98 Example 2, 1 mg/kg 44.29 ± 9.09  Example 2, 3 mg/kg 60.04 ± 12.00 Example 2, 10 mg/kg 63.47 ± 8.39 

TABLE-US-00006 TABLE 6 Effect of Compound A and Example 2 in Combination on Phosphate Absorption in Rats (Study 3) Absorption (vs. vehicle Treatment control, %, means ± SEM) Vehicle + Compound A, 1.2 mg/kg  71.00 ± 11.63 Example 2, 0.001 mg/kg ± 85.61 ± 5.16 Compound A, 1.2 mg/kg Example 2, 0.003 mg/kg ± 70.98 ± 7.27 Compound A, 1.2 mg/kg Example 2, 0.01 mg/kg ± 73.53 ± 7.29 Compound A, 1.2 mg/kg Example 2, 0.03 mg/kg + 61.82 ± 6.73 Compound A, 1.2 mg/kg Example 2, 0.1 mg/kg + 44.78 ± 6.09 Compound A, 1.2 mg/kg Example 2, 0.3 mg/kg + 35.56 ± 2.86 Compound A, 1.2 mg/kg Example 2, 1 mg/kg + 35.52 ± 8.67 Compound A, 1.2 mg/kg Example 2, 3 mg/kg + 26.94 ± 4.94 Compound A, 1.2 mg/kg Example 2, 10 mg/kg + 29.83 ± 3.72 Compound A, 1.2 mg/kg

TABLE-US-00007 TABLE 7 Example 2 ± 1.2 mg/kg Compound A Example 2 Compound A ED.sub.50 (mg/kg) No curve fit 0.041 0.056 % Inhibition at 18% 37% 70% the highest dose
Effect of Example 2 in Combination with an NPT2b Inhibitor (Compound A) on Phosphate Absorption and Intestinal Phosphate Retention in Rats

[0087] The purpose of this study is to investigate in rats the effect of Example 2, Compound A, and an Example 2/Compound A combination on phosphate absorption 15 min after dosing the compounds and the phosphate retained in the intestine 4.25 h after dosing the compounds. The latter is a measure of the oral phosphate load which is absorbed over an extended period.

[0088] Two vehicles are used in this study: 1) 1% HEC and 2) 0.46% poly-1-vinylpyrrolidone-co-vinyl acetate in water (PVP-VA vehicle). The vehicle control group in this study receives a 1:1 combination of the two vehicles.

[0089] The Example 2, Compound A and phosphate dosing solutions are prepared similarly as described above, except the doses are 0.4 mg/kg for Example 2 and 10 mg/kg for Compound A and they are prepared at a concentration where they would be dosed in a 5 mL/kg volume. The Example 2 and Compound A alone groups are mixed 1:1 with the vehicle of the other compound, while the Compound A and Example 2 are mixed 1:1 prior to dosing. The final dosing volume is 10 mL/kg in all cases.

[0090] Male Sprague Dawley rats are fasted 4 h and then administered appropriate vehicles, 10 mg/kg Compound A, 0.4 mg/kg Example 2, or a combination of both compounds. 15 min later, the animals are dosed with radiolabeled phosphate solution. 15 min later, blood is collected and plasma is prepared. Radioactivity in plasma is measured by scintillation counting and used to calculate the inhibition of phosphate absorption. While Example 2 (0.4 mg/kg) inhibits phosphate absorption by 29% (P=0.046 versus vehicle control) and Compound A (10 mg/kg) inhibits phosphate absorption by 35% (P=0.013 versus vehicle control), the combination of the two compounds inhibits phosphate absorption by 63% (P=0.0001 versus vehicle control). The 63% inhibition by the Example 2/Compound A combination exceeds the inhibition by either compound alone. Data are presented as mean±SEM with animal numbers equal to 8 for the groups. Statistical significance is determined by ANOVA with a Dunnett's comparison to the Example 2/Compound A combination using JMP 12.1.

[0091] 4 h after the radiolabeled phosphate solution is dosed, stomach, small intestine, large intestine, and feces are collected, weighed and digested with 1N NaOH overnight at 37° C. Radioactivity in each fraction is measured by scintillation counting.

[0092] The percent dose recovered in gastrointestinal tract is defined as radioactivity recovered in stomach, small intestine, large intestine and feces compared to the amount dosed. Data is presented as mean±SEM with animal numbers equal to 8 for the groups. Statistical significance is determined by ANOVA with a Dunnett's comparison to the Example 2/Compound A combination using JMP 12.1.

TABLE-US-00008 TABLE 8 The Percent Dose Recovered in Each Section of Gastrointestinal Tract. Treatment Stomach Small Intestine Large Intestine Feces Total Control, 5.50 ± 0.89 8.03 ± 0.54 11.83 ± 1.09 <0.1 25.36 ± 1.27 PVP + HEC Example 2,  3.9 ± 0.49 8.64 ± 0.32 18.16 ± 2.58 <0.1  30.7 ± 2.43 0.4 mg/kg Compound A, 4.86 ± 1.13 8.98 ± 0.48 18.43 ± 2.03 <0.1 32.27 ± 1.38 10 mg/kg Example 2 + 6.23 ± 0.80 10.76 ± 0.67  28.98 ± 1.93 <0.1 45.96 ± 1.33 Compound A
Percent Radiolabeled Phosphate Dose Recovered from each Section of the GI Tract, Mean±SEM

[0093] Total radioactivity recovered is greater in animals dosed with both Example 2 and Compound A (46%) than with either one alone (31% and 32%, respectively) (Table 8). Thus, the combination is more effective than either compound alone. The percent dose recovered in gastrointestinal tract data is further analyzed by a two-way ANOVA in JMP 12.1 to test for additivity of the effects of Example 2 and Compound A on inhibiting phosphate absorption. The test of Example 2/Compound A interaction is significant (p=0.0187) indicating a synergistic relationship between the compounds at the doses tested in this study. That is, the inhibitory effect of the combination is greater than the sum of the individual compound effects.