Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

Legislation on the Roles of the Pharmacist and Pharmacy in the Revision of the Pharmaceutical and Medical Device Act and the Pharmacists Act in Japan.

Japan's rapidly aging population has prompted a change in the country's pharmaceutical care framework from a drug-oriented to a patient-oriented approach. Pharmacies and pharmacists are expected to play central roles in this new patient-oriented approach by reconciling medication and managing polypharmacy, conducting home visits, and providing care to patients in collaboration with local clinics and hospitals. These additional roles are expected to lead to a reduction in the prevalence of adverse effects and improve the quality of life of patients in Japan's aging society. To promote patient-oriented care by pharmacists, these additional roles were legislated in a revision of the Japanese Pharmaceutical and Medical Device Act and other related Act in December 2019. Here, we overview how the revisions affect pharmacies, pharmacists, medical institutions, and outpatients.

Overview of the Premarketing and Postmarketing Requirements for Drugs Granted Japanese Conditional Marketing Approval.

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.

Radiolabeled cCPE Peptides for SPECT Imaging of Claudin-4 Overexpression in Pancreatic Cancer.

Overexpression of tight-junction protein claudin-4 has been detected in primary and metastatic pancreatic cancer tissue and is associated with better prognosis in patients. Noninvasive measurement of claudin-4 expression by imaging methods could provide a means for accelerating detection and stratifying patients into risk groups. Clostridium perfringens enterotoxin (CPE) is a natural ligand for claudin-4 and holds potential as a targeting vector for molecular imaging of claudin-4 overexpression. A glutathione S-transferases (GST)-tagged version of the C terminus of CPE (cCPE) was previously used to delineate claudin-4 overexpression by SPECT but showed modest binding affinity and slow blood clearance in vivo. Methods: On the basis of the crystal structure of cCPE, a series of smaller cCPE194-319 mutants with putatively improved binding affinity for claudin-4 was generated by site-directed mutagenesis. All peptides were conjugated site-specifically on a C-terminal cysteine using maleimide-diethylenetriamine pentaacetate to enable radiolabeling with 111In. The binding affinity of all radioconjugates was evaluated in claudin-4-expressing PSN-1 cells and HT1080-negative controls. The specificity of all cCPE mutants to claudin-4 was assessed in HT1080 cells stably transfected with claudin-4. SPECT/CT imaging of BALB/c nude mice bearing PSN-1 or HT1080 tumor xenografts was performed to determine the claudin-4-targeting ability of these peptides in vivo. Results: Uptake of all cCPE-based radioconjugates was significantly higher in PSN-1 cells than in HT1080-negative controls. All peptides showed a marked improvement in affinity for claudin-4 in vitro when compared with previously reported values (dissociation constant: 2.2 ± 0.8, 3 ± 0.1, 4.2 ± 0.5, 10 ± 0.9, and 9.7 ± 0.7 nM). Blood clearance of [111In]In-cCPE194-319, as measured by SPECT, was considerably faster than that of [111In]In-cCPE.GST (half-life, <1 min). All radiopeptides showed significantly higher accumulation in PSN-1 xenografts than in HT1080 tumors at 90 min after injection of the tracer ([111In]In-cCPE194-319, 2.7 ± 0.8 vs. 0.4 ± 0.1 percentage injected dose per gram [%ID/g], P < 0.001; [111In]In-S313A, 2.3 ± 0.9 vs. 0.5 ± 0.1 %ID/g, P < 0.01; [111In]In-S307A + N309A + S313A, 2 ± 0.4 vs. 0.3 ± 0.1 %ID/g, P < 0.01; [111In]In-D284A, 2 ± 0.2 vs. 0.7 ± 0.1 %ID/g, P < 0.05; [111In]In-L254F + K257D, 6.3 ± 0.9 vs. 0.7 ± 0.2 %ID/g, P < 0.001). Conclusion: These optimized cCPE-based SPECT imaging agents show great promise as claudin-4-targeting vectors for in vivo imaging of claudin-4 overexpression in pancreatic cancer.

A Method to Prepare Claudin-Modulating Recombinant Proteins.

