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  • br Discussion br PEPT is an attractive molecular target for


    4. Discussion
    PEPT1 is an attractive molecular target for the drug delivery into tumor cells in terms of cancer treatment and diagnosis. In normal tissues, the predominant expression of PEPT1 protein is restricted to the luminal membrane of enterocytes in the small intestine with lower level of expression in renal proximal tubules and bile ducts.26 In tumor cells, in contrast, high expression 1268720-28-0 of PEPT1 mRNA and protein has been reported in the cell lines of various tissue ori-gins.11e15 Although the information on the clinical specimens is limited, it is noted that PEPT1 is detectable in the tissue microarray from various types of human cancers (Fig. S4).27 In addition to the upregulation of PEPT1 in cancers, it is also of note that the pH of extracellular environment in solid tumors is often acidic, due to the increased excretion of metabolic acids from tumor cells with elevated glycolysis.28 Such acidic tumor microenvironment would assist the Hþ-coupled transport medicated by PEPT1.21 Moreover, the substrate specificity of PEPT1 is extremely broad, so that PEPT1 accepts numerous compounds including di/tri-peptides, peptide-mimetic drugs and nonpeptidic compounds.26 Several previous studies have, thus, proposed the possibility of PEPT1-targeted drug delivery for cancer detection and treatment.15,29
    In this study, we have characterized oligopeptide transporters of tumor cells in terms of boron delivery into tumor cells for BNCT. The results from this study have provided the first pharmacological evidences for the interaction of BPA-containing dipeptides with oligopeptide transporters. In the tumor cell lines, we have found that PEPT1 is the predominant oligopeptide transporter. We have confirmed that boron is successfully delivered into tumor cells using BPA-Tyr and Tyr-BPA via a PEPT1-mediated mechanism. In contrast, we have not been able to detect significant expression of PEPT2 protein even in the tumor cell lines reported to highly express PEPT2 mRNA. These results suggest that PEPT1-targeted boron delivery using BPA-containing dipeptide could be a promising approach to expand the indications and improve the efficacy of BNCT.
    In the study of multispecific nature of Ptr2p, a yeast member of the proton-coupled oligopeptide transporter family, it was shown that aromatic 1268720-28-0 (phenylalanine, tyrosine and trypto-phan) most frequently appeared in the high-affinity peptide group among 338 dipeptides examined.30 Although 3-D structure of
    [³H]Gly-Saruptake(pmol/mgprotein) 300
    Control -Tyr -BPA -Sar BPA Tyr Gly 
    [³H]Gly-Saruptake (pmol/mgprotein) 500
    Control -Tyr -BPA -Sar BPA Tyr Gly 
    Control -Tyr -BPA -Sar BPA Tyr Gly
    PEPT1 has not been solved yet, homology modeling of Ptr2p and human PEPT1 using the crystal structure of bacterial PepTso as a template suggested that the substrate recognition is shared among them.31 As expected, in genetic code study, BPA-Tyr and Tyr-BPA have been proved to be among the high affinity substrates of PEPT1 (Table 1), because the reported Ki values of known human PEPT1 substrates range from 80 mM to 22 mM.32
    As the novel boron delivery agents with a potential applica-tion to BNCT, BPA-containing dipeptides have beneficial chemical and biological characteristics. First, their solubility is significantly improved compared with BPA. Due to the relatively low solubility of BPA at physiological pH, BPA-fructose complex is used in the intravenous infusion in clinical. The solubility of BPA-Tyr in water is 276 mM (102.4 g/L) which is comparable to that of BPA in the form of fructose complex (about 100 g/L). Second, due to cancer cell-specific upregulation of PEPT1, BPA-Tyr and Tyr-BPA would be with less side effects when used in BNCT. In small intestine, where PEPT1 is highly expressed among normal tissues, PEPT1 is 
    localized in the luminal side of the epithelial cells.26 Thus, intravenously administered BPA-Tyr and Tyr-BPA would not reach PEPT1, avoiding side effects. Third, the broad substrate selectivity of PEPT1 offers a great potential for the structural modifications of BPA-containing dipeptides. The solubility, in vivo stability, biocompatibility, tumor accumulation, and tumor retention of BPA-containing dipeptides could be further improved by struc-tural optimization.
    In animal study, we have confirmed the boron accumulation in tumor xenograft with high tumor-to-blood ratio of boron concen-tration after intravenous injection of BPA-Tyr. The persistent boron retention in tumors suggests the promising property of BPA-Tyr as a boron delivery agent. Further studies are needed to optimize the injection dose, the route and method of administration, as well as the pre-conditioning treatment of animals to achieve a higher tumor-selective boron accumulation that could satisfy the re-quirements for clinical application (>20 ppm 10B in tumor and tumor-to-blood ratio of >~3).