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1.
Biochem Biophys Res Commun ; 616: 140-144, 2022 08 06.
Article in English | MEDLINE | ID: mdl-35679696

ABSTRACT

Although modulation of claudin-1-based tight junction (TJ) in stratum granulosum is an option for transdermal absorption of drugs, granular permeation enhancers have never been developed. We previously found that homoharringtonine (HHT), a natural alkanoid, weakened intestinal epithelial barrier with changing expression and cellular localization of TJ components such as claudin-1 and claudin-4. In the present study, we investigated whether HHT is an epidermal granular permeation enhancer. Treatment of normal human epidermal keratinocytes (NHEK) cells with HHT decreased claudin-1 and claudin-4 but not zonula occludens-1 and E-cadherin. HHT lowered TJ-integrity in NHEK cells, accompanied by permeation-enhancement of dextran (4 kDa) in a dose-dependent manner. Transdermal treatment of mice with HHT weakened epidermal barrier. HHT treatment enhanced transdermal absorption of dextran with a molecular mass of up to 10 kDa. Together, HHT may be a transdermal absorption enhancer.


Subject(s)
Dextrans , Homoharringtonine , Tight Junctions , Animals , Claudin-1/metabolism , Claudin-4/metabolism , Dextrans/metabolism , Homoharringtonine/pharmacology , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Tight Junctions/metabolism
2.
FEBS Lett ; 595(2): 220-229, 2021 01.
Article in English | MEDLINE | ID: mdl-33113151

ABSTRACT

Occludin (OCLN) is a tetraspan membrane component of epithelial tight junctions and a known receptor for hepatitis C virus (HCV). Previously, we established functional monoclonal antibodies (mAbs) that bind to each extracellular loop of OCLN and showed their ability to prevent in vitro and in vivo HCV infection. In this study, we converted these mAbs to corresponding monovalent antigen-binding fragments (Fabs) and single-chain variable fragment (scFv) antibodies. These Fab fragments and scFv antibodies demonstrate similar binding specificity and affinity to parental anti-OCLN mAbs. Moreover, Fab fragments and scFv antibodies inhibit in vitro HCV infection. The small functional monovalent OCLN-binding probes reported in our study have high potential as drug candidates and tools for biological and pharmaceutical studies of OCLN.


Subject(s)
Hepacivirus/physiology , Hepatitis C/metabolism , Immunoglobulin Fab Fragments/pharmacology , Occludin/metabolism , Single-Chain Antibodies/pharmacology , Antibody Affinity , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cell Line , Hepacivirus/drug effects , Hepatitis C/prevention & control , Humans , Immunoglobulin Fab Fragments/chemistry , Models, Biological , Occludin/chemistry , Single-Chain Antibodies/chemistry , Virus Internalization/drug effects , Virus Replication/drug effects
3.
J Biochem ; 166(4): 297-308, 2019 Oct 01.
Article in English | MEDLINE | ID: mdl-31077306

ABSTRACT

The tight junction protein occludin (OCLN) is a four-pass transmembrane protein with two extracellular loops (ELs), and also functions as a co-receptor for hepatitis C virus (HCV). Recently, we reported the establishment of monoclonal antibodies (mAbs) recognizing each intact EL domain of OCLN that can strongly prevent HCV infection in vitro and in vivo, and these mAbs were applicable for flow cytometric (FCM) analysis, immunocytochemistry (ICC) and cell-based enzyme-linked immunosorbent assay. In the present study, we further examined the application of these anti-OCLN mAbs and characterized their binding properties. All four mAbs were available for immunoprecipitation. The three first EL (EL1)-recognizing mAbs were applicable for immunoblotting, but the second EL (EL2)-recognizing one was not. Using site-directed mutagenesis, we also determined residues of OCLN critical for recognition by each mAb. Our findings showed that the small loop between two cysteines of the EL2 domain is essential for the binding to one EL2-recognizing mAb and that the recognition regions by three EL1-recognizing mAbs overlap, but are not the same sites of EL1. To obtain a deeper understanding of OCLN biology and its potential as a therapeutic target, specific mAbs to detect or target OCLN in intact cells should be powerful tools for future studies.