The epithelium forms tight junctions by sealing the paracellular space, and tight junctions prevent the free movement of solutes. Claudin is an important structural and functional component of tight junctions and contributes to the formation of paracellular pathways for different populations of size- and charge-selective solutes. Therefore, modulation of tight junctions is important to develop drug delivery strategies. Clostridium perfringens enterotoxin (CPE) causes food poisoning in humans and is a 35-kDa polypeptide, consisting of 319 amino acids and two functional regions. The C-terminal region of CPE (C-CPE) is not cytotoxic and binds to its receptor claudin, which in turn modulates the epithelial tight junction barrier. Thus, claudin binders, such as C-CPE, are useful tools for drug delivery targeting tight junctions. Here, we provide a protocol for the expression and purification of recombinant C-CPE proteins as claudin binders, an analysis method for C-CPE binding affinity, and a procedure for assessing the effect of modulating tight junction integrity.

Potential for Tight Junction Protein-Directed Drug Development Using Claudin Binders and Angubindin-1.

The tight junction (TJ) is an intercellular sealing component found in epithelial and endothelial tissues that regulates the passage of solutes across the paracellular space. Research examining the biology of TJs has revealed that they are complex biochemical structures constructed from a range of proteins including claudins, occludin, tricellulin, angulins and junctional adhesion molecules. The transient disruption of the barrier function of TJs to open the paracellular space is one means of enhancing mucosal and transdermal drug absorption and to deliver drugs across the blood-brain barrier. However, the disruption of TJs can also open the paracellular space to harmful xenobiotics and pathogens. To address this issue, the strategies targeting TJ proteins have been developed to loosen TJs in a size- or tissue-dependent manner rather than to disrupt them. As several TJ proteins are overexpressed in malignant tumors and in the inflamed intestinal tract, and are present in cells and epithelia conjoined with the mucosa-associated lymphoid immune tissue, these TJ-protein-targeted strategies may also provide platforms for the development of novel therapies and vaccines. Here, this paper reviews two TJ-protein-targeted technologies, claudin binders and an angulin binder, and their applications in drug development.

Lipin-1 is a novel substrate of protein phosphatase PGAM5.

Lipin-1 has multiple functions that regulate lipid and energy metabolism according to its subcellular localization. The subcellular localization of Lipin-1 is determined by kinase-dependent phosphorylation; however, the phosphatase that dephosphorylates and inactivates Lipin-1 has remained elusive. Using an immunoprecipitation and LC-MS/MS approach we have identified phosphoglycerate mutase family member 5 (PGAM5), a serine/threonine specific protein phosphatase, as a regulator of Lipin-1 activity. Treatment of human hepatocellular carcinoma cells with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which activates endogenous PGAM5, promoted dephosphorylation and nuclear accumulation of Lipin-1. Our findings further elucidate the molecular mechanisms that regulate Lipin-1.

The Clinical Innovation Network: a policy for promoting development of drugs and medical devices 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.

Anti-Claudin Antibodies as a Concept for Development of Claudin-Directed Drugs.

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.

Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening.

Splice-switching oligonucleotides (SSOs) that can modulate RNA splicing are used for the treatment of many genetic disorders. To enhance the efficacy of modulating splicing, it is important to optimize SSOs with regard to target sites, GC content, melting temperature (Tm value), chemistries, and lengths. Thus, in vitro assay systems that allow for the rapid and simple screening of SSOs are essential for optimizing SSO design. In this study, we established a novel tri-chromatic reporter cell line for SSO screening. This reporter cell line is designed to express three different fluorescent proteins (blue, green, and red) and was employed for high content screening (HCS, also known as high content analysis; HCA) for the evaluation of SSO-induced exon skipping by analyzing the expression levels of fluorescent proteins. The blue fluorescent protein is stably expressed throughout the cell and is useful for data normalization using cell numbers. Furthermore, both the green and red fluorescent proteins were used for monitoring the splicing patterns of target genes. Indeed, we demonstrated that this novel reporter cell line involving HCS leads to a more rapid and simple approach for the evaluation of exon skipping than widely used methods, such as RT-PCR, western blotting, and quantitative RT-PCR. Additionally, a brief screening of Locked nucleic acids (LNA)-based SSOs targeting exon 51 in DMD was performed using the reporter cell line. The LNA-based SSO cocktail shows high exon 51 skipping in a dose-dependent manner. Furthermore, the LNA-based SSO cocktails display high exon 51 skipping activities on endogenous DMD mRNA in human rhabdomyosarcoma cells.

