Mizuma Takashi, Fuseda Norihiko, Hayashi Masahiro
Department of Drug Absorption and Pharmacokinetics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
J Pharm Pharmacol. 2005 May;57(5):661-4. doi: 10.1211/0022357055948.
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.
糖苷酶活性影响糖苷的肠道吸收。我们之前在大鼠身上进行的研究表明，二糖缀合物可能是前体药物的原型，旨在通过钠/葡萄糖共转运体介导的前体药物（药物葡糖苷）转运作为一种新的吸收途径。关键因素之一是从二糖缀合物形成葡糖苷药物。由于代谢存在很大的种属差异，有必要检测源自人体组织的细胞和/或酶来证实这一概念。在本文中，我们对Caco-2细胞中二糖缀合物的糖苷酶活性进行了动力学表征。对硝基苯酚（p-NP）的二糖缀合物（p-NPβ-纤维二糖苷、p-NPβ-乳糖苷和p-NPβ-麦芽糖苷）被水解为p-NPβ-葡糖苷。β-葡糖苷酶或β-半乳糖苷酶（乳糖酶/根皮苷水解酶，LPH）以及α-葡糖苷酶（蔗糖酶-异麦芽糖酶）对二糖缀合物具有不同的pH依赖性活性。在中性pH条件下，LPH亲和力低、容量低，而蔗糖酶-异麦芽糖酶亲和力高、容量高；而在酸性pH条件下，LPH亲和力高、容量低，蔗糖酶-异麦芽糖酶亲和力低、容量高。用Vmax/Km计算的水解清除率表明，在Caco-2细胞中，无论是中性还是酸性pH条件下，蔗糖酶-异麦芽糖酶的活性都远高于LPH活性。由于二糖缀合物的水解速率高度依赖于pH值和糖苷键类型，在考虑这些差异后适当选择糖苷形式是设计糖缀合物前体药物的关键。
