Transcellular transport of a highly polar 3+net charge opioid tetrapeptide

Oligopeptides are generally thought to have poor permeability across biological membranes. Recent studies, however, suggest significant distribution of [Dmt(1)]DALDA (Dmt-D-Arg-Phe-Lys-NH2; Dmt is 2', 6'-dimethyltyrosine), a 3+ net charge opioid peptide, to the brain and spinal cord after subcutaneous administration. Peptide transporters (PEPT1 and PEPT2) play a major role in the uptake of di- and tripeptides across cell membranes, but their ability to transport tetrapeptides is not clear. The purpose of this study was to determine whether [Dmt(1)]DALDA can translocate across Caco-2 cell monolayers and whether PEPT1 plays a role in the uptake process. Our results show that [H-3][ Dmt(1)]DALDA can readily translocate across Caco-2 cells, with a permeability coefficient estimated to be 1.24x10(-5) cm/s. When incubated with Caco-2 cells, [H-3][Dmt(1)]DALDA was detected in cell lysates by 5 min. The internalization of [Dmt(1)]DALDA was confirmed visually with a fluorescent [Dmt(1)]DALDA analog (H-Dmt-D-Arg-Phe-dnsDap-NH2; dnsDap is beta-dansyl-L-alpha, beta-diaminopropionic acid). The uptake of [H-3][Dmt(1)]DALDA was concentration-dependent but temperature- and pH-independent. Treatment with diethylpyrocarbonate (DEPC) inhibited [C-14] glycine-sarcosine uptake but increased [H-3] [Dmt(1)]DALDA uptake 34-fold. These findings suggest that PEPT1 is not involved in [Dmt(1)]DALDA internalization. [Dmt(1)]DALDA uptake was also observed in SH-SY5Y, human embryonic kidney 293, and CRFK cells, and was independent of whether the cells expressed opioid receptors. The efflux of [H-3][Dmt(1)]DALDA from Caco-2 cells was temperature-dependent and was inhibited by DEPC, but was not affected by verapamil, an inhibitor of P-glycoprotein. These data show transcellular translocation of a highly polar 3+ charge tetrapeptide and suggest that [Dmt(1)]DALDA may not only distribute across the blood-brain barrier but also it may even have reasonable oral absorption.