Masuo Kondoh

Osaka University, Suika, Ōsaka, Japan

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Publications (137)398.26 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Claudins constitute a family of at least 27 proteins with four transmembrane domains, and play a pivotal role in maintaining tight-junctions seals in diverse epithelial tissues. The expression of claudin-4 often changes in intestinal tissues of inflammatory bowel disease and various human cancers. Therefore, claudin-4 is a promising target for treatment of these diseases. In our previous study, we established a reporter cell line to monitor claudin-4 expression on the basis of a functional claudin-4 promoter. Using this cell line, we have performed a cell-based screen of a library containing 2642 biologically active small-molecule compounds to identify modulators of claudin-4 expression. The screen identified 24 potential modulators of the claudin-4 promoter activity. Fourteen of these compounds (12 of them novel) induced endogenous claudin-4 expression. The identified compounds might serve as lead compounds targeting aberrant gene expression in inflammatory bowel disease.
    Biotechnology Letters 02/2015; DOI:10.1007/s10529-015-1791-7 · 1.74 Impact Factor
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    ABSTRACT: Hepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1). Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (mAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All mAbs produced by these hybridomas specifically bound to human CLDN1 with very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected mAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infection in vivo. Anti-CLDN1 mAbs may hence be promising candidates as novel anti-HCV agents. Safe and effective therapeutic entry inhibitors against Hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs such as direct-acting antivirals. In this study, we first showed the effective strategy to develop functional monoclonal antibodies (mAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), using parental cells expressing the intact target membrane protein and the target-defective cells. The established mAbs against CLDN1, which had very high affinity with intact CLDN1, efficiently inhibited in vitro and in vivo HCV infection. These anti-CLDN1 mAbs are promising leads for novel entry inhibitors against HCV. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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    ABSTRACT: Claudin-1 (CLDN1), a known host factor for hepatitis C virus (HCV) entry and cell-to-cell transmission, is a target molecule for inhibiting HCV infection. We previously developed 4 clones of mouse anti-CLDN1 monoclonal antibody (mAb) that prevented HCV infection in vitro. Two of these mAbs showed the highest anti-viral activity. Here, we optimized the anti-CLDN1 mAbs as candidates for therapeutics by protein engineering. Although Fab fragments of the mAbs prevented in vitro HCV infection, their inhibitory effects were much weaker than those of the whole mAbs. In contrast, human chimeric IgG1 mAbs generated by grafting the variable domains of the mouse mAb light and heavy chains inhibited in vitro HCV infection as efficiently as the parental mouse mAbs. However, the chimeric IgG1 mAbs activated Fcγ receptor, suggesting that cytotoxicity against mAb-bound CLDN1-expressing cells occurred through the induction of antibody-dependent cellular cytotoxicity (ADCC). To avoid ADCC-induced side effects, we prepared human chimeric IgG4 mAbs. The chimeric IgG4 mAbs did not activate Fcγ receptor or induce ADCC, but they prevented in vitro HCV infection as efficiently as did the parental mouse mAbs. These findings indicate that IgG4 form of human chimeric anti-CLDN1 mAb may be a candidate molecule for clinically applicable HCV therapy. The American Society for Pharmacology and Experimental Therapeutics.
    Journal of Pharmacology and Experimental Therapeutics 01/2015; DOI:10.1124/jpet.114.217653 · 3.86 Impact Factor
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    ABSTRACT: Homoharringtonine (HHT), a natural alkaloid produced by various Cephalotaxus species, has antileukemic activity in acute and chronic myelogenous leukemia. However, HHT can also induce unanticipated effects in the gastrointestinal tract, such as diarrhea and nausea/vomiting, but the mechanism behind these adverse effects has not been clarified. In the present study, we show that HHT affects the epithelial permeability of intestinal Caco-2 cell monolayers. HHT reduced the transepithelial electrical resistance (TER) of Caco-2 cells in a dose- and time-dependent manner. The HHT effect was reversible and no cytotoxicity was observed at the concentrations used. HHT simultaneously increased the paracellular flux of the 4 kDa and 40 kDa FITC-dextrans associated with the TER reduction. Immunoblotting analysis revealed that HHT decreased the protein expression of TJ components such as claudin-3, -5, and -7. However, the transcription levels of these claudins were not repressed by HHT treatment. HHT also disturbed the cellular localization of claudin-1 and -4. These changes coincided with the reduced barrier function. Our findings suggest that HHT enhances the paracellular permeability of Caco-2 cell monolayers by modulating the protein expression and localization of claudin isoforms; these actions might be responsible for the gastrointestinal effects of HHT.
