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Objective: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1. Methods: In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19. Results: C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126. Conclusion: These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 150,000Da and nasal absorption of dextran with a mass of 4000Da but not 150,000Da in rats. Although both binders showed similar nasal absorption-enhancing activity of dextran (4000Da), 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. [Copyright &y& Elsevier]

Abstract: Epithelium covers the body and, therefore, separates the inner body from the outside environment. Passage across the epithelium is the first step in drug absorption. Tight junctions (TJs) seal the space between adjacent epithelial cells and prevent the free movement of solutes through the paracellular space. Modulation of the epithelial barrier is the most important strategy for enhancing drug absorption. Development of the strategy has accelerated with progress in understanding of the biology of the TJ seal. The first-generation absorption enhancers were screened on the basis of their absorption-enhancing activity in vivo. However, TJs were not well understood initially. The identification of TJ components, including those based on occludin and claudins, has led to the development of new strategies for drug absorption. Accumulation of knowledge of claudins has provided new insights into the paracellular transport of drugs. This review examines the relationship between advances in understanding of TJ biology and paracellular transport of drugs and discusses progress in the development of mucosal absorption enhancers. [Copyright &y& Elsevier]

Abstract: Efficient delivery of antigen to mucosal immune tissues is an essential part of mucosal vaccination. Claudin-4 is expressed on the epithelial cells that cover the mucosal immune tissues. We previously found that claudin-4-targeting is a promising strategy for mucosal vaccination by using a claudin-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Substitution of Asn and Ser at positions 309 and 313, respectively, with alanine increased the affinity of C-CPE for claudin-4. However, application of the C-CPE mutant as a mucosal vaccine has never been tried. Here, we investigated whether the C-CPE mutant could serve as a mucosal vaccine. We used ovalbumin (OVA) as a model antigen and fused the C-CPE mutant to it. The resultant fusion protein was bound to claudin-4. When mice were immunized with the C-CPE mutant-fused OVA, OVA-specific serum IgG and nasal IgA increased relative to levels in mice immunized with a C-CPE-fused antigen. Immunization with the C-CPE mutant-fused OVA activated Th1- and Th2-type responses and led to increased anti-tumor activity against OVA-expressing thymoma cells relative to that of mice immunized with the C-CPE-fused antigen. These findings suggest that the alanine-substituted C-CPE mutant shows promise as a claudin-targeted mucosal vaccine. [Copyright &y& Elsevier]

Abstract: Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator. [Copyright &y& Elsevier]

Abstract: Mucosa-associated lymphoid tissue (MALT) plays pivotal roles in mucosal immune responses. Efficient delivery of antigens to MALT is a critical issue for the development of mucosal vaccines. Although claudin-4 is preferentially expressed in MALT in the gut, a claudin-4-targeting approach for mucosal vaccination has never been developed. In the present study, we found that claudin-4 is expressed in nasal MALT, and we prepared a fusion protein of ovalbumin (OVA) as a model antigen with a claudin-4-binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) (OVA-C-CPE). Nasal immunization with OVA-C-CPE, but not a mixture of OVA and C-CPE, induced the production of OVA-specific serum IgG and nasal, vaginal and fecal IgA. Deletion of the claudin-4-binding region in OVA-C-CPE attenuated the induction of the immune responses. OVA-C-CPE immunization activated both Th1 and Th2 responses, and nasal immunization with OVA-C-CPE showed anti-tumor activity in mice inoculated with OVA-expressing thymoma cells. These results indicate that the claudin-4-targeting may be a potent strategy for nasal vaccination. [Copyright &y& Elsevier]

Abstract: Claudin (CL)-4, a tight junction protein, is overexpressed in some human neoplasias, including ovarian, breast, pancreatic and prostate cancers. The targeting of CL-4 is a novel strategy for tumor therapy. We previously found that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to CL-4. In the present study, we genetically prepared a novel CL-4-targeting molecule (DTA–C-CPE) by fusion of C-CPE and diphtheria toxin fragment A (DTA). Although DTA is not toxic to CL-4-expressing L cells, even at 20μg/ml, DTA–C-CPE is toxic to CL-4-expressing L cells at 1μg/ml. DTA–C-CPE-induced cytotoxicity was attenuated by pretreatment of the cells with C-CPE but not bovine serum albumin, indicating that DTA–C-CPE may bind to CL-4-expressing L cells through its C-CPE domain. To evaluate the specificity of DTA–C-CPE, we examined its cytotoxic effects in L cells that express CL-1, -2, -4 or -5. We found that DTA–C-CPE was toxic to only CL-4-expressing L cells. Thus, C-CPE may be a promising ligand for the development of cancer-targeting systems. [Copyright &y& Elsevier]

Abstract: Biologics, such as peptides, proteins and nucleic acids, are emerging pharmaceuticals. Passage across the epithelium is the first step in the absorption of biologics. Tight junctions (TJ) function as seals between adjacent epithelial cells, preventing free movement of solutes across the epithelium. We previously found that modulation of a key TJ component, claudin-4, is a potent method to enhance jejunal absorption when we used dextran as a model drug and the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) as a claudin-4 modulator. Here, we investigated whether the claudin-4 modulator enhances jejunal, nasal and pulmonary absorption of a biologics human parathyroid hormone derivative, hPTH(1-34). The claudin-4 modulator enhanced nasal but not jejunal and pulmonary absorption of hPTH(1-34). C-CPE is hydrophobic with low solubility of less than 0.3mg/ml, but deletion of 10 amino acids at the N-terminal of C-CPE increased its solubility by 30-fold. Moreover, the N-terminal truncated C-CPE bound to claudin-4, modulated the TJ-barrier and enhanced jejunal absorption of dextran. The N-terminal-truncated C-CPE also enhanced jejunal and pulmonary absorption of hPTH(1-34). This report is the first to indicate that a claudin-4 modulator may be a promising enhancer of the jejunal, pulmonary and nasal absorption of a peptide drug. [Copyright &y& Elsevier]

