Structure-transport relationship for the intestinal small-peptide carrier: is the carbonyl group of the peptide bond relevant for transport?

PURPOSE: The objective of this study was to determine the influence of the peptide bond with emphasis on the carbonyl group on the interaction with and transport by the intestinal small-peptide carrier. Therefore enalapril, a known substrate for the small-peptide carrier, has been modified to an analogue with a reduced peptide bond, enamipril. The transport characteristics of both compounds have been determined.
METHODS: The in vitro transport studies were performed using rat ileum in Ussing chambers. The transport of enalapril and enamipril were measured in a concentration range from 0.5-8 mM in both directions across the ileum. in the presence and absence of inhibitors. The interaction with the small-peptide carrier was studied by evaluating the ability of enalapril and its analogue enamipril to inhibit the transport rate of amoxycillin.
RESULTS: Enalapril shows, besides passive diffusion (P(m)3.06+/-0.14 . 10(-6)cm/s), saturable transport kinetics (Jmax = 16+/-5 nmol/h.cm2, Km = 1.86+/-0.64 mM) which can be inhibited with 10 mM cephalexin. The analogue with a reduced peptide bond does not show saturable transport from the mucosal to the serosal side, and cephalexin does not inhibit the flux of enamipril. However, the transport of enamipril from the serosal to mucosal side of the intestinal membrane is saturable and can be inhibited by 100 microM verapamil. Although enamipril is not a substrate for the small-peptide carrier in contrast to enalapril, both enalapril and enamipril are able to inhibit the active transport of amoxycillin with a K(i) of 0.41+/-0.24 mM and 0.24+/-0.12 mM respectively.
CONCLUSIONS: The reduction of the peptide bond of enalapril results in a compound, enamipril, which does not show polarized and saturable transport from the mucosal to the serosal side of the intestinal tissue. Also because the transport of enamipril cannot be inhibited by cephalexin, the analogue with the reduced peptide bond is no longer a substrate for the intestinal small-peptide carrier. Therefore, it can be concluded that the carbonyl group is an essential structural requirement for transport by the small-peptide carrier. In contrast, the interaction with the small-peptide carrier is still present, shown by the inhibition of the fluxes of amoxycillin. Reduction of the peptide bond of enalapril resulted in a new substrate for the P-glycoprotein efflux pump.