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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Preparation of a Claudin-Targeting Molecule Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin

Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machida, Tokyo, Japan (C.E., M.K., N.H., M.H., M.F., Y.W.); Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan (H.M.); Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan (H.M.); and Department of Bacterial and Toxinology, Division of Infectious Diseases, Osaka University, Suita, Osaka, Japan (Y.H.)

Although most malignant tumors are epithelia-derived carcinomas, methods for specific and effective delivery of antitumor agents to carcinomas have not been developed. Recent reports indicate that epithelia overexpress claudin-3 and -4, which are integral membrane proteins of epithelial tight junctions. This suggests that claudins can be targeted for tumor therapy, but there is not currently a method for delivering drugs to claudin-expressing cells. In the present study, we evaluated whether a potent claudin-4-binding C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) would allow targeting to claudin-4-expressing cells. We fused C-CPE to the protein synthesis inhibitory factor (PSIF), which lacks the cell binding domain of Pseudomonas exotoxin. This fusion protein, C-CPE-PSIF, was cytotoxic to MCF-7 human breast cancer cells, which express endogenous claudin-4, but it was not toxic to mouse fibroblast L cells, which lack endogenous claudin-4. The cytotoxicity of C-CPE-PSIF was attenuated by pretreating the MCF-7 cells with C-CPE but not bovine serum albumin. Also, deletion of the claudin-4-binding region of C-CPE reduced the cytotoxicity of C-CPE-PSIF. Finally, we found that C-CPE-PSIF is toxic to L cells expressing claudin-4 but not to normal L cells or cells expressing claudin-1, -2, or -5. These results indicate that use of the C-CPE peptide may provide a novel way to target drugs to claudin-expressing cells.

Address correspondence to: Dr. Masuo Kondoh, Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machidashi, Tokyo 194-8543, Japan. E-mail: masuo{at}ac.shoyaku.ac.jp

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