D-cycloserine uses an active transport mechanism in the human intestinal cell line Caco 2

Abstract

In a previous study we have shown that cultured epithelial cell lines can be used to measure the transepithelial passage of antimicrobial agents across the intestine and to obtain information on the mechanisms of transport utilized and predict the bioavailability of the antimicrobial agents after oral administration. In particular, among the drugs investigated, D-cycloserine had been shown to be transported in a polarized manner only in the intestinal cells. In the present work, further characterization of the transport of D-cycloserine in the human intestinal cell line Caco 2 has shown that this occurs in the apical-to-basolateral direction by an active mechanism which is energy dependent but only partially sodium dependent. Competition studies have also indicated that the transport of D-cycloserine occurs via a carrier for imino acids, amino acids with aliphatic side chains (L-Ala, D-Ala, and iAla), and L-Trp, L-Tyr, L-Cys, and a-amino isobutyric acid. This system may correspond to a proton-dependent system for L-proline and 0-alanine recently described for Caco 2 cells. In contrast with the cephalosporins, which are taken up by the Caco 2 cells via a dipeptide carrier, D-cycloserine transport cannot be inhibited by either cephalexin (a member of the class of cephalosporins) or dipeptides.It has previously been shown by us and by others that intestinal cell lines in culture can constitute useful models for the study of the oral bioavailability of drugs (3,16,20,29,39). Several studies have determined the transport of drugs across cultured intestinal cells and attempted to relate it to the physicochemical characteristics and in vivo pharmacokinetics of the drugs (3-5, 29). The elucidation of the transport mechanisms increasingly suggests that cell lines could be used to predict potential oral bioavailability and thereby eliminate the need for animal models, at least in preliminary studies, although oral bioavailability will eventually need confirmation in vivo.Absorption of solutes from the intestinal lumen can occur passively through paracellular or transcellular routes and actively through the cellular pathways (33, 38). The active absorption of hexoses and amino acids has been studied in some detail and shown to exhibit wide species and regional differences (17 (29). Briefly, Caco 2 cells were routinely grown in Dulbecco modified minimum essential medium containing 25 mM glucose and 3.7 g of NaHCO3 liter-' and supplemented with 4 mM L-glutamine, 10% fetal calf serum, 1% nonessential amino acids, 100 U of penicillin ml-l, and 100 ,ug of streptomycin ml-1, while the MDCK cells were grown in the same medium without addition of nonessential amino acids and with 10% donor horse serum in place of fetal calf serum. At confluency, the cells were passaged by detachment with 0.25% trypsin (1:250) and 10 mM EDTA in calcium-free and magnesium-free phosphate-buffered saline.

D‐Cycloserine transport in human intestinal epithelial (Caco‐2) cells: mediation by a H + ‐coupled amino acid transporter

“…The stoichiometry of the cotransport was 1:1 for Hb +/L-alanine cotransport (Thwaites et at., 1994b). The rheogenic nature of the H +a-coupled dipeptide transport has been established in intestine (Ganapathy & Leibach, 1985), in oocytes where the cloned transporter (PepTi) has been expressed (Fei et at., 1994), and in Caco-2 epithelia (Thwaites et at., 1993d (Ranaldi et al, 1994) Competition studies of D-cycloserine absorption in Caco-2 epithelia (Ranaldi et al, 1994) are in broad agreement with the present data; those amino acids capable of competitive inhibition include L-alanine, /3-alanine, D-alanine, L-proline, hydroxy-L-proline and a-aminoisobutyric acid. Those amino acids not capable of inhibition of D-cycloserine absorption included leucine, isoleucine, valine, phenylalanine, methionine, asparagine, glutamine, histidine, arginine, lysine, glutamate and D-aspartate.…”

“…Those amino acids not capable of inhibition of D-cycloserine absorption included leucine, isoleucine, valine, phenylalanine, methionine, asparagine, glutamine, histidine, arginine, lysine, glutamate and D-aspartate. Those amino acids where there is inhibition of D-cycloserine absorption but which are not substrates for H + /amino acid symport are threonine and cysteine (Ranaldi et al, 1994). Thus in broad terms there is good agreement that Dcycloserine absorption is mediated via the H+/amino acid transporter expressed in the apical membrane of intestinal Caco-2 cells.…”

Transport of Pharmacologically Active Proline Derivatives by the Human Proton-Coupled Amino Acid Transporter hPAT1

“…The currently accepted suggestion is that PAT1 and system IMINO are structurally and functionally identical (Chen et al, 2003). Most results gained on PAT1 confirm the early reports of H ϩ gradient driven uptake of amino acids such as proline, glycine, and many others and that of amino acid derived drugs such as D-serine, D-cycloserine, GABA, and APSA at the apical membrane of Caco-2 cells (Thwaites et al, 1993a(Thwaites et al, ,b, 1995a(Thwaites et al, ,b, 2000Ranaldi et al, 1994;Thwaites and Stevens, 1999). In our study, we found that Caco-2 cells take up L-proline at their apical membrane in a strongly H ϩ -dependent manner via a system with an affinity constant of 2 mM.…”

Applying Biopharmaceutical Classification System (BCS) Criteria to Predict Oral Absorption of Drugs in Dogs: Challenges and Pitfalls

“…Despite their poor membrane permeability, both compounds undergo active transport via peptide transporter 1 (PEPT1) (79)(80)(81). Thus, enterocyte penetration via facilitated/active rather than passive processes will result in an extent of absorption that cannot be predicted solely on the basis of BCS classification criteria.…”