Abstract: Nano-sized silica is a promising material for disease diagnosis, cosmetics and drugs. For the successful application of nano-sized material in bioscience, evaluation of nano-sized material toxicity is important. We previously found that nano-sized silica particles with a diameter of 70nm showed acute liver failure in mice. Here, we performed histological analysis of major organs such as the liver, spleen, lung, kidney, brain and heart in mice, chronically injected with 70-nm silica particles for 4weeks. Histological analysis revealed hepatic microgranulation and splenic megakaryocyte accumulation in these 70-nm silica particles treated mice, while the kidney, lung, brain and heart remained unaffected. Thus, liver and spleen appear to be the major target organs for toxicity by the chronic administration of the 70-nm silica particles. [Copyright &y& Elsevier]

Abstract: Nano-size materials are increasingly used in cosmetics, diagnosis, imaging and drug delivery, but the toxicity of the nano-size materials has never been fully investigated. Here, we investigated the relationship between particle size and toxicity using silica particles with diameters of 70, 300 and 1000nm (SP70, SP300, and SP1000) as a model material. To evaluate acute toxicity, we first performed histological analysis of liver, spleen, kidney and lung by intravenous administration of silica particles. SP70-induced liver injury at 30mg/kg body weight, while SP300 or 1000 had no effect even at 100mg/kg. Administration of SP70 dose-dependently increased serum markers of liver injury, serum aminotransferase and inflammatory cytokines. Repeated administration of SP70 twice a week for 4weeks, even at 10mg/kg, caused hepatic fibrosis. Taken together, nano-size materials may be hepatotoxic, and these findings will be useful for future development in nanotechnology-based drug delivery system. [Copyright &y& Elsevier]

Abstract: Primary cultured hepatocytes are widely used in the studies of basic and clinical hepatology. Finding an efficient method for gene transfer into primary hepatocytes will be an important advance for these studies. In the present study, we evaluated the activity of an adenovirus vector including promoters for the Rous sarcoma virus (RSV), elongation factor 1α, and cytomegalovirus (CMV) as well as the β-actin promoter/CMV enhancer (CA) using β-galactosidase as a reporter gene. Although RSV and elongation factor 1α promoters had low transcriptional activity in hepatocytes, the CA and CMV promoters had high activity. The CA promoter was the most active, mediating 50.3- and 204.4-fold more activity than the RSV promoter in mouse and rat hepatocytes, respectively. Dose-response studies revealed that transgene activity can be controlled by as much as 1000-fold, by selection of the promoter and the number of infectious particles per cell. These findings should help in the construction of adenovirus vectors for expressing genes of interest in rodent primary cultured hepatocytes. [Copyright &y& Elsevier]

The choice of drug target and the ability to deliver drug to those targets are pivotal in drug development. Most druggable targets are membrane proteins, such as G-protein-coupled receptors, channels and transporters. However, little attention has been paid to potential druggable targets in the membrane proteins of tight junctions (TJs), through which adjacent cell membranes contact one another. Recent progress in the cell biology of TJs provides new insights into the barrier and fence functions of TJs, suggesting that TJ components are promising candidates for drug discovery. In this review, we summarize the cell biology of TJs and discuss the TJ-based drug discovery. [Copyright &y& Elsevier]

Abstract: We previously reported that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a novel type of absorption enhancer that interacts with claudin-4 and that Tyr306 of C-CPE plays a role in ability of C-CPE to modulate barrier of tight junctions. In the current study, to investigate effects of Tyr306 on the C-CPE activity, we prepared some C-CPE mutants substituted Tyr306 with Trp (Y306W), Phe (Y306F) and Lys (Y306K). We found that Y306W and Y306F mutants of C-CPE had claudin-4 binding affinities and effects on the barrier function of tight junctions, whereas both of these properties were greatly reduced with the Y306K mutant. Finally, the Y306K but not the Y306F and Y306W mutants had reduced abilities to enhance absorption in rat jejunum. These results indicate that aromatic and hydrophobic properties, not hydrogen bonding potential, of Tyr306 are involved in the interaction of C-CPE with claudin-4 and in the modulation of the tight junction barrier function by C-CPE. [Copyright &y& Elsevier]

Abstract: The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) modulates the barrier function of claudin-4 via its C-terminal 16 amino acids. In the current study, we investigated the roles of tyrosine residues (Y306, Y310 and Y312) in this region in the modulation of TJs by C-CPE. Single mutations of Y306, Y310 and Y312 to alanine resulted in partial reduction of claudin-4 binding. We also prepared double mutants of C-CPE to further evaluate the roles of these tyrosine residues. Replacement of Y310 and Y312 with alanine (Y310A/Y312A) partly reduced the ability of C-CPE to bind to claudin-4. Double mutants Y306A/Y310A and Y306A/Y312A, however, lost the ability to bind to claudin-4 and to modulate the TJ barrier. We also found that a triple mutant (Y306A/Y310A/Y312A) lost the ability to bind claudin-4, modulate the TJ barrier, and enhance jejunal absorption in rats. These results indicate that tyrosines 306, 310, and 312 are critical for the interaction of C-CPE with claudin-4 and for the modulation of TJ barrier function by C-CPE. This study provides information that should help in the development of claudin modulators based on C-CPE. [Copyright &y& Elsevier]