    European Journal of Pharmaceutics and Biopharmaceutics 12/2014; 89. DOI:10.1016/j.ejpb.2014.12.012 · 4.25 Impact Factor
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    ABSTRACT: Most malignant tumors are derived from epithelium, and claudin (CLDN)-3 and -4 are frequently overexpressed in such tumors. Although antibodies have potential in cancer diagnostics and therapy, development of antibodies against CLDNs has been difficult because the extracellular domains of CLDNs are too small and there is high homology among human, rat, and mouse sequences. Here, we created a monoclonal antibody that recognizes human CLDN-3 and -4 by immunizing rats with a plasmid vector encoding human CLDN-4. A hybridoma clone that produced a rat monoclonal antibody recognizing both CLDN-3 and -4 (clone 5A5) was obtained from a hybridoma screen by using CLDN-3- and -4-expressing cells; 5A5 did not bind to CLDN-1, -2, -5, -6, -7 or -9-expressing cells. Fluorescence-conjugated 5A5 injected into xenograft mice bearing human cancer MKN74 or LoVo cells could visualize the tumor cells. The human-rat chimeric IgG1 monoclonal antibody (xi5A5) activated FcgammaRIIIa in the presence of CLDN-3- or -4-expressing cells, indicating that xi5A5 may exert antibody-dependent cellular cytotoxicity. Administration of xi5A5 attenuated tumor growth in xenograft mice bearing MKN74 or LoVo cells. These results suggest that 5A5 shows promise in the development of a diagnostic and therapeutic antibody for cancers.
    Journal of Pharmacology and Experimental Therapeutics 08/2014; DOI:10.1124/jpet.114.216911 · 3.86 Impact Factor
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    ABSTRACT: Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells using either the transcription activator-like effector nuclease (TALEN) or shRNA-lentivirus, and achieved complete, long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of hepatocyte nuclear factor 4α (HNF4α) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV.
    Journal of Virology 06/2014; 88(16). DOI:10.1128/JVI.01428-14 · 4.65 Impact Factor
  • Masuo Kondoh, Jun Kunisawa
    YAKUGAKU ZASSHI 01/2014; 134(5):613. DOI:10.1248/yakushi.14-00006-F · 0.31 Impact Factor
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    ABSTRACT: Most pathogens invade body through the mucosal epithelium, which is a primary target to prevent the infectious diseases. Mucosal vaccine has been considered to be an effective strategy to establish immunosurveillance against pathogens by the induction of antigen-specific immune responses at both mucosal and systemic immune compartments. The development of antigen delivery system and mucosal adjuvants are required for the sufficient induction of protective immunity in the development of mucosal vaccine. In this review, we shed light on the recent advances in the development of antigen delivery system using microbial functions for mucosal vaccines.
    YAKUGAKU ZASSHI 01/2014; 134(5):629-634. DOI:10.1248/yakushi.14-00006-3 · 0.31 Impact Factor
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    ABSTRACT: Epithelium plays pivotal roles in biological barrier separating the inside of body and the outside environment. Ninety percent of malignant tumors are derived from epithelium. Most pathological microorganisms invade into the body from mucosal epithelium. Thus, epithelium is potential targets for drug development. Claudins (CLs), a family of tetra-transmembrane protein consisting of over 20 members, are structural and functional components of tight junction-seals in epithelium. Modulation of CL-seals enhanced mucosal absorption of drugs. CLs are often over-expressed in malignant tumors. CL-4 expression is increased in the epithelial cells covering the mucosal immune tissues. Very recently, CLs are also expected to be targets for traumatic brain injury and regenerative therapy. In this review, we overview the past, the present and the future of CLs-targeted drug development.
