title: Transcellular transport of a highly polar 3+ net charge opioid tetrapeptide.
abstract: 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 x 10(-5) 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 beta-dansyl-L-alpha,beta-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.
authors.affiliation: Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021, USA. hhszeto@med.cornell.edu
authors.forename: Kesheng
authors.initials: K
authors.lastname: Zhao
authors.forename: Guoxiong
authors.initials: G
authors.lastname: Luo
authors.forename: Guo-Min
authors.initials: GM
authors.lastname: Zhao
authors.forename: Peter W
authors.initials: PW
authors.lastname: Schiller
authors.forename: Hazel H
authors.initials: HH
authors.lastname: Szeto
mesh_terms: D001693:Biological Transport, Active; D001812:Blood-Brain Barrier; D018938:Caco-2 Cells; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism; D004734:Energy Metabolism / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007408:Intestinal Absorption; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000378:metabolism* / Q000493:pharmacokinetics; D018847:Opioid Peptides / Q000378:metabolism* / Q000493:pharmacokinetics; D000074011:Peptide Transporter 1; D011869:Radioligand Assay; D027981:Symporters; D014700:Verapamil / Q000494:pharmacology
chemical_list: C424424:2',6'-dimethyltyrosyl-arginyl-phenylalanyl-lysinamide; D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D004151:Dipeptides; D009842:Oligopeptides; D018847:Opioid Peptides; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; C004194:glycylsarcosine; D014700:Verapamil; D004047:Diethyl Pyrocarbonate