The oligopeptide transporter PEPT1 (SLC15A1) accepts di-and tripeptides as endogenous substrates 1,2) and also transports various peptide-mimetic therapeutic agents (e.g., cephalexin).3) PEPT1 is primarily expressed in apical membrane of small intestinal epithelial cells. 4) PEPT1 is thought to be involved in gastrointestinal absorption of its substrates. Involvement of PEPT1 may explain the difference in bioavailability between valacyclovir (PEPT1 substrate) and acyclovir (non-substrate) in humans. 5,6) We have recently reported that rab8-null mice, which exhibit minimal expression of PEPT1 on apical membranes of small intestine, lack saturable uptake of a prototypical PEPT1 substrate glycylsarcosine (Gly-Sar).7) This may indicate a predominant role of PEPT1 in the peptide absorption. In addition to small intestine, PEPT1 is functionally expressed in some human cancer cell lines [8][9][10] and therefore would be a promising target for tumor detection. Indeed, a positron emitting probe targeted to PEPT1, 11 C-glycylsarcosine, was recently demonstrated to be able to distinguish between tumor and inflamed tissues. . 12-14) This D-cephalexin is now clinically used for chemotherapy. PEPT1 is presumably responsible for membrane permeation of D-cephalexin in small intestine, because small intestinal uptake of D-cephalexin assessed with an everted sac method was greatly reduced in rab8-null mice.15) The L-diastereomer of cephalexin (Lcephalexin) is also probably transported by PEPT1 in Caco-2 cells.16) However, intact L-cephalexin was not found in vesicles or proteoliposomes prepared from brush border membrane of intestinal epithelial cells. 17) Although, L-cephalexin was not detected, and the hydrolysis product, 7-aminodesacetoxycephalosporanic acid (7-ADCA), appeared very rapidly in serum and urine after oral administration in rats.18) This observation suggests that L-cephalexin can be absorbed from gastrointestinal tract. However, there has been no direct demonstration that L-cephalexin is transported by PEPT1 protein.We recently proposed that orally administered Lcephalexin might be transported and metabolized by PEPT1.13) Indeed, hydrolysis of L-cephalexin was markedly increased by exogenous transfection of PEPT1 gene into cultured cell lines, and metabolism of cephalexin was observed even in membrane-permeabilized cells expressing PEPT1, in which intracellular accumulation of L-cephalexin was negligible.13) This observation may imply that L-cephalexin hydrolysis is mediated by PEPT1 itself, although possible involvement of endogenous peptidases cannot be excluded, because these experimental systems utilized mammalian cells.The purpose of the present study was to clarify whether or not PEPT1 transports and/or metabolizes L-cephalexin. We performed L-cephalexin metabolism and uptake experiments using a highly purified PETP1 protein-expressing system, budded baculovirus 19) which lacks endogenous mammalian metabolic enzymes. MATERIALS AND METHODS MaterialsThe L-diastereoisomer of cephalexin was ob- The oligopepti...