    YAKUGAKU ZASSHI 01/2014; 134(5):641-645. DOI:10.1248/yakushi.14-00006-5 · 0.31 Impact Factor
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    ABSTRACT: We previously found that claudin (CL) is a potent target for cancer therapy using a CL-3 and -4-targeting molecule, namely the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Although CL-3 and -4 are expressed in various normal tissues, the safety of this CL-targeting strategy has never been investigated. Here, we evaluated the tissue distribution of C-CPE in mice. Ten minutes after intravenous injection into mice, C-CPE was distributed to the liver and kidney (24.0% and 9.5% of the injected dose, respectively). The hepatic level gradually fell to 3.2% of the injected dose by 3 h post-injection, whereas the renal C-CPE level gradually rose to 46.5% of the injected dose by 6 h post-injection and then decreased. A C-CPE mutant protein lacking the ability to bind CL accumulated in the liver to a much lesser extent (2.0% of the dose at 10 min post-injection) than did C-CPE, but its renal profile was similar to that of C-CPE. To investigate the acute toxicity of CL-targeted toxin, we intravenously administered C-CPE-fused protein synthesis inhibitory factor to mice. The CL-targeted toxin dose-dependently increased the levels of serum biomarkers of liver injury, but not of kidney injury. Histological examination confirmed that injection of CL-targeted toxin injured the liver but not the kidney. These results indicate that potential adverse hepatic effects should be considered in C-CPE-based cancer therapy.
    European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 11/2013; 52. DOI:10.1016/j.ejps.2013.10.018 · 3.01 Impact Factor
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    ABSTRACT: Platinum nanoparticles are being utilized in various industrial applications, including in catalysis, cosmetics, and dietary supplements. Although reducing the size of the nanoparticles improves the physicochemical properties and provides useful performance characteristics, the safety of the material remains a major concern. The aim of the present study was to evaluate the biological effects of platinum particles less than 1 nm in size (snPt1). In mice administered with a single intravenous dose of snPt1, histological analysis revealed necrosis of tubular epithelial cells and urinary casts in the kidney, without obvious toxic effects in the lung, spleen, and heart. These mice exhibited dose-dependent elevation of blood urea nitrogen, an indicator of kidney damage. Direct application of snPt1 to in vitro cultures of renal cells induced significant cytotoxicity. In mice administered for 4 weeks with twice-weekly intraperitoneal snPt1, histological analysis of the kidney revealed urinary casts, tubular atrophy, and inflammatory cell accumulation. Notably, these toxic effects were not observed in mice injected with 8-nm platinum particles, either by single- or multiple-dose administration. Our findings suggest that exposure to platinum particles of less than 1 nm in size may induce nephrotoxicity and disrupt some kidney functions. However, this toxicity may be reduced by increasing the nanoparticle size.
    Nanoscale Research Letters 09/2013; 8(1):395. DOI:10.1186/1556-276X-8-395 · 2.52 Impact Factor
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    ABSTRACT: Nano-sized materials are widely used in consumer products, medical devices and engineered pharmaceuticals. Advances in nanotechnology have resulted in materials smaller than the nanoscale, but the biologic safety of the sub-nanosized materials has not been fully assessed. In this study, we evaluated the toxic effects of sub-nanosized platinum particles (snPt) in the mouse liver. After intravenous administration of snPt (15 mg/kg body weight) into mice, histological analysis revealed acute hepatic injury, and biochemical analysis showed increased levels of serum markers of liver injury and inflammatory cytokines. In contrast, administration of nano-sized platinum particles did not produce these abnormalities. Furthermore, snPt induced cytotoxicity when directly applied to primary hepatocytes. These data suggest that snPt have the potential to induce hepatotoxicity. These findings provide useful information on the further development of sub-nanosized materials.
    Pharmazie 03/2013; 68(3):178-82. DOI:10.1691/ph.2013.2141 · 1.00 Impact Factor
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    ABSTRACT: Hepatitis C virus (HCV) is a hepatotropic member of the Flaviviridae family and contains a 9.6 kb positive-sense RNA genome. Approximately 170-million people are infected with HCV worldwide. These people face increased risks of chronic hepatitis, cirrhosis and hepatocellular carcinoma compared with the general population. Transduction of the HCV genome into hepatocytes is essential for understanding the mode of action of HCV infection, and for preparing HCV, evaluating HCV replication, and screening anti-HCV drugs. Although electroporation of in vitro-synthesized HCV genome and transduction of plasmid vectors containing the HCV genome are widely used in HCV research, a more convenient system with higher transduction efficiency is needed. Among viral transduction systems, adenovirus (Ad) vector is one of the most efficient and convenient systems; Ad vector has been widely used in clinical gene therapies. Therefore, Ad vector is a promising system for the delivery of the HCV genome; however, an Ad vector expressing the HCV genome has never been developed. We here describe the preparation of an Ad vector expressing the HCV genome, and outline future directions of HCV research using this vector system.