4.
Biochem Biophys Res Commun ; 514(3): 785-790, 2019 06 30.
Article in English | MEDLINE | ID: mdl-31079928

ABSTRACT

Occludin (OCLN), an integral tetra-spanning plasma membrane protein, is a host entry factor essential for hepatitis C virus (HCV) infection, making it a promising host-targeting molecule for HCV therapeutic intervention. We previously generated rat anti-OCLN monoclonal antibodies (mAbs) that strongly prevented HCV infection in vitro and in vivo. In the present study, we attempted to improve the druggability of the extracellular loop domain-recognizing anti-OCLN mAbs, namely clones 1-3 and 37-5, using genetic engineering. To avoid adverse reactions induced by antibody-dependent cellular cytotoxicity and enhance the antibody stability, we developed human-rat chimeric immunoglobulin G4 S228P mutant (IgG4m) forms of clones 1-3 and 37-5 (named Xi 1-3 and Xi 37-5, respectively) by grafting the variable regions of the light and heavy chains of each rat anti-OCLN mAb into those of human IgG4m. The constructed Xi 1-3 and Xi 37-5 chimeras demonstrated levels of affinity and specificity similar to each parental rat anti-OCLN mAb, and the Fcγ receptor Ⅲa was not activated by the antigen-bound chimeric mAbs, as expected. Both chimeric mAbs inhibited in vitro infection with various HCV genotypes. These results indicate that the IgG4m forms of human-rat chimeric anti-OCLN mAbs may be potential candidate molecules of host-targeting antivirals with pan-genotypic anti-HCV activity.


Subject(s)
Antibodies, Monoclonal/pharmacology , Hepacivirus/drug effects , Hepatitis C/virology , Occludin/immunology , Animals , Cell Line , Humans , Immunoglobulin G/metabolism , Inhibitory Concentration 50 , Jurkat Cells , Protein Domains , Protein Structure, Secondary , Rats , Receptors, IgG/metabolism
5.
Yakugaku Zasshi ; 139(2): 285-297, 2019.
Article in Japanese | MEDLINE | ID: mdl-30713241

ABSTRACT

This review reflects back over almost 40 years of the author's basic research conducted at Graduate School of Pharmaceutical Sciences, Osaka University, Japan. After performing postdoctoral research in USA, the author became a research associate at Prof. Yoshiharu Miura's lab and started research on Biochemical Engineering in 1984. At that time, the main research purpose was to solve global environmental issues for maintaining human health. The author's achievements included novel useful material production system under inorganic conditions and genetically engineered whole-cell bacterial sensors detecting arsenite by naked eye without a detecting device. Another theme in the lab was to construct bioartificial liver support system. Various scaffolds for hepatocytes were newly prepared for constructing the compact reactor. Besides the bioreactor study, the author conducted cell transplantation research for the treatment of chronic liver diseases. It was shown that mesenchymal stem cells derived from third molars (wisdom teeth) could differentiate into hepatocytes and exhibit therapeutic effects in liver-damaged animals. After 2006, the lab started research on drug delivery systems, including noninvasive delivery of drugs such as peptides and nucleic acids by regulating epithelial tight junctions. Many substances enabling drug delivery through "paracellular" route were newly prepared. The author started basic research on Biochemical Engineering in the 1970s. Although these studies eventually shifted into the pharmaceutical field, the underlying concept was based on "engineering" throughout a 40-year research period. The author cordially thanks all colleagues for supporting engineering research in our lab.


