Transport of the phosphonodipeptide alafosfalin by the H + /peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells

The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H + /peptide cotransporters was studied in Caco‐2 cells, expressing the low‐affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high‐affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [ 14 C]glycylsarcosine with K i values of 0.19 ± 0.01 m m and 0.07 ± 0.01 m m for PEPT1 and PEPT2, respectively. Saturation kinetic studies revealed that in both cell types alafosfalin affected only the affinity constant ( K t ) but not the maximal velocity ( V max ) of glycylsarcosine (Gly‐Sar) uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon‐type experiments. Caco‐2 cells and SKPT cells were also cultured on permeable filters: apical uptake and transepithelial apical to basolateral flux of [ 14 C]Gly‐Sar across Caco‐2 cell monolayers were reduced by alafosfalin (3 m m ) by 73%. In SKPT cells, uptake of [ 14 C]Gly‐Sar but not flux was inhibited by 61%. We found no evidence for an inhibition of the basolateral to apical uptake or flux of [ 14 C]Gly‐Sar by alafosfalin. Alafosfalin (3 m m ) did not affect the apical to basolateral [ 14 C]mannitol flux. Determined in an Ussing‐type experiment with Caco‐2 cells cultured in Snapwells™, alafosfalin increased the short‐circuit current through Caco‐2 cell monolayers. We conclude that alafosfalin interacts with both H + /peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H + ‐symport, explaining its oral availability. The results also demonstrate that dipeptides where the C‐terminal carboxyl group is substituted by a phosphonic function represent high‐affinity substrates for mammalian H + /peptide cotransporters.