Abstract: During pregnancy, the zinc level in fetal serum is up to two-fold higher than that in the maternal serum at the end of pregnancy, but the mechanism of zinc release from the placenta into fetal circulation is not well understood. In this study, we determined the expression profiles of zinc transporters in human trophoblast BeWo cells, a representative human trophoblast cell line. Zn transporter 1–8 (ZnT1–8), Zrt/IRT-like protein 1 and Zn transporter-like transporter 1 were detected in BeWo cells by reverse transcription-polymerase chain reaction. Forskolin (FK) is a representative inducer of differentiation of BeWo cells cytotrophoblast into syncytiotrophoblast. Treatment of BeWo cells with FK resulted in morphological changes of BeWo cells into syncytiotrophoblast cells and secretion of human chorionic gonadotropin, which is a characteristic of syncytiotrophoblast cells. Treatment of BeWo cells with FK elevated ZnT1, 2 and 4 mRNA levels. These data about expression profiles of ZnTs may be useful for further investigation of placental biology. [Copyright &y& Elsevier]

Abstract: Claudin family proteins, which contain 4 transmembrane domains, play a pivotal role in the barrier function of tight junctions (TJs) in epithelial sheets. We previously found that a modulator of claudin-4, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is a potent enhancer of jejunal drug absorption in rats. But the effects of C-CPE on the barrier function of TJs have never been fully understood. In the present study, we investigated the effects of C-CPE on the barrier function of TJs in Caco-2 monolayer and characterized the functional domain of C-CPE that is responsible for interaction with claudin-4. To evaluate the effects of C-CPE on the barrier function of TJs, we measured transepithelial electric resistance (TER) in Caco-2 monolayer cells seeded onto polycarbonate filters. Treatment of Caco-2 cells with C-CPE resulted in a decrease in TER. But, deletion of the 30 C-terminal amino acids of C-CPE, which is the putative binding domain for claudin, attenuated the decrease in TER values. Moreover, ablation of the 16 C-terminal amino acids of C-CPE also resulted in attenuation of the decrease in TER values. The C-terminal-deleted C-CPEs did not interact with claudin-4 or the extracellular domain 2 of claudin-4, which is the C-CPE binding site. These results suggest that the 16 C-terminal amino acids of C-CPE are responsible for the interaction of C-CPE and claudin-4 following the disruption of TJ barrier function. [Copyright &y& Elsevier]

Abstract: Novel cytotoxic rearranged kaurene- and ent-kaurene-type diterpenoids against HL-60 cells have been isolated from the unidentified New Zealand liverwort Jungermannia species together with previously known ent-kaurane-type diterpenoids. Their structures were determined by extensive NMR techniques, chemical degradation and X-ray crystallographic analysis. [Copyright &y& Elsevier]

Zinc is a vital metal that is a structural and functional component of many proteins. The precise mechanism of zinc transport in the placenta remains unclear. In this study, we investigated the expression of zinc transporters (ZnTs) in the mouse placenta and in two rat trophoblast cell lines, TR-TBT cells, which are syncytiotrophoblast cells of the labyrinth zone, and Rcho-1 cells, which retain trophoblast cell features and differentiate into trophoblast giant cells of the junctional zone. All of the ZnTs that have been identified in mice (ZnT1–7) were detected in the mouse placenta by RT-PCR. The expression profiles of ZnTs in the placenta during pregnancy were different. The mRNA levels of ZnTs, with the exception of ZnT7, did not change during pregnancy. The ZnT7 mRNA level in placenta was elevated during pregnancy. In TR-TBT cells, ZnT1, ZnT3 and ZnT4 were detected by RT-PCR analysis. In Rcho-1 cells, all of the ZnTs that have been identified in rats (ZnT1–4) were detected by RT-PCR analysis. There were no differences between the mRNA expression levels of ZnT family members in undifferentiated Rcho-1 cells and differentiated Rcho-1 cells. This is the first report of expression profiles of ZnTs during differentiation of the placenta in the mouse placenta and rat placental cell models. [Copyright &y& Elsevier]

Mercurial compounds modulate immunologic functions by inducing cytotoxicity. Although mercury chloride (HgCl2) is known to induce apoptosis in various immune system cells, the mechanism of the induction of apoptosis is poorly understood. In this study, we examined the activation of caspase-3, an important cysteine aspartic protease, during HgCl2-induced apoptosis in a human leukemia cell line (HL-60 cells). Both DNA fragmentation, a characteristic of apoptotic cells, and proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, occurred at 6 h after HgCl2 treatment in HL-60 cells. These results suggest that the activation of caspase-3 was involved in HgCl2-induced apoptosis. The release of cytochrome c (Cyt c) from mitochondria into the cytosol, which is an initiator of the activation of caspase cascades, was also observed in HgCl2-treated HL-60 cells. Moreover, the release of Cyt c from mitochondria was observed in HgCl2-treated mitochondria isolated from mice liver, and this was followed by mitochondrial permeability transition (PT). The PT was inhibited by cyclosporin A (CsA), a potent inhibitor of PT. CsA also suppressed the occurrence of DNA fragmentation induced by HgCl2 treatment in HL-60 cells. Taken together, these findings indicate that HgCl2 is a potent inducer of apoptosis via Cyt c release from the mitochondria in HL-60 cells. [Copyright &y& Elsevier]

Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HL-60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HL-60 cells with Zn for 6 h in the presence of Py (1 µm) exhibited cytotoxicity in a Zn dose-dependent manner (25–200 µm). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn dose-dependent fashion (50–100 µm), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method, peaked at 25 µm, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N ,N ,N ′,N ′-tetrakis (2-pyridylmethyl)ethylenediamine, a cell-permeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1 µm Py and 25 µm Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADP-ribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogen-activated protein kinase (p38MAPK ), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. Z-Asp-CH2 -DCB, a broad-spectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPK -specific inhibitor SB203580 also inhibited induction... [ABSTRACT FROM AUTHOR]

It is known that metallothionein (MT) synthesis occurs in the liver in various stressful situations such as immobilization and fasting. However, the mechanism of MT synthesis in stressful situations is not fully understood. In this study, we examined the involvement of leptin, the obese gene product, in MT synthesis induced by fasting stress. Despite an increase in hepatic MT levels induced by 24-hr fasting in wild-type mice, both wild-type and MT-null mice showed decreases in plasma leptin levels after 24 hr of fasting. Hepatic MT levels increased to levels comparable with that in control mice in ICR, C3H, 129Sv and Balb/c mice fasted for 24 hr, and plasma leptin levels decreased significantly. Repetition of fasting and feeding in turn every 24 hr caused a gradual decrease in hepatic MT levels after the fasting period. In contrast, the reduced plasma leptin levels increased after the fasting period with repetition of fasting-feeding cycles. The findings indicate that there is an adaptation to starvation. On the other hand, subcutaneous leptin infusion in fasted mice via an osmotic pump resulted in increases in hepatic MT levels compared to the levels in vehicle-treated mice after 24 hr of fasting. Only leptin infusion had no effect on hepatic MT levels. These results suggest that MT synthesis in fasting stress is not correlated with decrease in plasma leptin levels but that leptin itself is a potent inducer of MT in a fasting situation. [Copyright &y& Elsevier]

The blood–brain barrier (BBB) plays pivotal roles in synaptic and neuronal functioning by sealing the space between adjacent microvascular endothelial cells. BBB breakdown is present in patients with mild cognitive impairment (MCI) or Alzheimer disease (AD). Claudin-5 (CLDN-5) is a tetra-spanning protein essential for sealing the intercellular space between adjacent endothelial cells in the BBB. In this study, we developed a blood-based assay for CLDN-5 and investigated its diagnostic utility using 100 cognitively normal (control) subjects, 100 patients with MCI, and 100 patients with AD. Plasma CLDN-5 levels were increased in patients with AD (3.08 ng/mL) compared with controls (2.77 ng/mL). Plasma levels of phosphorylated tau (pTau181), a biomarker of pathological tau, were elevated in patients with MCI or AD (2.86 and 4.20 pg/mL, respectively) compared with control subjects (1.81 pg/mL). In patients with MCI or AD, plasma levels of CLDN-5—but not pTau181—decreased with age, suggesting some age-dependent BBB changes in MCI and AD. These findings suggest that plasma CLDN-5 may a potential biochemical marker for the diagnosis of AD. [ABSTRACT FROM AUTHOR]

• Modulation of tight junctions may overcome the blood–brain barrier (BBB). • Claudin-5 is the main component of BBB tight junctions. • The composition of BBB tight junctions is not fully known. • The ROCK and VE-cadherin/β-catenin pathways are targets for BBB modulation. • RNAi agents and direct-binding molecules may enable size-selective BBB passage. Overcoming the blood–brain barrier (BBB) to enable the treatment of central nervous system (CNS) diseases is an active field of research. Modulating or opening the tight junctions (TJs) in brain endothelial cells is one method to enable a range of small-molecular-weight drugs to cross the BBB via the paracellular route. Over the past 2 decades, the molecular understanding of TJ proteins in the BBB has significantly improved, and several agonists and antagonists have been tested for modulation of the TJs. In this review, we discuss the composition of TJ proteins in the BBB and introduce indirect pharmacological TJ modulators, which target regulators of TJ proteins, and direct TJ modulators, which can selectively inhibit functions of TJ proteins. [ABSTRACT FROM AUTHOR]

Highlights • The total costs of development of new drugs and medical devices increases. • The increasing costs puts pressure on the budgets of countries. • Seventy to ninety percent of these costs are associated with clinical trials. • The cost-effective registry-oriented clinical trials has been developed in Japan. The continuous increase in the costs of developing new drugs and medical devices drives increases in medical expenses. Seventy to ninety percent of these costs are associated with clinical trials. Therefore, the development of cost-effective methods to perform clinical trials remains a challenge. One approach is to use patient registries, collections of data related to patients with a specific diagnosis, condition, or procedure. Patient registries are used in Denmark, Sweden, and the USA for the enrollment of patients into clinical trials, and to evaluate endpoints. In Japan, a national project for registry-oriented clinical research, termed the 'Clinical Innovation Network' (CIN), was initiated in 2016. Here, we provide an overview of the CIN and discuss its impact on drug and device development in Japan. [ABSTRACT FROM AUTHOR]

