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2843
W.; Gaillard, R. C.; Grossman, A. B.; Kola, B.; Lacroix,
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11b-HSD1 enzyme inhibition was assessed in HEK293
cells stably transfected with the full length human
hsd11b1 gene. HEK293 cells were plated in 96-well
poly-D-Lys coated flat-bottomed microplates in
DMEM containing 1% glutamine, 1% penicillin and
streptomycin. Compounds were added to plates such
that the final concentration of DMSO was 1%. Tritiated
cortisone was added at a final concentration of 20nM
and the cells incubated at 37 °C in 5% CO2, 95% O2 for
2 h. The assay solutions were transferred to a scintil-
lation microplate and mixed with a solution of anti-
mouse YSi SPA beads and anti-cortisol antibody in
assay buffer (50mM Tris.HCl, pH 7.0; 300 mM NaCl,
1mM EDTA, 5% glycerol). The plate was incubated for
2 h at room temperature and read on a scintillation
counter. The percentage inhibition was determined
relative to a non-inhibited control and the median
inhibitory concentration (IC50) determined by plotting
fractional inhibition against log compound concentra-
tion. Data were fitted to the four parameter logistic
equation. Murine 11b-HSD1 enzyme inhibition was
assessed in CHO cells stably transfected with the full
length murine hsd11b1 gene. Enzyme inhibition was
determined as described for human 11b-HSD1 follow-
ing a 4 h incubation of cells, compound and substrate.
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14. Male C57BL/6 mice (25–30 g in weight) were group
housed and allowed free access to food and water.
Compounds were dissolved in 5% DMSO, 3% ethanol,
4 mM cyclodextrin. Animals (n = 2 per group) were
dosed intraperitoneally with vehicle or compound at
12-hourly intervals. At 1 h following the third dose
mice were euthanized by cervical dislocation. Liver,
adipose, kidney and brain samples were removed and
frozen until analysis was performed. Inhibition of 11b-
HSD1 in each tissue was determined by incubating
homogenates with 20 nM tritiated corticosterone, 2mM
NADP+ and 0.2% glucose in Krebs buffer, pH 7.4.
Dehydrocorticosterone and corticosterone levels were
measured by HPLC and the percentage inhibition
determined relative to vehicle treated tissue.