Compounds and methods for inhibiting phosphate transport
By inhibiting small intestinal phosphate uptake using non-NHE3-binding compounds such as guanylyl cyclase C receptor agonists and pH regulators in the gastrointestinal tract, the hypercalcemia problem of existing phosphate binders to treat hyperphosphatemia is solved. symptoms and gastrointestinal discomfort, achieving safe and efficient regulation of serum phosphorus levels and alleviation of renal failure.
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Embodiment 1
[0621] Increased intracellular pH results in decreased phosphate uptake in cells
[0622] Experiments were performed to test the relationship between changes in intracellular pH and the uptake of phosphate ions (Pi) in human embryonic kidney cells (HEK-293 cells).
[0623] HEK-293 cells were seeded into 96-well plates at 25,000 cells / well and cultured overnight. Cells were then transfected with rat or human NaP2b cDNA, or mock-transfected (no DNA) using Lipofectamine 2000 (Invitrogen). Cells were brought near confluency during the second overnight incubation.
[0624] The intracellular pH was lowered from about 7.4 to about 6.8 using an ammonium pulse program. Aspirate the culture medium from the well and wash with NaCl-HEPES buffer (100mM NaCl, 50mM HEPES, 10mM glucose, 5mM KCl, 2mM CaCl 2 , 1mM MgCl 2 , pH 7.4) washed the cells twice, and then mixed with NH containing 5 μM BCECF-AM at room temperature 4 Cl-HEPES buffer (20mM NH 4 Cl, 80mM NaCl, 50mM HEPES, 5mM KCl, 2mM...
Embodiment 2
[0629] Guanylate cyclase C (GC-C) receptor agonists reduce phosphate uptake
[0630] Experiments were performed to determine whether guanylate cyclase C (GC-C) receptor agonists could reduce the 33 Phosphate absorption / uptake in the small intestine measured by P uptake. use 33 P and linaclotide were administered to rats simultaneously as follows:
[0631] 1. Vehicle (N=5 / group)
[0632] 2. Linaclotide at 0.1 mg / kg (N=6 / group)
[0633] 3. Linaclotide at 0.3mg / kg (N=4 / group)
[0634] exist 33 Blood was collected 5, 15, 30, 45 and 60 min after P administration and plasma scintillation counts were performed. The results are shown in Figures 1A-1B middle. Figure 1A The results of a two-way ANOVA using repeated measures followed by Dunnett's multiple comparison test are shown, and Figure 1B Results of one-way ANOVA followed by Dunnett's multiple comparison test are shown. These results showed that both doses of linaclotide decreased phosphate absorption from the gastroi...
Embodiment 3
[0636] I1 receptor agonists and adenylyl cyclase agonists reduce phosphate uptake
[0637] Conduct experiments to determine whether other classes of drugs can reduce 33 Phosphate absorption / uptake in the small intestine measured by P uptake. use 33 P and imidazoline subtype 1 (I 1 ) receptor agonist (moxonidine) or adenylyl cyclase agonist (water-soluble forskolin analogue NKH477) were administered to rats simultaneously as follows:
[0638] 1. Medium
[0639] 2. Moxonidine at 2mg / kg
[0640] 3. Moxonidine at 6mg / kg
[0641] 4. NKH477 at 1mg / kg
[0642] 5. NKH477 at 3mg / kg
[0643] exist 33 Blood was collected 5, 15, 30, 45 and 60 min after P administration and plasma scintillation counts were performed. The results are shown in Figure 2A-2B middle. Figure 2A Shown are the results of a two-way ANOVA using repeated measures, followed by Dunnett's multiple comparison test, and Figure 2B Results of one-way ANOVA followed by Dunnett's multiple comparison test are sh...
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