Functional Expression of Stereoselective Metabolism of Cephalexin by Exogenous Transfection of Oligopeptide Transporter PEPT1

Abstract

Gastrointestinal absorption of the beta-lactam antibiotic cephalexin (CEX) is highly stereoselective: l- and d-CEX are both taken up by intestinal epithelial cells through the brush-border membrane, most likely via oligopeptide transporter PEPT1, but l-CEX is not found in serum or urine after administration p.o. because of its rapid intestinal metabolism, whereas d-CEX is well absorbed in the unchanged form. We examined the contribution of PEPT1 to the stereoselective uptake and metabolism of CEX. We observed stereoselective metabolism of CEX after exogenous transfection of PEPT1 alone into mammalian cell lines: l-CEX, but not d-CEX, was metabolized to 7-aminodesacetoxycephalosporanic acid (7-ADCA) in HeLa and human embryonic kidney 293 cells stably and transiently expressing human PEPT1, respectively, whereas such metabolism was minor in cells expressing the vector alone. The formation rate of 7-ADCA depended on the amount of PEPT1 cDNA transfected. l-CEX metabolism was rapid because only 7-ADCA was found inside and outside the cells during incubation with l-CEX. The characteristics of PEPT1-mediated metabolism of l-CEX were similar, but not identical, to those of PEPT1-mediated transport. PEPT1-mediated metabolism was also observed in permeabilized cells expressing PEPT1, in which PEPT1-mediated intracellular substrate accumulation was negligible, suggesting that the increase in l-CEX metabolism by PEPT1 transfection cannot be fully explained by an increase in uptake and subsequent exposure to intracellular hydrolases. The present findings show that stereoselectivity in CEX absorption can be fully explained in terms of PEPT1, implying that the l-CEX hydrolase is PEPT1 itself or is induced by PEPT1.

Control of metabolite efflux in microbial cell factories: current advances and future prospects

“…[266,267]) have been carried out on SLC15 [268] members (previously known as PEPT1 and PEPT2), and responsible e.g. for the intestinal uptake of penicillins [269] and cepaholsporins [270,271].…”

Direct Evidence for Efficient Transport and Minimal Metabolism of L-Cephalexin by Oligopeptide Transporter 1 in Budded Baculovirus Fraction

“…13,16,17) These observations and the fact that a metabolite of L-cephalexin, 7-ADCA, was detected immediately after oral dosing 18) imply that PEPT1 itself may mediate L-cephalexin hydrolysis, although to date, only we 13) have suggested that hydrolytic enzyme activity may be associated with PEPT1. In the present study using budded baculovirus, which expresses PEPT1, but not any endogenous mammalian enzyme, PEPT1-mediated transport of L-cephalexin was clearly observed, since substantial amount of intact Lcephalexin was detected in PEPT1-expressing budded baculovirus (Fig.…”

“…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.…”

“…13) Hydrophilic Interaction Chromatography (HILIC) packed column, COSMOSIL HILIC 4.6 mm I.D.ϫ250 mm (Nacalai Tesque, Inc., Kyoto Japan) was used as an analytical column, and the UV detector was set at 260 nm. The mobile phase was 10 mM ammonium acetate/acetonitril (30 : 70, v/v).…”

“…[12][13][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.…”