UPTAKE AND TRANSEPITHELIAL TRANSPORT OF THE ORALLY ABSORBED CEPHALOSPORIN CEPHALEXIN, IN THE HUMAN INTESTINAL-CELL LINE, CACO-2

Cephalexin (CPX) uptake in cultures of the human colon adenocarcinoma cell lines, Caco-2 and HT-29 has been shown to involve the di-/tripeptide transporter (DPT). However, little is known about the mechanism mediating the transepithelial (TE) transport of CPX either in vivo or in cultured cells. In this study, uptake and TE transport of CPX were investigated in Caco-2 monolayers grown on microporous membranes. Caco-2 cells did not show net TE transport of CPX when the pH of both apical (AP) and basolateral (BL) bathing solutions was 7.4. When the pH of the AP bathing solution was decreased from 7.4 to 6.0, while maintaining the pH of the BL bathing solution at 7.4, AP-BL transport of CPX (0.1 mM) increased from 0.1 to 0.23% h(-1) cm(-2). Reversal of the pH gradient across the monolayer (AP, pH 7.4; BL, pH 6.0) did not alter the BL-AP flux of CPX. Manipulation of the AP or BL pH between 5.5 and 7.4 affected neither the AP-BL nor the BL-AP flux of mannitol (both similar to 0.1% h(-1) cm(-2)), an internal marker of passive, paracellular diffusion. The pH-dependent AP uptake and AP-BL flux of CPX were time-, concentration- and temperature-dependent. Apparent half-maximal transport concentration (K-t) and maximal transport velocity (V-t) were 2.9 mM and 1.0 nmol min(-1) mg protein(-1) for AP uptake, and 4.7 mM and 0.13 nmol min(-1) cm(-2) (0.30 nmol min(-1) mg protein(-1)) for AP-BL transport. The carrier-mediated AP uptake and AP-BL transport of CPX were inhibited by Gly-Pro, Pro-Gly, cephradine, cefadroxil, benzylpenicillin and ampicillin, but not by proline, glycine, valine, lysine or aspartic acid. In addition, CPX uptake was not inhibited by the nucleoside, adenosine, or the bile acid, taurocholic acid, suggesting that the uptake and TE transport of CPX involves the DPT but not other carriers present in the intestinal mucosa. We confirmed that the AP uptake of CPX involves mainly the DPT and conclude the following: (a) AP-BL transport of CPX is predominantly transcellular and involves a carrier, probably the DPT; (b) the rate-limiting step in AP-BL transport of cephalexin appears to be BL efflux and not AP uptake; (c) interaction with the DPT alone may be a poor predictor of substrate transport via this carrier; and (c) the Caco-2 culture system is a good model for studying mucosal uptake and TE transport of small peptides and peptidomimetic drugs.