Subject(s)
Biochemistry/trends , Biopharmaceutics/trends , Chemical Engineering/trends , Research/trends , Biosensing Techniques , Biotransformation , Cell Transplantation , Drug Delivery Systems , Humans , Japan , Liver, Artificial , Polymers , Time Factors , United States
6.
J Pharmacol Exp Ther ; 368(2): 179-186, 2019 02.
Article in English | MEDLINE | ID: mdl-30530622

ABSTRACT

Claudin (CLDN) proteins, a tetra-transmembrane family containing over 20 members, have been identified as key structural and functional components of intercellular seals, tight junctions (TJs). CLDNs are involved in the barrier and fence functions of TJs. Loosening the TJ barrier is one strategy for increasing drug absorption and delivery to the brain. Due to aberrant CLDN expression, the TJ fence function is frequently dysregulated in carcinogenesis. In addition, CLDN-1 is a co-receptor for the hepatitis C virus. Together these characteristics indicate CLDNs as promising targets for drug development, and CLDN binders are potential candidates for delivering drugs, treating cancer, and preventing viral infection. Before 2008, a receptor-binding fragment of Clostridium perfringens enterotoxin was the only CLDN binder available. Since then, several challenges regarding the generation of monoclonal antibodies against CLDNs have been surmounted, leading to breakthroughs in CLDN-targeted drug development. Here, we provide an overview of the recent progress in technology using created CLDN binders-anti-CLDN monoclonal antibodies.


Subject(s)
Autoantibodies/metabolism , Claudins/antagonists & inhibitors , Claudins/metabolism , Drug Development/trends , Pharmaceutical Preparations/metabolism , Amino Acid Sequence , Animals , Autoantibodies/genetics , Claudins/genetics , Humans
7.
Sci Rep ; 8(1): 8383, 2018 05 30.
Article in English | MEDLINE | ID: mdl-29849184

ABSTRACT

The production of antibodies against the extracellular regions (ECR) of multispanning membrane proteins is notoriously difficult because of the low productivity and immunogenicity of membrane proteins due to their complex structure and highly conserved sequences among species. Here, we introduce a new method to generate ECR-binding antibodies utilizing engineered liposomal immunogen prepared using a wheat cell-free protein synthesis system. We used claudin-5 (CLDN-5) as the target antigen, which is a notoriously difficult to produce and poorly immunogenic membrane protein with two highly conserved extracellular loops. We drastically improved the productivity of CLDN-5 in the cell-free system after suppressing and normalizing mRNA GC content. To overcome its low immunogenicity, two engineered antigens were designed and synthesized as proteoliposomes: a human/mouse chimeric CLDN-5, and a CLDN-5-based artificial membrane protein consisting of symmetrically arranged ECRs. Intraperitoneal immunization of both engineered CLDN-5 ECR antigens induced ECR-binding antibodies in mice with a high success rate. We isolated five monoclonal antibodies that specifically recognized CLDN-5 ECR. Antibody clone 2B12 showed high affinity (<10 nM) and inhibited CLDN-5-containing tight junctions. These results demonstrate the effectiveness of the methods for monoclonal antibody development targeting difficult-to-produce membrane proteins such as CLDNs.


Subject(s)
Antibodies, Monoclonal/immunology , Claudin-5/genetics , Claudin-5/immunology , Extracellular Space/metabolism , Protein Engineering , Amino Acid Sequence , Animals , Antibody Specificity , Claudin-5/chemistry , Conserved Sequence , Humans , Immunization , Male , Mice
8.
J Control Release ; 283: 126-134, 2018 08 10.
Article in English | MEDLINE | ID: mdl-29753959

ABSTRACT

Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders.


Subject(s)
Bacterial Toxins/pharmacology , Blood-Brain Barrier/drug effects , Oligonucleotides, Antisense/metabolism , Tight Junctions/metabolism , Animals , Bacterial Toxins/administration & dosage , Blood-Brain Barrier/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Enterotoxins/administration & dosage , Female , Mice, Inbred C57BL , Oligonucleotides, Antisense/administration & dosage , RNA, Long Noncoding/genetics , Rats , Receptors, Lipoprotein/metabolism
9.
Sci Rep ; 8(1): 2904, 2018 02 13.
Article in English | MEDLINE | ID: mdl-29440671