Discovery of peroxisome proliferator-activated receptor α (PPARα) activators with a ligand-screening system using a human PPARα-expressing cell line.

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator-responsive elements (PPREs) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of >12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both in vitro and in vivo Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose-fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia.

Degradation of human Lipin-1 by BTRC E3 ubiquitin ligase.

Lipin-1 has dual functions in the regulation of lipid and energy metabolism according to its subcellular localization, which is tightly controlled. However, it is unclear how Lipin-1 degradation is regulated. Here, we demonstrate that Lipin-1 is degraded through its DSGXXS motif. We show that Lipin-1 interacts with either of two E3 ubiquitin ligases, BTRC or FBXW11, and that this interaction is DSGXXS-dependent and mediates the attachment of polyubiquitin chains. Further, we demonstrate that degradation of Lipin-1 is regulated by BTRC in the cytoplasm and on membranes. These novel insights into the regulation of human Lipin-1 stability will be useful in planning further studies to elucidate its metabolic processes.

Analysis of the subcellular localization of the human histone methyltransferase SETDB1.

SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol.

Facilitation of adipocyte differentiation of 3T3-L1 cells by debrominated tetrabromobisphenol A compounds detected in Japanese breast milk.

Tetrabromobisphenol A (TeBBPA) is widely used type of brominated flame retardant. In this study, we newly synthesized materials for the debrominated congeners, 2,2',6-tribromobisphenol A (TriBBPA), 2,2'-dibromobisphenol A (2,2'-DiBBPA), 2,6-dibromobisphenol A (2,6-DiBBPA), and 2-monobromobisphenol A (MoBBPA) and evaluated the actual extent of contamination with bisphenol A (BPA), TeBBPA and debrominated congeners in Japanese breast milk samples. TriBBPA was detected at higher levels than that of TeBBPA, while DiBBPA and MoBBPA were detected at lower levels than that of TeBBPA. This observation suggested that humans are exposed to debrominated congeners, which might cause adverse effects. Contamination of the congeners in breast milk was concern about risk infant health, having vulnerable defense system. As pilot study by in vitro experiment, we assessed the toxic potency of debrominated congeners by studying their effect on adipocyte differentiation in 3T3-L1 cells. We observed 2,6-DiBBPA, TriBBPA and TeBBPA elevated the lipid accumulation and adipocyte-specific protein 2 expression in a manner dependent on the number of substituted bromines. Moreover, PPARγ transcriptional activities increased in a dose-dependent manner in the presence of 2,6-DiBBPA and TriBBPA as well as TeBBPA. Our study clarified that TeBBPA and its debrominated congeners accumulated in breast milk and the debrominated congeners promoted adipocyte differentiation, showing that a comprehensive evaluation of the influences of these compounds including the debrominated congeners of TeBBPA on health in infants is necessary.

PPARß/δ activation of CD300a controls intestinal immunity.

Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPARß/δ (peroxisome proliferator-activated receptor ß/δ) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a-/- mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a-/- macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis.

Human mannose-binding lectin 2 is directly regulated by peroxisome proliferator-activated receptors via a peroxisome proliferator responsive element.

Human mannose-binding lectin (MBL) is encoded by the MBL2 gene and is a key player in innate immunity. However, the mechanism of the transcriptional regulation of MBL2 is largely unknown. The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that play an important role in a number of biological responses, including lipid homeostasis, immune function and adipogenesis. In this study, we showed that PPARα and PPARγ up-regulate the expression of human MBL2. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that PPARs regulate the expression of human MBL2 via the peroxisome proliferator responsive element (PPRE). On the other hand, MBL2 mRNA expression was not affected by the PPARα ligand both in vivo in rat liver and in vitro in rat H4IIE hepatoma cells. Thus, there is a species difference in regulation of MBL2 gene expression by PPARs between humans and rodents. We also show that the species differences in response to PPAR could be due in part to sequence-specific differences in the PPRE in the promoter region of MBL2. These results indicate that human, but not rat, MBL2 expression is regulated by PPARs via a PPRE.

