Inhibition of binding of an enzymatically stable thrombin inhibitor to lumenal proteases as an additional mechanism of intestinal absorption enhancement.

PURPOSE

The objective of the study was to investigate the mechanisms behind increased bioavailability of an enzymatically stable thrombin inhibitor, inogatran, after coadministration with a trypsin inhibitor, aprotinin.

METHODS

Rat jejunum, ileum and colon segments were stripped and mounted in modified Ussing chambers, and the permeability to inogatran was determined both in the presence and absence of aprotinin. Inogatran and aprotinin were also coadministered intraduodenally to conscious rats. Competitive binding of inogatran to trypsin was studied using kinetic dialysis and was compared to aprotinin. The fraction of free (unbound) trypsin probe, in the absence of trypsin inhibitors was determined by performing experiments without pancreatine and without inhibitors, respectively.

RESULTS

A 3-fold increased permeability to inogatran in the presence of aprotinin was seen in vitro, in some cases correlated with changed barrier properties of the intestinal segments. The in vitro results were well correlated with the in vivo results. There was a 5-fold increase in the bioavailability of inogatran in the presence of aprotinin. The binding of a trypsin probe was inhibited by both the presence of inogatran and aprotinin. Aprotinin showed a several fold higher displacement than inogatran. The results indicate both an effect of aprotinin on the epithelial membrane and an inhibition of binding of the thrombin inhibitor to trypsin or other serine proteases in the gut.

CONCLUSIONS

The coadministration of aprotinin with enzymatically stable peptides, like thrombin inhibitors, may improve their absorption after oral administration. This suggests a new additional mechanism for intestinal absorption enhancement of peptide drugs.