title: Kinetic characterization of glycosidase activity from disaccharide conjugate to monosaccharide conjugate in Caco-2 cells.
abstract: Glycosidase activity influences the intestinal absorption of glycosides. Our previous study in rats suggested that disaccharide conjugates might be prototypes for pre-prodrugs aiming at the Na(+)/glucose co-transporter-mediated transport of prodrugs (drug glucoside) as a novel absorption pathway. One of the crucial factors is the formation of a glucoside drug from the disaccharide conjugate. Since there is a large species difference in metabolism, it is necessary to examine the cells and/or enzymes derived from human tissue to confirm this concept. In this paper, we kinetically characterized the glycosidase activity of disaccharide conjugates in Caco-2 cells. Disaccharide conjugates of p-nitrophenol (p-NP) (p-NP beta-cellobioside, p-NP beta-lactoside and p-NP beta-maltoside) were hydrolysed to p-NP beta-glucoside. beta-glucosidase or beta-galactosidase (lactase/phloridzin hydrolase, LPH) and alpha-glucosidase (sucrase-isomaltase) had different pH-dependent activities for disaccharide conjugates. At neutral pH, LPH has low affinity and low capacity, and sucrase-isomaltase has high affinity and high capacity, whereas at acid pH, LPH has high affinity and low capacity, and sucrase-isomaltase has low affinity and high capacity. The hydrolysis clearance calculated with Vmax/Km indicated that sucrase-isomaltase activity is much higher than LPH activity at either neutral or acid pH in Caco-2 cells. Since the hydrolysis rate of the disaccharide conjugate was highly dependent on the pH value and type of glycoside linkage, the appropriate selection of a glycoside form after consideration of these differences is the key to designing a sugar-conjugate prodrug.
authors.affiliation: Department of Drug Absorption and Pharmacokinetics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. mizuma@ps.toyaku.ac.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Mizuma
authors.forename: Norihiko
authors.initials: N
authors.lastname: Fuseda
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hayashi
mesh_terms: D018938:Caco-2 Cells; D005960:Glucosides / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics
chemical_list: D005960:Glucosides; D006027:Glycosides; C036988:4-nitrophenylmaltoside; C029976:4-aminophenyl beta-lactoside; C025193:4-nitrophenyl beta-D-glucoside; C034170:4-nitrophenyl beta-cellobioside; D006026:Glycoside Hydrolases