Lipolysis-stimulated lipoprotein receptor (LSR), a lipid metabolism-related factor localized in tricellular tight junctions (tTJs), plays an important role in maintaining the epithelial barrier. LSR is highly expressed in well-differentiated endometrial endometrioid carcinoma (EEC), and its expression decreases during malignancy. Angubindin-1, a novel LSR ligand peptide, regulates tTJs without cytotoxicity, enhances paracellular permeability, and regulates epithelial barrier via c-Jun N-terminal kinase (JNK)/cofilin. In this study, we investigated the immune-modulatory roles of an anti-LSR antibody in the treatment of EEC in vitro compared to those of angubindin-1. We prepared an antibody against the extracellular N-terminal domain of human LSR (LSR-N-ab) and angubindin-1. EEC cell-line Sawano cells in 2D and 2.5D cultures were treated with 100 μg/ml LSR-N-ab or 2.5 μg/ml angubindin-1 with or without protein tyrosine kinase 2β inhibitor PF431396 (PF43) and JNK inhibitor SP600125 (SP60) at 10 μM. Treatment with LSR-N-ab and angubindin-1 decreased LSR at the membranes of tTJs and the activity of phosphorylated LSR and phosphorylated cofilin in 2D culture. Treatment with LSR-N-ab and angubindin-1 decreased the epithelial barrier measured as TEER values in 2D culture and enhanced the epithelial permeability of FD-4 in 2.5D culture. Treatment with LSR-N-ab, but not angubindin-1, induced apoptosis in 2D culture. Pretreatment with PF43 and SP60 prevented all the changes induced by treatment with LSR-N-ab and angubindin-1. Treatment with LSR-N-ab and angubindin-1 enhanced the cell metabolism measured as the mitochondrial respiration levels in 2D culture. LSR-N-ab and angubindin-1 may be useful for therapy of human EEC via enhanced apoptosis or drug absorption. [ABSTRACT FROM AUTHOR]

Given that most malignant tumors are derived from epithelium, developing a strategy for treatment of epithelium-derived cancers (i.e., carcinomas) is a pivotal issue in cancer therapy. Carcinomas, including ovarian, breast, prostate, and pancreatic cancers, are known to overexpress various claudins (CLDNs); in particular, CLDN-3 and -4 are frequently overexpressed in malignant case. The generation of CLDN binders is a key for expanding CLDN-targeted cancer therapy but has been delayed due to the small size of CLDN extracellular domains (approximately 50 amino acids for the first domain and 15 amino acids for the second) and their high homology among species. Interestingly, however, the receptors for Clostridium perfringens enterotoxin (CPE), a foodborne toxin in humans, happen to be identical to CLDN-3 and -4. Thus, the first CLDN binder, CPE, has provided us CLDN-targeted cancer therapy from a concept into a potential reality. In this review, we describe roles of CPE technology in cancer therapy and discuss future directions in the CLDN-targeting concept-to-therapy process. [ABSTRACT FROM AUTHOR]

Claudin-4 (CLDN4) is a key component of tight junctions (TJs) in epithelial cells. CLDN4 is overexpressed in many epithelial malignancies and correlates with cancer progression. Changes in CLDN4 expression have been associated with epigenetic factors (such as hypomethylation of promoter DNA), inflammation associated with infection and cytokines, and growth factor signaling. CLDN4 helps to maintain the tumor microenvironment by forming TJs and acts as a barrier to the entry of anticancer drugs into tumors. Decreased expression of CLDN4 is a potential marker of epithelial-mesenchymal transition (EMT), and decreased epithelial differentiation due to reduced CLDN4 activity is involved in EMT induction. Non-TJ CLDN4 also activates integrin beta 1 and YAP to promote proliferation, EMT, and stemness. These roles in cancer have led to investigations of molecular therapies targeting CLDN4 using anti-CLDN4 extracellular domain antibodies, gene knockdown, clostridium perfringens enterotoxin (CPE), and C-terminus domain of CPE (C-CPE), which have demonstrated the experimental efficacy of this approach. CLDN4 is strongly involved in promoting malignant phenotypes in many epithelial cancers and is regarded as a promising molecular therapeutic target. [ABSTRACT FROM AUTHOR]

Entry of the hepatitis C virus (HCV) into host cells is a multistep process mediated by several host factors, including a tight junction protein claudin-1 (CLDN1). We repeatedly passaged HCV-JFH1-tau, an HCV substrain with higher infectivity, on Huh7.5.1-8 cells. A multi-passaged HCV-JFH1-tau lot was infectious to CLDN1-defective S7-A cells, non-permissive to original HCV-JFH1-tau infection. We identified a single mutation, M706L, in the E2 glycoprotein of the HCV-JFH1-tau lot as an essential mutation for infectivity to S7-A cells. The pseudovirus JFH1/M706L mutant could not infect human embryonic kidney 293 T (HEK293T) cells lacking CLDN family but infected HEK293T cells expressing CLDN1, CLDN6, or CLDN9. Thus, this mutant virus could utilize CLDN1, and other CLDN6 and CLDN9, making HCV possible to infect cells other than hepatocytes. iPS cells, one of the stem cells, do not express CLDN1 but express CLDN6 and other host factors required for HCV infection. We confirmed that the HCV-JFH1-tau-derived mutant with an M706L mutation infected iPS cells in a CLDN6-dependent manner. These results demonstrated that a missense mutation in E2 could broaden the CLDN member specificity for HCV infection. HCV may change its receptor requirement through a single amino acid mutation and infect non-hepatic cells. [ABSTRACT FROM AUTHOR]

