2.2.2. DPP4 inhibition assay
Inhibitory activities against human DPP4 were measured using a
fluorogenic DPP4 Assay Kit (BPS Bioscience, San Diego, CA). Test
compound solution, DPP substrate, and human recombinant DPP4
were mixed to initiate the enzyme reaction. After a 10 min reaction
at room temperature, the fluorescence intensity was measured
using a microplate reader (Molecular Devices, Sunnyvale, CA).
Inhibitory activities against rat DPP4 were measured using
serum collected from 7-week-old male SpragueeDawley (SD) rats
(Charles River Japan, Yokohama, Japan). Rat serum and Glycyl-Lproline
4-methylcoumaryl-7-amide (MCA) were mixed with PBS
containing 0.003% Brij-35 to initiate the enzyme reaction, as previously
described (18). The fluorescence intensity of MCA was
measured using a microplate reader after a 1-h incubation at 37 C.