(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxyb-D-glucopyranosyl)-a-D-glucopyranoside (CS-1036), which is an a-amylase inhibitor, exhibited biphasic and sustained elimination with a long t 1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t 1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t 1/2 , the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (K D ) of 8.95 and 27.2 nM, and maximal binding capacity (B max ) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (K D 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (£10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro K D and B max , the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.