Exposure of humans to aflatoxin B1 (AFB1) via ingestion of contaminated agricultural products is a major concern for human health throughout the world because epoxidized AFB1, biotransformed from AFB1 by hepatic CYP3A4, is strongly hepatotoxic and hepatocarcinogenic. Intestinal epithelial cells serve as a physical and physiological barrier against xenobiotics via their intercellular tight junction (TJ) seals and the metabolizing enzyme CYP3A4. However, the effect of AFB1 on the intestinal barrier remains unclear. Here, we investigated the influence of AFB1 on these physical and physiological intestinal barriers by means of an in vitro human intestinal model utilizing doxycycline-inducible CYP3A4-expressing Caco-2 cells, in which CYP3A4 activity is comparable to that in the adult human intestine. Cellular toxicity of AFB1 in induced Caco-2 cells (i.e., cells in which expression of CYP3A4 is induced by doxycycline) was approximately 5 times that of uninduced Caco-2 cells. Exposure to 16 µM AFB1 did not decrease the transepithelial electric resistance (TEER; a measure of TJ barrier integrity) in monolayers of uninduced Caco-2 cells to 95.8 % of that in vehicle-treated cells; in contrast, in induced Caco-2 cells, TEER was reduced to 28.8 %. Exposure to 16 µM AFB1 increased paracellular permeation of 4- and 20-kDa dextrans (paracellular permeation markers) through monolayers of induced Caco-2 cells to 5.4 and 5.2 times that through uninduced Caco-2 cells. These results together show that ingested AFB1 can modulate the intestinal barrier, and that inducible CYP3A4-expressing Caco-2 cells are a promising tool for evaluating the safety of food contaminants in the human intestine. • Aflatoxin B1 decreased tight junction-integrity in CYP3A4-Caco-2 cells. • Aflatoxin B1 enhanced paracellular permeation of macromolecules in CYP3A4-Caco-2 cells. • Intestinal CY3A4 may be a risk factor for disruption of intestinal barrier by aflatoxin B1. • The CYP3A4-Caco-2 cells are a promising in vitro model of the human intestine. [ABSTRACT FROM AUTHOR]

Claudins (CLDNs) are a 27-member family of tetra-transmembrane proteins that have pivotal roles in maintaining cellular polarity and sealing the spaces between adjacent cells. Deregulation of their functions is often associated with pathological conditions, including carcinogenesis and inflammation. Some CLDNs are co-receptors for hepatitis C virus. Because CLDN-driven regulation of intercellular seals might be manipulated to enhance drug absorption, CLDNs are attractive targets for drug development. Monoclonal antibodies recognizing the extracellular domain of CLDNs are the first choice for therapeutics, but their development has been delayed. Here, we overview recent advances in the creation of anti-CLDN antibodies and discuss CLDNs as drug development targets. [ABSTRACT FROM AUTHOR]

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. • Homoharringtonine decreased epidermal barrier in vitro and in vivo. • Homoharringtonine enhanced transdermal absorption of solutes (up to 10000 Da). • Homoharringtonine is a transdermal absorption enhancer. [ABSTRACT FROM AUTHOR]

The blood–brain barrier is a major obstacle to the delivery of drugs to the central nervous system. In the blood–brain barrier, the spaces between adjacent brain microvascular endothelial cells are sealed by multiprotein complexes known as tight junctions. Among the many components of the tight junction, claudin‐5 has received the most attention as a target for loosening the tight‐junction seal and allowing drugs to be delivered to the brain. In mice, transient knockdown of claudin‐5 and the use of claudin‐5 binders have been shown to enhance the permeation of small molecules from the blood into the brain without apparent adverse effects. However, sustained knockdown of claudin‐5 in mice is lethal within 40 days, and administration of an anti‐claudin‐5 antibody induced convulsions in a nonhuman primate. Here, we review the safety concerns of claudin‐5–targeted technologies with respect to their clinical application. [ABSTRACT FROM AUTHOR]

The tight junction (TJ) protein claudin-4 (CLDN4) is overexpressed in bladder urothelial carcinoma (BUC) and correlates with cancer progression. However, the mechanism of CLDN4 upregulation and promotion of malignant phenotype is not clear. Here, we analyzed 157 cases of BUC and investigated the hypomethylation of CpG island in the CLDN4 promoter DNA and its correlation with cancer progression. In hypomethylated cases, CLDN4 expression, cell proliferation, stemness, and epithelial-mesenchymal transition were increased. Treatment of three human BUC cell lines with the demethylating agent aza-2′-deoxycytidine (AZA) led to excessive CLDN4 expression, and, specifically, to an increase in CLDN4 monomer that is not integrated into the TJ. The TJ-unintegrated CLDN4 was found to bind integrin β1 and increase stemness, drug resistance, and metastatic ability of the cells as well as show an anti-apoptosis effect likely via FAK phosphorylation, which reduces upon knockdown of CLDN4. Thus, CLDN4 is overexpressed in BUC by an epigenetic mechanism and the high expression enhances the malignant phenotype of BUC via increased levels of TJ-unintegrated CLDN4. CLDN4 promoter DNA methylation is expected to be a novel indicator of BUC malignant phenotype and a new therapeutic target. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Abstract: Claudins (CL) are a family of tetra-transmembrane proteins that are the structural and functional components of tight junctions (TJ). CLs are promising targets for drug development because of their role in mucosal drug absorption and cancer. However, CL-targeted drug development has been delayed because CLs have low antigenicity and preparing CL proteins is difficult. We developed a CL binder by using the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) and a baculoviral display system. After screening CL binders from a C-CPE mutant-displaying library by using CL-displaying budded baculovirus (BV) we isolated a C-CPE mutant called m19, which bound to CL1, CL2, CL4 and CL5. A 3-dimensional analysis showed that m19 has a structural backbone similar to C-CPE. The charge density of the CL-binding domains of m19 and C-CPE differed, suggesting that electrostatic interactions may occur between m19 and CLs. Treatment of epithelial cells with m19 decreased the paracellular but not transcellular integrity, and m19 enhanced jejunal absorption. Thus, we successfully created a CL binder with broad specificity. These findings will contribute to future preparation of CL binders for CL-targeted drug development. [Copyright &y& Elsevier]

