6 -Benzylphenyl-2 - sulfurterahydropyran-3, 4, 5 -triol derivatives as inhibitors of sodium -glucose cotrans porters 1 and 2 for use in diabetic patients
A dual inhibitor, patient technology, which can be used in medical preparations containing active ingredients, metabolic diseases, drug combinations, etc., and can solve the problems of glucose-galactose absorption disorder, high urinary sugar excretion, etc.
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[0258] In vitro human SGLT2 inhibition assay
[0259] Human sodium / glucose cotransporter type 2 (SGLT2; accession number P31639; GI:400337) was cloned in the pIRESpuro2 vector for mammalian expression (construct: HA-SGLT2-pIRESpuro2).
[0260] HEK293 cells were transfected with the human HA-SGLT2-pIRESpuro2 vector, and the main stable cell line was selected in the presence of 0.5 μg / ml puromycin. Human HA-SGLT2 cells were maintained in DMEM medium containing 10% FBS, 1% GPS and 0.5 μg / ml puromycin.
[0261] HEK293 cells expressing human HA-SGLT2 were seeded in 384-well plates (30,000 cells / well) in DMEM medium containing 10% FBS, 1% GPS, and 0.5 μg / ml puromycin, and then incubated at 37 °C and 5 %CO 2 Incubate overnight. Cells were then washed with uptake buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10mM HEPES, 5mM Tris, 1mg / ml bovine serum albumin (BSA), pH 7.3) for washing. 20 microliters of uptake buffer with or without test compound was added to the c...
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