Vol. 304, Issue 1, 425-432, January 2003

Transcellular Transport of a Highly Polar 3+ Net Charge Opioid Tetrapeptide

Kesheng Zhao, Guoxiong Luo, Guo-Min Zhao, Peter W. Schiller and Hazel H. Szeto

Department of Pharmacology, Weill Medical College of Cornell University, New York, New York (K.Z., G.L., G.-M.Z., H.H.S.); and Clinical Research Institute of Montreal, Montreal, Quebec, Canada (P.W.S.)

Oligopeptides are generally thought to have poor permeability across biological membranes. Recent studies, however, suggest significant distribution of [Dmt¹]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 [Dmt¹]DALDA can translocate across Caco-2 cell monolayers and whether PEPT1 plays a role in the uptake process. Our results show that [³H][Dmt¹]DALDA can readily translocate across Caco-2 cells, with a permeability coefficient estimated to be 1.24 × 10⁵ cm/s. When incubated with Caco-2 cells, [³H][Dmt¹]DALDA was detected in cell lysates by 5 min. The internalization of [Dmt¹]DALDA was confirmed visually with a fluorescent [Dmt¹]DALDA analog (H-Dmt-D-Arg-Phe-dnsDap-NH₂; dnsDap is -dansyl-L-,-diaminopropionic acid). The uptake of [³H][Dmt¹]DALDA was concentration-dependent but temperature- and pH-independent. Treatment with diethylpyrocarbonate (DEPC) inhibited [¹⁴C]glycine-sarcosine uptake but increased [³H][Dmt¹]DALDA uptake 34-fold. These findings suggest that PEPT1 is not involved in [Dmt¹]DALDA internalization. [Dmt¹]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 [³H][Dmt¹]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 [Dmt¹]DALDA may not only distribute across the blood-brain barrier but also it may even have reasonable oral absorption.