Abstract: Host tropism of hepatitis C virus (HCV) is limited to human and chimpanzee. HCV infection has never been fully understood because there are few conventional models for HCV infection. Human induced pluripotent stem cell-derived hepatocyte-like (iPS-Hep) cells have been expected to use for drug discovery to predict therapeutic activities and side effects of compounds during the drug discovery process. However, the suitability of iPS-Hep cells as an experimental model for HCV research is not known. Here, we investigated the entry and genomic replication of HCV in iPS-Hep cells by using HCV pseudotype virus (HCVpv) and HCV subgenomic replicons, respectively. We showed that iPS-Hep cells, but not iPS cells, were susceptible to infection with HCVpv. The iPS-Hep cells expressed HCV receptors, including CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin; in contrast, the iPS cells showed no expression of SR-BI or claudin-1. HCV RNA genome replication occurred in the iPS-Hep cells. Anti-CD81 antibody, an inhibitor of HCV entry, and interferon, an inhibitor of HCV genomic replication, dose-dependently attenuated HCVpv entry and HCV subgenomic replication in iPS-Hep cells, respectively. These findings suggest that iPS-Hep cells are an appropriate model for HCV infection. [Copyright &y& Elsevier]

Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. CCPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4- displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator. [ABSTRACT FROM AUTHOR]

Oxidative stress accelerates adipocyte differentiation and lipid accumulation, leading to endoplasmic reticulum (ER) stress, which causes insulin resistance. Because metallothionein (MT) has a role in prevention of oxidative and ER stress, we examined the effects of MT on the development of obesity induced by 27 wk of a high-fat diet (HFD) in female MT-I- and MT-II-null (MT-/-) and wild-type (MT-/-) mice. Body weight, fat mass, and plasma cholesterol increased at a greater rate in MT-/- mice fed an HFD than in MT-/- mice fed a control diet (CD) and MT-/- mice fed an HFD, indicating that MT-/- mice fed an HFD became obese and hypercholesterolemic and that MT could prevent HFD-induced obesity. The observed increases in the levels of plasma leptin and leptin mRNA in the white adipose tissue of MT-/- mice fed the HFD suggested a leptin-resistant state. Enhanced expression of a mesoderm-specific transcript, which regulates the enlargement of fat cells, was accompanied by enlarged adipocytes in the white adipose tissue of young MT-/- mice before obesity developed after 3 and 8 wk of feeding the HFD. Thus, MT may have a preventive role against HFD-induced obesity by regulating adipocyte enlargement and leptin signaling. [ABSTRACT FROM AUTHOR]

N-Methyl-2-pyrrolidone (NMP) increased the skin permeation of estradiol (E2) in Yucatan micropig epidermis using a modified Franz-type diffusion cell. The addition of NMP significantly increased the fluxes of E2 from water and soybean oil. The flux and skin concentration of E2 were higher from soybean oil than from water and increased with increasing NMP concentrations in soybean oil. Correlation was observed with E2 flux and skin concentration (R2=0.804). NMP enhanced E2 skin permeation because NMP made E2 skin concentration higher. Thus, NMP (10%) was added to the oily gel made by isocetyl isostearate and hydrogenated phospholipid. E2 permeation from the gel without NMP was the same as that from soybean oil suspension. The flux of E2 from the gel with NMP was 0.6 μg/h per cm2 and might be sufficient for estrogen replacement therapy. [Copyright &y& Elsevier]

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well‐differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction‐mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium. [ABSTRACT FROM AUTHOR]

Claudin-5 (CLDN-5) is an essential component of the tight junction seal in the blood–brain barrier. Previously, we showed that CLDN-5 modulation in vitro via an anti-CLDN-5 monoclonal antibody (mAb) may be useful for increasing the permeability of the blood–brain barrier for drug delivery to the brain. Based on these findings, here we examined the safety and efficacy of the anti-CLDN-5 mAb in a non-human primate. Cynomolgus monkeys were intravenously administered the anti-CLDN-5 mAb followed by fluorescein dye (376 Da), and the concentrations of the dye in the cerebrospinal fluid was examined. When the mAb was administered at 3.0 mg/kg, the concentration of dye in the cerebrospinal fluid was increased, and no behavioral changes or changes in plasma biomarkers for inflammation or liver or kidney injury were observed. However, a monkey that received the mAb at 6 mg/kg experienced convulsions, and subsequent histopathological examination of this animal revealed vasodilation in the liver, lung, and kidney; hemorrhage in the lung; and edema in the brain. Together, our data indicate that CLDN-5 might be a potential target for enhancing drug delivery to the brain, but also that the therapeutic window of the anti-CLDN-5 mAb may be narrow for separating efficacy and toxicity. [Display omitted] [ABSTRACT FROM AUTHOR]

For drugs that are intended to fill unmet medical needs, such as the treatment of rare diseases or a subtype of cancer, it can take a long time to conduct confirmatory clinical trials due to limited patient availability. Delayed access to these drugs increases the risk of mortality of patients with these diseases. To address this issue, the Ministry of Health, Labour, and Welfare of Japan has decided to implement the Conditional Early Approval System with issuing the Ministry Notification in 2017. Drugs eligible for conditional early approval are those that are indicated for the treatment of a serious disease, have proven safety and efficacy, and cannot be examined easily by confirmatory clinical trials. When the benefit of immediate availability outweighs the risk of having less comprehensive data with which to confirm the clinical benefit of a product in the premarketing phase, products can be approved under the Conditional Early Approval System, accompanied by postmarketing regulatory requirements to manage postmarketing risks and, if needed, conduct postmarketing confirmatory clinical studies. Overview of the pre‐approval and post‐approval regulatory considerations will promote to more efficiently develop pharmaceutical products that fill unmet medical needs, leading to the prompt delivery of safe and effective drugs to patients who often have few therapeutic options available. As of March 2020, four drugs had been approved under the Conditional Early Approval System. In this review, we describe the premarketing and postmarketing requirements of these drugs and discuss the regulatory landscape around the Conditional Early Approval System. [ABSTRACT FROM AUTHOR]

