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

• Published in: The Journal of pharmacology and experimental therapeutics 2003-01, Vol.304 (1), p.425-432
• Main Authors: Zhao, Kesheng, Luo, Guoxiong, Zhao, Guo-Min, Schiller, Peter W, Szeto, Hazel H
• Format: Article
• Language: English
• Subjects: Biological Transport, Active; Blood-Brain Barrier; Caco-2 Cells; Calcium Channel Blockers - pharmacology; Carrier Proteins - metabolism; Cell Membrane - metabolism; Diethyl Pyrocarbonate - pharmacology; Dipeptides - metabolism; Energy Metabolism - physiology; Humans; Hydrogen-Ion Concentration; Intestinal Absorption; Microscopy, Confocal; Oligopeptides - metabolism; Oligopeptides - pharmacokinetics; Opioid Peptides - metabolism; Opioid Peptides - pharmacokinetics; Peptide Transporter 1; Radioligand Assay; Symporters; Verapamil - pharmacology
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.102.040147

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-NH 2 ; 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 [ 3 H][Dmt 1 ]DALDA can readily translocate across Caco-2 cells, with a permeability coefficient estimated to be 1.24 × 10 −5 cm/s. When incubated with Caco-2 cells, [ 3 H][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-NH 2 ; dnsDap is β-dansyl- l -α,β-diaminopropionic acid). The uptake of [ 3 H][Dmt 1 ]DALDA was concentration-dependent but temperature- and pH-independent. Treatment with diethylpyrocarbonate (DEPC) inhibited [ 14 C]glycine-sarcosine uptake but increased [ 3 H][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 [ 3 H][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.