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18. (a) Yang, Z. Q.; Lorenz, J. C.; Shi, Y. Tetrahedron Lett. 1998, 39, 8621; (b) Lorenz, J.
C.; Long, J.; Yang, Z. Q.; Xue, S.; Xie, Y.; Shi, Y. J. Org. Chem. 2004, 69, 327.
19. Kulinkovich, O. G.; Sviridov, S. V.; Vasilevski, D. A. Synthesis 1991, 234.
high selectivity toward SGLT2, and some of them elicited a high
level of glucosuria from laboratory animals.
20.
A plasmid bearing the human full-length SGLT1 coding sequence in the
pDream 2.1 mammalian expression vector was purchased from GenScript
Corporation. A full-length human SGLT2 cDNA (GenScript Corporation) was
cloned into the pEAK15 mammalian expression vector. Human SGLT1
expression plasmid DNA was transfected into COS-7 cells (American Type
Culture Collection) using Lipofectamine 2000 (Invitrogen Corporation).
Acknowledgments
We gratefully acknowledge Ying Chen for in vitro activity test-
ing and the analytical group of Egret Pharma (Shanghai) Limited
for analytical service.
Transfected cells were evaluated for SGLT1 activity in methyl-a-D-
[U-14C]glucopyranoside (AMG) uptake assay and cryopreserved until use.
Plasmid containing human SGLT2 was linearized and stably transfected into
293.ETN cells. SGLT2-expressing clones were selected based on resistance to
puromycin (Invitrogen Corporation) and activity in AMG uptake assay. Cells
expressing SGLT1 or SGLT2 were seeded on 96-well ScintiPlates (PerkinElmer,
References and notes
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twice with 150 lL of either sodium buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM
CaCl2, 1.2 mM MgCl2, 10 mM tris(hydroxymeth-yl)aminomethane/N-2-
hydroxyethylpiperazine-N0-ethanesulfonic acid [Tris/Hepes], pH 7.2) or
sodium-free buffer (137 mM N-methyl-glucamine, 5.4 mM KCl, 2.8 mM
CaCl2, 1.2 mM MgCl2, 10 mM Tris/Hepes, pH 7.2). Test compound in 50 lL
each of sodium or sodium-free buffer containing 40 lCi/mL methyl-a-D-
[U-14C]glucopyranoside (Amersham Biosciences/GE Healthcare) and 25%
human plasma was added per well of a 96-well plate and incubated at 37 °C
with shaking for either 2 h (SGLT1 assay) or 1.5 h (SGLT2 assay). Cells were
washed twice with 150
10 mM Tris/Hepes, pH 7.2) and methyl-
l
L
of wash buffer (137 mM N-methylglucamine,
a-D
-[U-14C]glucopyranoside uptake
was quantitated using a TopCount scintillation counter (PerkinElmer, Inc.).
Compounds were initially screened at 3 different concentrations in triplicate
measurements. Potent and selective SGLT2 inhibitors were further evaluated at
8 concentrations in triplicates. Sodium-dependent glucopyranoside uptake
was calculated by subtracting the values obtained with sodium-free buffer
from those obtained using sodium buffer. In general, ratios of sodium-
dependent to sodium-independent AMG uptake in SGLT1 and SGLT2
expressing cells were 10–15 and 15–20 respectively. Results of AMG uptake
were analyzed using GraphPad Prism (Intuitive Software for Science). IC50
calculations were performed using nonlinear regression with variable slope. As
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6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol was routinely included in
the assays. In 26 independent evaluations, the reference compound inhibited
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lM
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overnight-fasted normal male SD rats by gavage at the dose level of 1 mg/kg.
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collected within metabolic cages from 0 to 24 h post-dosing for urine volume
and glucose measurement. Food was removed 16 h before dosing and then
provided 4 h after dosing. Water was supplied ad libitum. The concentration of
urinary glucose was determined at 24 h post-dose using an Integra 400 Plus
Automatic Biochemistry Analyzer (Roche).
23. Each test compound was dissolved in 10% PEG400 and administered orally to
overnight-fasted normal female Beagle dogs by gavage at the dose level of
0.03 mg/kg. Control dogs were given 10% PEG400 only. One hour post dosing,
glucose solution (2 g/kg, 5 mL/kg) was administered by oral gavage. Urine was
collected within metabolic cages from 0 to 24 h post-dosing for urine volume
and glucose measurement. Food was removed 16 h before dosing and then
provided 3 h after dosing. Water was supplied ad libitum. The concentration of
urinary glucose was determined at 24 h post-dose on the Integra 400 Plus
Automatic Biochemistry Analyzer (Roche).
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