A fluorescent hPept1 transporter substrate for uptake screening

• Published in: Pharmaceutical research 2003-11, Vol.20 (11), p.1738-1745
• Main Authors: Landowski, Christopher P, Han, Hyo-kyung, Lee, Kyung-Dall, Amidon, Gordon L
• Format: Article
• Language: English
• Subjects: Amino acids; Bioavailability; Caco-2 Cells; Cancer; Carrier Proteins - analysis; Carrier Proteins - chemistry; Carrier Proteins - pharmacokinetics; Cell culture; Drugs; Fluorescein-5-isothiocyanate - analysis; Fluorescein-5-isothiocyanate - chemistry; Fluorescein-5-isothiocyanate - metabolism; Fluorescent Dyes - analysis; Fluorescent Dyes - chemistry; Fluorescent Dyes - pharmacokinetics; HeLa Cells; Humans; Peptide Transporter 1; Peptides; Permeability; Reverse Transcriptase Polymerase Chain Reaction - methods; Symporters
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1023/B:PHAM.0000003369.64891.51

To synthesize fluorescent analogues of hPept1 substrates, FITC-Val-OCH3, Lys-FITC-OH, and Lys-FITC-OCH3, and to characterize their hPept1 transporter-mediated uptake. FITC analogues of amino acids were synthesized using established synthetic procedures, and the extent of their [3H]Gly-Sar uptake inhibition in HeLa/hPept1 cells was determined. The uptake of Lys-FITC-OCH3 was evaluated in HeLa, HeLa/hPept1, and Caco-2 cells in the presence and absence of Gly-Sar using a fluorescence microscopy-based assay. The uptake and transport of the Lys-FITC analogues were also determined in Caco-2 cells using HPLC assays. In HeLa/hPept1 cells, [3H]Gly-Sar uptake was significantly inhibited by Lys-FITC-OCH3 (74%) but not by FITC-Val-OCH3 (22%). The uptake of Lys-FITC-OCH3 (100 microM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, Lys-FITC-OCH3 (100 microM) uptake in HeLa/hPept1 and Caco-2 cells was reduced by 77% and 80%, respectively, in the presence of 1 mM Gly-Sar. Dipeptides and cephalexin significantly reduced Lys-FITC-OCH3 uptake in Caco-2 cells. The apical permeability of Lys-FITC-OCH3 (1.5 x 10(6) cm/s) in Caco-2 cells was significantly lowered in the presence of Gly-Sar. Fluorescence micrographs revealed that this analogue was localized in the cytoplasm and in the nucleus. The combined results indicate that Lys-FITC-OCH3 is recognized and transported by hPept1 in HeLa/hPept1 and by peptide transporters in Caco-2 cells. The results also suggest that Lys-FITC-OCH3 might be a useful fluorescent substrate for rapid assessment of peptide transporter activity in cells of interest.