Improvement of Intestinal Absorption of Peptide Drugs by Glycosylation: Transport of Tetrapeptide by the Sodium Ion-Dependent D-Glucose Transporter

Abstract

□ A tetrapeptide (Gly-Gly-Tyr-Arg, GGYR), which is not transported by di- or tripeptide transporters, was glycosylated with p-(succinylamido)phenyl α- or β-D-glucopyranoside (α,β-SAPG) to investigate whether these glycosylated molecules are transported by the Na+-dependent D-glucose transporter. Their uptake into brush-border membrane vesicles (BBMVs) and transport through the intestinal membrane were examined using the rapid filtration technique and the everted sac method. It was observed that glycosylation at the α-amino position of GGYR increased resistance to aminopeptidase activity and inhibited its degradation. When α- and β-SAPG-GGYR were incubated with BBMVs, overshoot uptake was observed about 2min after the start of incubation in the presence of an inward Na+ gradient. This uptake remained unaffected by the addition of GGYR while it was significantly inhibited when Na+ was replaced with K+ or α- and β-SAPG-GGYR were incubated with BBMVs at 4°C. Uptake was also markedly inhibited either with 1mM phloridzin or 10mM D-glucose. These findings suggested that the Na+-dependent glucose transporter (SGLT-1) played an important role in the uptake of both α- and β-SAPG-GGYR into BBMVs. A comparison of α- with β-SAPG-GGYR revealed that the amount of β-SAPG-GGYR taken up was greater than that of α-SAPG-GGYR. From the everted sac method data, it was shown that the elimination clearance from the mucosal side, CLel, and permeation clearance to the serosal side, CLp, were 15.82±6.83 and 0.83±0.06μL/min/cm for α-SAPG-GGYR and 44.52±3.61 and 3.50±0.81μL/min/cm for β-SAPG-GGYR, respectively, and that α-SAPG-GGYR was more resistant to enzymatic degradation than β-SAPG-GGYR. Permeation of both α-and β-SAPG-GGYR was inhibited in the presence of D-glucose and in the absence of a Na+ gradient, suggesting that both α- and β-SAPG-GGYR were transported by the Na+-dependent D-glucose transporter. The permeation clearance transported by the Na +-dependent D-glucose transporter, (CLp)Na+, of β-SAPG-GGYR was about 5 times greater than that for α-SAPG-GGYR. This result may be ascribable to the fact that the β-form of glucose has higher affinity to SGLT-1 than the α-form. The results of the present study encourage further investigations on improvements in intestinal absorption of peptide drugs by glycosylation.