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 behin d 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 wer e also coadministered intraduodenally to conscious rats. Competitive bindin g of inogatran to trypsin was studied using kinetic dialysis and was compar ed to aprotinin. The fraction of free (unbound) trypsin probe, in the absen ce of trypsin inhibitors was determined by performing experiments without p ancreatine and without inhibitors, respectively. Results. A 3-fold increased permeability to inogatran in the presence of ap rotinin was seen in vitro, in some cases correlated with changed barrier pr operties of the intestinal segments. The in vitro results were well correla ted with the in vivo results. There was a 5-fold increase in the bioavailab ility of inogatran in the presence of aprotinin. The binding of a trypsin p robe was inhibited by both the presence of inogatran and aprotinin. Aprotin in showed a several fold higher displacement than inogatran. The results in dicate both an effect of aprotinin on the epithelial membrane and an inhibi tion of binding of the thrombin inhibitor to trypsin or other serine protea ses in the gut. Conclusions. The coadministration of aprotinin with enzymatically stable pe ptides, like thrombin inhibitors, may improve their absorption after oral a dministration. This suggests a new additional mechanism for intestinal abso rption enhancement of peptide drugs.