    YAKUGAKU ZASSHI 01/2013; 133(3):305-11. DOI:10.1248/yakushi.12-00237-3 · 0.31 Impact Factor
  • Fuminori Sakurai, Masuo Kondoh
    YAKUGAKU ZASSHI 01/2013; 133(3):289. DOI:10.1248/yakushi.12-00237-F · 0.31 Impact Factor
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    ABSTRACT: The control of permeability through the paracellular route has been paid great attention to for enhanced bioavailability of macromolecular and hydrophilic drugs. The paracellular permeability is controlled by tight junctions (TJ), and claudins are the major constituents of TJ. Despite numerous studies on TJ modulation, the dynamics is not well understood, although it could be crucial for clinical applications. Here, we studied the time (t) course of electrical conductivity (Σ) in a monolayer of Madin-Darby canine kidney (MDCK) and Caco-2 cells upon treatment with modulators, the C-terminus fragments of Clostridium perfringens enterotoxin (C-CPE) and sodium caprate (C10). For C-CPE treatment, Σ remains approximately constant, then starts increasing at t=t (percolation threshold). For C10, on the other hand, Σ increases to 1.6-2.0 fold of the initial value, stays constant, and then starts increasing again for both MDCK and Caco-2 cells at t=t. We find that this behavior can be explained within a framework of percolation, where Σ shows a logarithmic dependence on t-t with the power of μ; μ denotes the critical exponent. We obtain μ=1.1-1.2 regardless of cell type or modulator. Notably, μ depends only on the dimensionality (d) of the system, and these values correspond to those for d=2. Percolation is thus the operative mechanism for the increase in Σ through TJ modulation. The findings provide fundamental knowledge, not only on controlled drug delivery, but also on bio-nanotechnologies including the fabrication of biological devices.
    Biological & Pharmaceutical Bulletin 01/2013; 36(3):384-9. DOI:10.1248/bpb.b12-00786 · 1.78 Impact Factor
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    ABSTRACT: Most malignant tumors are derived from epithelium, and pathologic microorganisms often invade the body through the mucosal epithelium. Thus epithelial tissues are potent targets for drug delivery. The tight junction (TJ) is the intercellular seal in epithelial cell sheets. Claudins (CLs) are a family of tetratransmembrane proteins with a molecular mass of approximately 23 kDa. CLs are key structural and sealing components of TJs. CLs are often overexpressed in malignant tumors. CL-4 is highly expressed in the epithelial cells covering mucosal immune tissues. Therefore CLs may be potent targets for drug delivery, cancer therapy, and mucosal vaccination. Herein, we overview a series of our studies using the C-terminal fragment of Clostridium perfringens enterotoxin to target and bind CLs; we also discuss the efficacy of CL-targeted drug delivery.
    Biological & Pharmaceutical Bulletin 01/2013; 36(5):708-14. DOI:10.1248/bpb.b13-00014 · 1.78 Impact Factor
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    ABSTRACT: In this study, we evaluated a baculoviral display system for analysis of viral entry by using a recombinant adenovirus (Ad) carrying a luciferase gene and budded baculovirus (BV) that displays the adenoviral receptor, coxsackievirus and adenovirus receptor (CAR). CAR-expressing B16 cells (B16-CAR cells) were infected with luciferase-expressing Ad vector in the presence of BV that expressed or lacked CAR (CAR-BV and mock-BV, respectively). Treatment with mock-BV even at doses as high as 5 µg/mL failed to attenuate the luciferase activity of B16-CAR cells. In contrast, treatment with CAR-BV with doses as low as 0.5 µg/mL significantly decreased the luciferase activity of infected cells, which reached 65% reduction at 5 µg/mL. These findings suggest that a receptor-displaying BV system could be used to evaluate viral infection.