The migration of activated T cells across the blood–brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or proteinbased targeting of their interactions in pMBMECmonolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dossiers on approved pharmaceutical products must be kept updated and current during the products' life cycles. The coalition, merger and acquisition along with corporate strategy that pursues efficiency and profitability of pharmaceutical companies have led to the globalization of supply chains for pharmaceutical ingredients and instruments in the post-marketing phase, and progress in manufacturing technologies can improve manufacturing processes during this phase. Regulatory requirements for post-marketing management of pharmaceutical products sometimes differ among countries around the world depending on national/regional policies or situations, even though the basic concepts of each regulation are the same. Therefore, an understanding of up-to-date region specific regulatory management frameworks is important for the optimal provision of pharmaceutical products by pharmaceutical industries. The amendment of the Japanese Pharmaceutical and Medical Device Act (Act No. 63 of 2019) was promulgated in December 2019, and will be enforced from September 2020 onwards. The amended Act sets out regulatory frameworks for post-marketing management systems, including inspection for good manufacturing practice of drugs, quasi-drugs, and gene-, cell-, and tissue-based products; and post-approval change-management protocols. Here, we review these new Japanese post-marketing management frameworks. [ABSTRACT FROM AUTHOR]

Angulin-1/LSR is a tricellular tight junction molecule, that plays an important role in maintaining the epithelial and endothelial barriers. The actin cytoskeleton at tricellular contacts also contributes to the maintenance of the epithelial barrier. Loss of angulin-1/LSR enhances the migration of various cancer cells. Angubindin-1 is a novel binder to angulin-1/LSR and angulin-3. It is a peptide generated from the angulin-1 binding site of Clostridium perfringens iota toxin, which affects the actin cytoskeleton and decreases the epithelial and endothelial barrier functions. However, its regulatory mechanisms are not well understood. To investigate the regulatory mechanisms of the epithelial barrier dysfunction and cell migration induction by angubindin-1, we used human endometrial cancer cell line Sawano, which has high LSR expression and the epithelial barrier function. Angubindin-1 decreased LSR expression and the epithelial barrier function and increased cell migration. It inhibited the recovery of the epithelial barrier function in a Ca-switch model. At tricellular contacts, sinking of the membrane and an increase of actin fibers near the junctions were caused by angubindin-1. It dynamically changed F-actin from lines to dot-like structures at tricellular contacts. Angubindin-1 transiently increased the phosphorylation of cofilin and JNK, which are involved in the regulation of the intracellular actin cytoskeleton. Furthermore, knockdown of JNK and the JNK inhibitor SP600125 prevented the decrease of the epithelial barrier function and the increase of cell migration induced by angubindin-1. These findings suggest that angubindin-1 might reversibly regulate the epithelial barrier and cell migration at tricellular contacts via JNK/cofilin/actin cytoskeleton dynamics. [ABSTRACT FROM AUTHOR]

Lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 is a crucial molecule of tricellular contacts in the epithelial barrier of normal cells and the malignancy of cancer cells. To investigate whether LSR/angulin-1 affects the epithelial barrier and malignancy in human pancreatic cancer, human pancreatic cancer cell line HPAC was used. Treatment with EGF or TGF-β increased the expression of LSR, but not tricellulin (TRIC), and induced the localization of LSR and TRIC to bicellular tight junctions from tricellular tight junctions. TGF-β receptor type-1 inhibitor EW-7197 prevented changes of the distribution and the barrier function of LSR by TGF-β. Knockdown of LSR increased cell migration, invasion, proliferation and EGF ligand amphiregulin expression and decreased the epithelial barrier. Treatment with amphiregulin induced cell migration and invasion and knockdown of amphiregulin prevented the increases of cell migration, invasion and proliferation caused by knockdown of LSR. Treatment with LSR ligand peptide angubindin-1 decreased the epithelial barrier and the expression of LSR, but not TRIC, and increased cell invasion. Knockdown of TRIC decreased cell migration and the epithelial barrier. In immunohistochemical analysis of human pancreatic cancer tissues, LSR and TRIC were found to be localized at the cell membranes of normal pancreatic ducts and well-differentiated pancreatic ductal adenocarcinomas (PDAC), whereas in poorly differentiated PDAC, LSR was weakly detected in the cytoplasm. Amphiregulin was highly expressed in the cytoplasm of well- and poorly differentiated PDAC. In pancreatic cancer, LSR contributes to the epithelial barrier and malignancy via growth factors and may be a potential targeting molecule in the therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Image 1 • Creation of human-rat chimeric anti-occludin monoclonal antibodies (Xi anti-OCLN mAbs). • Xi anti-OCLN mAbs had no antibody-dependent cellular cytotoxicity activity. • Xi anti-OCLN mAbs displayed high HCV-inhibiting activities in vitro. • Xi anti-OCLN mAbs can serve as a potential candidate for anti-HCV therapy. [ABSTRACT FROM AUTHOR]