The Reseach On The LAT1-targeting Antitumor Agents And Its Pharmacokenetics

The L-type amino acid transporter 1(LAT1) is a predicted 12-transmembrane protein a Na-independent neutral amino acid transporter. LAT1 preferentially transports large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine and histidine in a Na-independent manner1-3. It is a major route through which living cells take up branched or aromatic amino acids from extracellμlar fluids. Because of its broad substrate selectivity, LAT1 transports not only naturally occurring amino acids but also amino acid-related compounds.LAT1 overexpressed on membrane of various tumor cells, are potential targets for tumor-targeting therapy. This study aimed to develop a LAT1-mediated drug delivery to target chemotherapeutic agents to LAT1-overexpressed tumor cells in vitro and in vivo. Here the aspartate(Asp) was attached to the N-terminal of doxorubicin(DOX) via the amido bond with a free carboxyl and a free amino group on the α-carbon atom of the Asp residue. The product Asp-DOX was characterized by HPLC spectroscopy and mass spectrometry. In vitro, the Asp-DOX exerted stronger inhibition to the cancer cells(HepG2, HCT116 and MCF7) overexpressing LAT1 and the uptake of Asp-DOX awas about 3.5-fold higher than that of DOX in HepG2 cells. Data of pharmacokinetics showed that Asp-DOX expressed a longer circμlation time(t1/2 = 49.14 min) and larger AUC(in the blood compared to DOX(t1/2 = 15.12 min). Biodistribution study in HepG2 and HCT116 tumor-bearing mice showed that Asp-DOX achieved 3.1-fold and 6.4-fold accumμlation of drug in tumor tissue than those of free unmodified formμlations. More importantly, treatment of tumor-bearing mice with Asp-DOX showed a significantly stronger inhibition in tumor growth than mice treated with free DOX in HepG2 tumor models. Furthermore, the bidirectional transport assay in the MDR1-overexpressed MDCK cell lines suggested that after Asp modification, Asp-DOX was not yet the substrate of P-gp. These resμlts suggested that this Asp-modified drug may provide a new treatment strategy for tumors which overexpress LAT1 and MDR1 and that LAT1 offered an efficient target for the targeting therapy and therapeutic indicator.The experimental and semiempirical computational analyses showed that, for an aromatic amino acid or its analogs to be a LAT1 substrate, it must have properties as follows: 1) it must have three binding sites: a carboxyl binding sites, an amino binding sites and a hydrophobic site; 2) the substrates must be neural in the media; 3) the hydrophobic interaction and ion affinity between the substrate side chain and the substrate binding site of LAT1 seems to be crucial for the substrate binding.