    Biological & Pharmaceutical Bulletin 01/2013; 36(11):1867-9. DOI:10.1248/bpb.b13-00627 · 1.78 Impact Factor
  • Akihiro Watari, Kiyohito Yagi, Masuo Kondoh
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    ABSTRACT: Claudin-4, a member of a tetra-transmembrane protein family that comprises 27 members, is a key functional and structural component of the tight junction-seal in mucosal epithelium. Modulation of the claudin-4-barrier for drug absorption is now of research interest. Disruption of the claudin-4-seal occurs during inflammation. Therefore, claudin-4 modulators (repressors and inducers) are promising candidates for drug development. However, claudin-4 modulators have never been fully developed. Here, we attempted to design a screening system for claudin-4 modulators by using a reporter assay. We prepared a plasmid vector coding a claudin-4 promoter-driven luciferase gene and established stable reporter gene-expressing cells. We identified thiabendazole, carotene and curcumin as claudin-4 inducers, and potassium carbonate as a claudin-4 repressor by using the reporter cells. They also increased or decreased, respectively, the integrity of the tight junction-seal in Caco-2 cells. This simple reporter system will be a powerful tool for the development of claudin-4 modulators.
    Biochemical and Biophysical Research Communications 08/2012; 426(4):454-60. DOI:10.1016/j.bbrc.2012.08.083 · 2.28 Impact Factor
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    ABSTRACT: Claudins (CLs) are a family of tetra-integral membrane proteins that are a key structural and functional component of tight junctions. CLs are overexpressed in some malignant tumors. Claudin-4 is highly expressed in the epithelial cells covering mucosal immune tissues. CLs may therefore be a potential target for improving drug absorption, treating cancer, and developing mucosal vaccine. Research using Clostridium perfringens enterotoxin has resulted in proofs of concept for CL-targeted drug development. A platform for the creation of CL binders, such as immunization of CL and preparation of CL proteins, is now beginning to be established.
    Annals of the New York Academy of Sciences 07/2012; 1258(1):65-70. DOI:10.1111/j.1749-6632.2012.06503.x · 4.31 Impact Factor
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    ABSTRACT: Intercellular spaces between adjacent mucosal epithelial cells are sealed by tight junctions (TJs) that prevent the free movement of solutes across the epithelium. Claudins (CLs), a family of 27 integral membrane proteins, are essential components for TJ seals. We previously used a CL-3/-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), to show that CL modulation is a promising method to enhance mucosal absorption. Recently, by using a C-CPE mutant library, we developed a CL binder (m19) with broad specificity to CL-1, -2, -4, and -5. Here, we compared the mucosal absorption-enhancing activity of C-CPE and m19. Both CL binders enhanced jejunal absorption of dextran with a molecular mass of 4000 and 150,000 Da and nasal absorption of dextran with a mass of 4000 Da but not 150,000 Da in rats. Although both binders showed similar nasal absorption-enhancing activity of dextran (4000 Da), m19 exhibited a more potent jejunal absorption-enhancing effect than that of C-CPE. These findings suggest that mucosal absorption-enhancing activity may be modified by modulating CL specificity.
    Biochemical and Biophysical Research Communications 05/2012; 423(2):229-33. DOI:10.1016/j.bbrc.2012.05.060 · 2.28 Impact Factor

Publication Stats

2k Citations
398.26 Total Impact Points

Institutions

  • 1998–2014
    • Osaka University
      • • Graduate School of Pharmaceutical Sciences
      • • Laboratory of Bio-Function Molecular Chemistry
      • • Division of Molecular Pharmaceutical Science
      Suika, Ōsaka, Japan
  • 2002–2013
    • Showa Pharmaceutical University
      Machida, Tōkyō, Japan
  • 1998–2011
    • Osaka University of Pharmaceutical Sciences
      • • Graduate School of Pharmaceutical Sciences
      • • Faculty of Pharmaceutical Sciences
      Ōsaka, Ōsaka, Japan
  • 2000–2010
    • Tokushima Bunri University
      • Faculty of Pharmaceutical Sciences
      Tokusima, Tokushima, Japan
  • 2006
    • Kobayashi Pharmaceutical Co., Ltd
      Kobayashi-chō, Miyazaki, Japan