Drug pharmacophores covalently linked to the red cell surface are active without prior release. Drug targeting of renin with a synthetic ligand conjugated to red blood cells.
Red blood cells have been labeled with an anti-renin pharmacophore using the activated labeling agent Boc-Phe-His-ACHPA-Ile-6-NH(CH2)5CO-NHS (4) and the corresponding sulfo-NHS-ester (5). Renin inhibition by labeled cells varies according to the concentrations of 4 or 5 used in the labeling protocols, and with the densities of the red cells employed. Flow cytometry measurements using specific polyclonal antibodies toward the anti-renin pharmacophore confirm that red cells are labeled on their outer surfaces with anti-renin pharmacophores. Inhibitory activity of labeled red cells is clearly associated with the cells themselves, and does not require prior release of an inhibitory entity: renin inhibition increases as a function of the concentration of NHS-ester used to label cells suspended in buffer, and with cell density; on the other hand, the separated supernatant portions of the medium make only minor contributions to the observed inhibitory activities. Renin inhibition also increases with increasing concentrations of ghosts derived from labeled red cells, firmly establishing that activity is intimately associated with cell membranes. Thus, the composite evidence is strongly supportive of inhibitory activity specific to the extracellular surface of red cells, which has been modified by the introduction of anti-renin pharmacophores. This study of inhibitory activity by drug/red blood cell-conjugates represents one of the few examples of a red cell-bound ligand of synthetic origin capable, without prior release, of specifically blocking the activity of its target enzyme. As well, it demonstrates the feasibility of exploiting the activity of covalently bound pharmacophores, free from interference of their carriers, for drug targeting.