ABSTRACT

Vaccine delivery is an essential element for the development of mucosal vaccine, but it remains to be investigated how physical barriers such as mucus and cilia affect vaccine delivery efficacy. Previously, we reported that C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) targeted claudin-4, which is expressed by the epithelium associated with nasopharynx-associated lymphoid tissue (NALT), and could be effective as a nasal vaccine delivery. Mice lacking tubulin tyrosine ligase-like family, member 1 (Ttll1-KO mice) showed mucus accumulation in nasal cavity due to the impaired motility of respiratory cilia. Ttll1-KO mice nasally immunized with C-CPE fused to pneumococcal surface protein A (PspA-C-CPE) showed reduced PspA-specific nasal IgA responses, impaired germinal center formation, and decreased germinal center B-cells and follicular helper T cells in the NALT. Although there was no change in the expression of claudin-4 in the NALT epithelium in Ttll1-KO mice, the epithelium was covered by a dense mucus that prevented the binding of PspA-C-CPE to NALT. However, administration of expectorant N-acetylcysteine removed the mucus and rescued the PspA-specific nasal IgA response. These results show that the accumulation of mucus caused by impaired respiratory cilia function is an interfering factor in the C-CPE-based claudin-4-targeting nasal vaccine.


Subject(s)
Bacterial Proteins/immunology , Claudin-4/metabolism , Immunoglobulin A/immunology , Mucus/metabolism , Nasal Cavity , Vaccination/methods , Animals , Bacterial Proteins/metabolism , Enterotoxins/metabolism , Epithelium/metabolism , Gene Knockout Techniques , Mice , Peptide Synthases/deficiency , Peptide Synthases/genetics
10.
Eur J Pharm Sci ; 117: 161-167, 2018 May 30.
Article in English | MEDLINE | ID: mdl-29448044

ABSTRACT

Claudin-2 (CLDN-2), a pore-forming tight junction protein with a tetra-transmembrane domain, is involved in carcinogenesis and the metastasis of some cancers. Although CLDN-2 is highly expressed in the tight junctions of the liver and kidney, whether CLDN-2 is a safe target for cancer therapy remains unknown. We recently generated a rat monoclonal antibody (mAb, clone 1A2) that recognizes the extracellular domains of human and mouse CLDN-2. Here, we investigated the safety of CLDN-2-targeted cancer therapy by using 1A2 as a model therapeutic antibody. Because most human therapeutic mAbs are IgG1 subtype that can induce antibody-dependent cellular cytotoxicity, we generated a human-rat chimeric IgG1 form of 1A2 (xi-1A2). xi-1A2 activated Fcγ receptor IIIa in the presence of CLDN-2-expressing cells, indicating that xi-1A2 likely exerts antibody-dependent cellular cytotoxicity. At 24 h after its intravenous injection, xi-1A2 was distributed into the liver, kidney, and tumor tissues of mice bearing CLDN-2-expressing fibrosarcoma cells. Treatment of the xenografted mice with xi-1A2 attenuated tumor growth without apparent adverse effects, such as changes in body weight and biochemical markers of liver and kidney injury. These results support xi-1A2 as the lead candidate mAb for safe CLDN-2-targeted cancer therapy.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Claudin-2/immunology , Neoplasms/drug therapy , Protein Domains/immunology , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Female , Humans , Immunoglobulin G/immunology , Jurkat Cells , Kidney/metabolism , Liver/metabolism , Mice, Inbred BALB C , Neoplasms/immunology , Neoplasms/metabolism , Rats , Receptors, IgG/genetics , Receptors, IgG/immunology
11.
J Virol ; 92(8)2018 04 15.
Article in English | MEDLINE | ID: mdl-29437969