Fenofibrate suppresses growth of the human hepatocellular carcinoma cell via PPARα-independent mechanisms.

Fenofibrate, a peroxisome proliferator-activated receptor (PPAR) α agonist, is a hypolipidemic drug. Although several studies have explored the fenofibrate-induced antiproliferative effect in cultured human cells, it is not clear which role PPARα plays in this antiproliferative effect. Therefore, we investigated the antiproliferative mechanism of fenofibrate in Huh7 (human hepatoma cell line). Cell viability was measured by the WST-8 assay and cell proliferation was assessed using the BrdU incorporation assay. The cell cycle was analyzed by flow cytometry. The cyclins, tumor suppressor proteins and regulators of the AKT signaling pathway were analyzed by immunoblotting. Using flow cytometry, we showed that fenofibrate blocks entry into the S phase of the cell cycle. We certified that this G1 arrest is caused by the reduction of cyclin A and E2F1 and the accumulation of the cyclin-dependent kinase inhibitor p27. Interestingly, the antiproliferative effect of fenofibrate was not affected by the PPARα antagonist (GW6471) or by PPARα-specific siRNA. These results suggest that fenofibrate suppresses Huh7 cell growth through a PPARα independent mechanism. Furthermore, we showed that treatment of Huh7 cells with fenofibrate leads to suppression of AKT phosphorylation. We also found for the first time that fenofibrate increased the C-terminal modulator protein (CTMP), which inhibits AKT phosphorylation. Our data suggest that fenofibrate inhibits the proliferation of Huh7 cells by blocking Akt activation, and that CTMP is one of the key players for this antiproliferative property of fenofibrate in Huh7 cells.

Sterol-regulatory-element-binding protein 2 and nuclear factor Y control human farnesyl diphosphate synthase expression and affect cell proliferation in hepatoblastoma cells.

FDPS (farnesyl diphosphate synthase) catalyses the formation of farnesyl diphosphate, a key intermediate in the synthesis of cholesterol and isoprenylated cellular metabolites. FDPS is also the molecular target of nitrogen-containing bisphosphonates, which are used as bone-antiresorptive drugs in various disorders. In the present study, we characterized the sterol-response element and NF-Y (nuclear factor Y)-binding site in the human FDPS promoter. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of the FDPS gene, and that its transcriptional activation is mediated by SREBP-2 (sterol-regulatory-element-binding protein 2) and NF-Y. We also investigated whether sterol-mediated FDPS expression is involved in the cell proliferation induced by zoledronic acid, an FDPS inhibitor. We show that the SREBP-2- and NF-Y-mediated regulation of FDPS gene transcription modulates cell proliferation. These results suggest that SREBP-2 and NF-Y are required to trigger cell proliferation through the induction of FDPS expression and that the pharmacological action of zoledronic acid is involved in this pathway.

Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells.

Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial beta-oxidation. The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor that plays an important role in the regulation of beta-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPARalpha and found that PPARalpha induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPARalpha regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

Sterol-mediated regulation of human lipin 1 gene expression in hepatoblastoma cells.

Lipin 1 plays a crucial role in lipid metabolism in adipose tissue, skeletal muscle, and liver. Its physiological role involves two cellular functions: regulation of phosphatidate phosphatase activity and regulation of fatty acid oxidation. In this study, we have demonstrated that lipin 1 gene (LPIN1) expression is regulated by cellular sterols, which are key regulators of lipid metabolism. We have also characterized the sterol-response element and nuclear factor Y-binding sites in the human LPIN1 promoter. Using a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of LPIN1 gene, mediated by SREBP-1 (sterol regulatory element-binding protein 1) and nuclear factor Y. Furthermore, we investigated whether lipin 1 is involved in lipogenesis by transfection of LPIN1 small interfering RNA. We infer that sterol-mediated regulation of lipin 1 gene transcription modulates triglyceride accumulation. This modulation involves changes in the activity of phosphatidate phosphatase.