ABSTRACT

Hepatitis C virus (HCV) entry into host cells is a multistep process requiring various host factors, including the tight junction protein occludin (OCLN), which has been shown to be essential for HCV infection in in vitro cell culture systems. However, it remains unclear whether OCLN is an effective and safe target for HCV therapy, owing to the lack of binders that can recognize the intact extracellular loop domains of OCLN and prevent HCV infection. In this study, we successfully generated four rat anti-OCLN monoclonal antibodies (MAbs) by the genetic immunization method and unique cell differential screening. These four MAbs bound to human OCLN with a very high affinity (antibody dissociation constant of <1 nM). One MAb recognized the second loop of human and mouse OCLN, whereas the three other MAbs recognized the first loop of human OCLN. All MAbs inhibited HCV infection in Huh7.5.1-8 cells in a dose-dependent manner without apparent cytotoxicity. Additionally, the anti-OCLN MAbs prevented both cell-free HCV infection and cell-to-cell HCV transmission. Kinetic studies with anti-OCLN and anti-claudin-1 (CLDN1) MAbs demonstrated that OCLN interacts with HCV after CLDN1 in the internalization step. Two selected MAbs completely inhibited HCV infection in human liver chimeric mice without apparent adverse effects. Therefore, OCLN would be an appropriate host target for anti-HCV entry inhibitors, and anti-OCLN MAbs may be promising candidates for novel anti-HCV agents, particularly in combination with direct-acting HCV antiviral agents.IMPORTANCE HCV entry into host cells is thought to be a very complex process involving various host entry factors, such as the tight junction proteins claudin-1 and OCLN. In this study, we developed novel functional MAbs that recognize intact extracellular domains of OCLN, which is essential for HCV entry into host cells. The established MAbs against OCLN, which had very high affinity and selectivity for intact OCLN, strongly inhibited HCV infection both in vitro and in vivo Using these anti-OCLN MAbs, we found that OCLN is necessary for the later stages of HCV entry. These anti-OCLN MAbs are likely to be very useful for understanding the OCLN-mediated HCV entry mechanism and might be promising candidates for novel HCV entry inhibitors.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antiviral Agents/pharmacology , Carcinoma, Hepatocellular/prevention & control , Disease Models, Animal , Hepatitis C/prevention & control , Liver Neoplasms/prevention & control , Occludin/antagonists & inhibitors , Animals , Carcinoma, Hepatocellular/virology , Hepacivirus/isolation & purification , Hepatitis C/virology , Humans , Liver Neoplasms/virology , Male , Mice , Occludin/immunology , Rats, Wistar , Tight Junctions , Tumor Cells, Cultured , Virus Internalization
12.
J Nat Med ; 72(2): 448-455, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29275476

ABSTRACT

Lentinula edodes mycelia solid culture extract (MSCE) is used as a medical food ingredient and provides beneficial effects to patients with cancer and chronic type C hepatitis. Low molecular weight lignin (LM-lignin), which is an active component of MSCE, exhibits hepatoprotective, antitumor, antiviral, and immunomodulatory effects. In this study, we investigated the effect of LM-lignin/lignosulfonic acid on intestinal barrier function. Lignosulfonic acid enhanced transepithelial membrane electrical resistance in human intestinal Caco-2 cell monolayers. In Caco-2 cells treated with lignosulfonic acid, expression of claudin-2, which forms high conductive cation pores in tight junctions (TJs), was decreased. Lignosulfonic acid also attenuated the barrier dysfunction that is caused by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. TNF-α- and IFN-γ-induced activation of NF-κB, such as translocation of NF-κB p65 into the nucleus and induction of gene expression, was inhibited by lignosulfonic acid treatment. Furthermore, lignosulfonic acid decreased the TNF-α- and IFN-γ-induced increase in interleukin (IL)-1ß and IL-6 expression in Caco-2 cells. These results suggest that lignosulfonic acid not only enhances TJ barrier function but also restores TJ barrier integrity impaired by inflammatory cytokines. Therefore, lignosulfonic acid may be beneficial for the treatment of inflammation-induced intestinal barrier dysfunction observed in inflammatory bowel disease.


Subject(s)
Epithelial Cells/drug effects , Intestinal Mucosa/drug effects , Lignin/analogs & derivatives , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , Caco-2 Cells , Cell Line, Tumor , Humans , Lignin/pharmacology , Lignin/therapeutic use , Signal Transduction , Transfection
13.
Sci Rep ; 7(1): 14514, 2017 11 06.
Article in English | MEDLINE | ID: mdl-29109448

ABSTRACT

Claudins are key functional and structural components of tight junctions (TJs) in epithelial cell sheets. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to claudin-4 and reversibly modulates intestinal TJ seals, thereby enhancing paracellular transport of solutes. However, the use of C-CPE as an absorption enhancer is limited by the molecule's immunogenicity and manufacturing cost. Here, we developed a high-throughput screening system based on the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method to identify claudin-4 binders in a library collection of 32,560 compounds. Thiostrepton, identified from the screen, decreased transepithelial electrical resistance and increased flux of 4-kDa fluorescein isothiocyanate-labelled dextran (FD-4) in Caco-2 cell monolayers, a model of intestinal epithelium. Thiostrepton changed the expression, but not the localisation, of TJ components. Treatment of rat jejunum with thiostrepton increased the absorption of FD-4 without tissue toxicity, indicating that thiostrepton is a novel claudin-4 binder that enhances intestinal permeability. The screening system may therefore be a useful tool for identifying claudin-4 binders to enhance drug absorption in mucosa.


Subject(s)
Claudin-4/metabolism , Enterotoxins/pharmacology , Thiostrepton/pharmacology , Tight Junctions/drug effects , Tight Junctions/metabolism , Animals , Caco-2 Cells , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Electric Impedance , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Humans , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Jejunum/drug effects , Jejunum/metabolism , L-Lactate Dehydrogenase/metabolism , Male , Rats, Wistar , Recombinant Proteins/metabolism
14.
J Pharmacol Exp Ther ; 363(3): 444-451, 2017 12.
Article in English | MEDLINE | ID: mdl-28928120

ABSTRACT

Disruption of the gastrointestinal epithelial barrier is a hallmark of chronic inflammatory bowel diseases (IBDs). The transmembrane protein claudin 2 (CLDN2) is a component of epithelial tight junctions (TJs). In the intestines of patients with IBDs, the expression of the pore-forming TJ protein CLDN2 is upregulated. Although CLDN2 is involved in these leaky barriers, whether it can be a target to enhance TJ integrity is unknown because a CLDN2-specific inhibitor has not been developed. Here, we used DNA immunization to generate a monoclonal antibody (mAb) that recognized an extracellular loop of CLDN2. Treatment of epithelial cell monolayers with the mAb increased barrier integrity. In addition, the anti-CLDN2 mAb attenuated the decrease in TJ integrity induced by the proinflammatory cytokine tumor necrosis factor-α (TNF-α), and cotreatment of cells with anti-TNF-α mAb and anti-CLDN2 mAb showed additive attenuating effects. These findings indicate that CLDN2 may be a target for enhancing TJ integrity, and CLDN2 binder may be an enhancer of mucosal barrier integrity and a potential therapeutic option for IBDs.


Subject(s)
Claudins/metabolism , Inflammatory Bowel Diseases/metabolism , Tight Junctions/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Cell Line, Tumor , Claudins/immunology , Female , Humans , Inflammatory Bowel Diseases/therapy , Intestinal Mucosa/metabolism , Mice , Mice, Inbred BALB C , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
15.
J Pharmacol Exp Ther ; 363(2): 275-283, 2017 11.
Article in English | MEDLINE | ID: mdl-28819070

ABSTRACT

A current bottleneck in the development of central nervous system (CNS) drugs is the lack of drug delivery systems targeting the CNS. The intercellular space between endothelial cells of the blood-brain barrier (BBB) is sealed by complex protein-based structures called tight junctions (TJs). Claudin-5 (CLDN-5), a tetra-transmembrane protein is a key component of the TJ seal that prevents the paracellular diffusion of drugs into the CNS. In the present study, to investigate whether CLDN-5 binders can be used for delivery of drugs to the CNS, we generated monoclonal antibodies (mAbs) specific to the extracellular domains of CLDN-5. In an in vitro model of the BBB, the anti-CLDN-5 mAbs attenuated trans-epithelial/endothelial electrical resistance and enhanced solute permeation. These anti-CLDN-5 mAbs are potential leads for the development of novel drug delivery systems targeting the CNS.


Subject(s)
Blood-Brain Barrier/metabolism , Claudin-5/metabolism , Animals , Antibodies, Monoclonal/immunology , Cell Line, Tumor , Claudin-5/chemistry , Claudin-5/immunology , Extracellular Space/metabolism , Female , Humans , Male , Mice , Permeability , Protein Domains , Tight Junctions/metabolism
16.
J Control Release ; 260: 1-11, 2017 08 28.
Article in English | MEDLINE | ID: mdl-28528740

ABSTRACT

A limiting barrier for mucosal absorption of drugs is the tight junction (TJ). TJs exist between two adjacent cells (bicellular TJ, bTJ) and at the sites where three cells meet (tricellular TJ, tTJ). We present a novel approach which employs a physiologically regulated pathway for the passage of large molecules through the tTJ. Main barrier-relevant tTJ proteins are tricellulin and angulin-1 to -3. We developed an angulin binder from Clostridium perfringens iota-toxin (Ib) whose receptor is angulin-1. An Ib fragment corresponding to amino acids 421-664 (Ib421-664) of iota-toxin proved to bind in cells expressing angulin-1 and -3, but not angulin-2. This binding led to removal of angulin-1 and tricellulin from the tTJ which enhanced the permeation of macromolecular solutes. Ib421-664 enhanced intestinal absorption in rats and mice. Our findings indicate that Ib421-664, which we designate angubindin-1, is a modulator of the tTJ barrier and that modulation of that barrier qualifies for a new strategy of developing a mucosal absorption enhancer.


Subject(s)
ADP Ribose Transferases/chemistry , Bacterial Toxins/chemistry , Intestinal Absorption , MARVEL Domain Containing 2 Protein/metabolism , Peptide Fragments/pharmacology , Receptors, Cell Surface/metabolism , Animals , Cell Line , Humans , Male , Mice , Rats, Wistar , Tight Junctions/metabolism
17.
Cell Physiol Biochem ; 41(5): 1924-1934, 2017.
Article in English | MEDLINE | ID: mdl-28391269

ABSTRACT

BACKGROUND/AIMS: Although proinflammatory cytokine-induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α. METHODS: To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER) and paracellular permeability were measured. Production levels and localization of tight junction (TJ) proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC) and MLC kinase (MLCK) mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively. RESULTS: Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition. CONCLUSION: Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation.


Subject(s)
Carbazoles/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Intestinal Mucosa/enzymology , Myosin-Light-Chain Kinase/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Caco-2 Cells , Checkpoint Kinase 1/metabolism , Claudin-5/biosynthesis , Enzyme Activation/drug effects , Humans , Myosin Light Chains/metabolism , Phosphorylation/drug effects , Tight Junctions/enzymology
18.
Ann N Y Acad Sci ; 1397(1): 5-16, 2017 06.
Article in English | MEDLINE | ID: mdl-28415141

ABSTRACT

The 27-member family of tetraspan membrane proteins known as claudins (CLDNs) is a major component of tight junctions. A series of studies elucidating the relationship between CLDNs and various pathological conditions has provided new insights into drug development. For instance, CLDN-1 may be a potent target for epidermal absorption of drugs and for treating hepatitis C virus (HCV) infection. CLDN-4 may be a target for treating cancer. Because CLDNs are also expressed in various normal tissues, safety and efficacy evaluations are critical for translational research. We previously developed several anti-CLDN antibodies and have established proof of concept for CLDN-targeted drug development using these reagents. Here, we provide an overview of CLDN-1 as a target for improving epidermal drug absorption and preventing HCV infection and of CLDN-4 as a target for anticancer therapeutics.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Claudin-1/metabolism , Claudin-4/metabolism , Hepacivirus/drug effects , Hepatitis C/drug therapy , Neoplasms/drug therapy , Animals , Claudin-1/immunology , Claudin-4/immunology , Epidermis/metabolism , Hepacivirus/physiology , Hepatitis C/virology , Humans , Tight Junctions/metabolism
19.
Int Immunopharmacol ; 45: 43-52, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28187312

ABSTRACT

Lipid A comprises the active region of lipopolysaccharide (LPS), and its phosphate group is required for LPS activities. Additionally, it is essential for effects of inhibitors of LPS-induced coagulation activity in limulus amebocyte lysate (LAL) tests. Lipid A has phosphorylated glucosamine residues, which are structurally similar to glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P). This study focused on the antagonistic effects of glucose phosphates on the action of protein or non-protein inhibitors against LAL coagulation, LPS-LPS-binding protein (LBP) interaction, and LPS bioactivities. These effects of glucose phosphates were evaluated and compared with those of other charged sugars such as fructose 6-phosphate and glucuronic acid by LAL tests, ELISA-based LPS-LBP binding assay, cell-based assay, and using a mouse endotoxin shock model. G6P neutralized the interfering actions of drug substances and plasma proteins on LPS coagulation activity in LAL tests. Compared to other sugars, G6P more strongly inhibited LPS binding to LBP, leading to significant inhibition of LPS-induced cellular responses in human umbilical vein endothelial cells and in the THP-1 human leukemic line. Consistent herewith, G6P inhibited inflammatory cytokine release and decreased serum alanine aminotransferase and hepatic caspase-3/7 activities and mortality in LPS-stimulated d-galactosamine-sensitized mice. These data indicated that the structural properties of G6P, such as its glucose moiety and phosphorylation on carbon 6, are important for suppressing the interaction of proteins with LPS. Therefore, G6P is useful to improve sensitivity and accuracy of plasma and drug LPS assays, and such structural property is more suitable to antagonize LPS activities.


Subject(s)
Acute-Phase Proteins/metabolism , Carrier Proteins/metabolism , Glucose/metabolism , Lipid A/metabolism , Lipopolysaccharides/metabolism , Membrane Glycoproteins/metabolism , Nephelometry and Turbidimetry/methods , Animals , Carbon , Caspase 3/metabolism , Glucose/chemistry , Horseshoe Crabs , Human Umbilical Vein Endothelial Cells , Humans , Male , Mice , Mice, Inbred C57BL , Phosphates/chemistry , Phosphorylation , Protein Binding , Tumor Necrosis Factor-alpha/metabolism
20.
Biochem Biophys Res Commun ; 484(3): 543-549, 2017 03 11.
Article in English | MEDLINE | ID: mdl-28130106

ABSTRACT

When considering the safety of ingested nanomaterials, it is important to quantitate their transfer across intestinal cells; however, little information exists about the effects of nanomaterial size or exposure side (apical versus basolateral epithelial surface) on nanomaterial transfer. Here, we examined cellular internalization and transcellular transport, and the effects of nanomaterials on Caco-2 monolayers after apical or basolateral exposure to Ag or Au nanoparticles with various sizes. After apical treatment, both internalization and transfer to the basolateral side of the monolayers were greater for smaller Ag nanoparticles than for larger Ag nanoparticles. In contrast, after basolateral treatment, larger Ag nanoparticles were more internalized than smaller Ag nanoparticles, but the transfer to the apical side was greater for smaller Ag nanoparticles. Au nanoparticles showed different rules of internalization and transcellular transport compared with Ag nanoparticles. Furthermore, the paracellular permeability of the Caco-2 monolayers was temporarily increased by Ag nanoparticles (5 µg/mL; diameters, ≤10 nm) following basolateral but not apical exposure. We conclude that the internalization, transfer, and effects of nanomaterials in epithelial cell monolayers depend on the size and composition of nanomaterials, and the exposure side.


Subject(s)
Cell Membrane Permeability/drug effects , Cell Membrane/chemistry , Cell Membrane/drug effects , Gold/chemistry , Metal Nanoparticles/chemistry , Silver/chemistry , Subcellular Fractions/chemistry , Caco-2 Cells , Diffusion , Gold/administration & dosage , Humans , Metal Nanoparticles/administration & dosage , Metal Nanoparticles/ultrastructure , Particle Size , Respiratory Tract Absorption , Silver/administration & dosage , Subcellular Fractions/drug effects
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