ABSTRACT Organosilanes constitute a useful class of compounds in various fields of research in modern organic chemistry. Discovery of unexplored reactivity and utility of organosilanes is therefore highly important to further enhance their values. In this context, a palladium‐catalyzed regioselective synthesis of benzylsilanols from Kobayashi‐type reagents has been developed through a mechanistically distinct approach involving 1,4‐palladium migration followed by intramolecular transmetalation instead of conventional aryne formation. The reaction can be combined with various prior transition‐metal‐catalyzed processes and the resulting benzylsilanols have been utilized as effective benzyl nucleophiles for the Hiyama–Denmark cross‐coupling reactions with aryl halides under simple conditions. The mechanistic experiments have also been carried out, showing that the 1,4‐palladium migration goes through a palladacycle intermediate via turnover‐limiting deprotonative C(sp 3 )─H bond activation.

Modulation of claudin-based bicellular tight junction (TJ) and angulin-based tricellular TJ seals has been shown to enhance mucosal permeation of macromolecules, by using the receptor-binding fragments of Clostridium perfringens enterotoxin (C-CPE194, C-CPEmt, and C-CPEm19) and Clostridium perfringens iota-toxin (angubindin-1) as claudin modulators and an angulin modulator, respectively. Here, we compared the activity of these modulators on the TJ in human intestinal Caco-2 cells. All the claudin modulators loosened TJ integrity more potently compared to angubindin-1 with the order of potency being C-CPEm19 > C-CPE194 > C-CPEmt, and results for permeation enhancement were similar. Treatment with C-CPEmt and C-CPE194 at 100 µg/mL for 48 h enhanced the permeation of dextran sized 20 kDa and 70 kDa, respectively. Treatment with C-CPEm19 at 30 µg/mL for 48 h enhanced permeation of dextran with a molecular mass of up to 150 kDa. Furthermore, co-treatment of bicellular TJ modulators, such as C-CPEmt, C-CPE194, and C-CPEm19, and tricellular TJ modulators, such as angubindin-1, showed additive TJ-loosening and permeation-enhancing activities compared with individual treatments; specifically, C-CPEm19 and angubindin-1 co-treatment increased permeation of large molecules (70 kDa and 150 kDa). These findings indicate that TJ modulators may be used as size-selective permeation enhancers.

The blood-brain barrier (BBB) protects the brain from harmful substances, but it also limits drug delivery, hindering the treatment of brain diseases. Claudin 5, a tight junction protein in endothelial cells, plays a key role in maintaining the BBB integrity. Although modulation of Claudin 5 is a promising strategy for transiently opening the BBB, the currently available modulators often cause adverse effects due to strong or prolonged activity. Thus, we aimed to develop a moderate Claudin 5 modulator that enables safe and transient opening of the BBB. We identified a Claudin 5-binding small molecule (CL5B), capable of binding Claudin 5 via a high-throughput screening of 9600 compounds using a Claudin 5 antibody and proteoliposomes. CL5B increased endothelial permeability and tracer permeation across the endothelial monolayer by altering the localization of Claudin 5 without affecting its expression. In mice, CL5B transiently opened the BBB for less than 30 min, delivering drugs specifically to the brain. Notably, CL5B-facilitated delivery of methylscopolamine, a drug with limited brain penetration, alleviated seizure symptoms in a mouse epilepsy model. These findings demonstrate that CL5B is a safe BBB modulator that enhances brain drug delivery and holds therapeutic potential for brain diseases.

Tight junctions (TJs) are formed where two or three cells meet and are therefore categorized, respectively, into bicellular TJs (bTJs) and tricellular TJs (tTJs). Angubindin-1 is the first tTJ modulator enhancing intestinal macromolecule permeation via binding to the key tTJ proteins, angulin-1 and angulin-3. It is a fragment (amino acids 421-664) derived from domain IV of Clostridium perfringens iota toxin. Here, we identified critical residues (L562, L598, E638, V640, Y643, K644) of angubindin-1 to be essential for binding to angulins by alanine scanning. Mutants substituting these amino acids with alanine exhibited reduced binding to angulin-expressing cells. Simultaneous substitution of all these amino acids lost binding to angulins and resulted in the loss of tTJ-modulating functions of angubindin-1. These insights highlight crucial residues for the tTJ-modulating activity of angubindin-1, which may hold promise in the design of noninvasive, targeted therapeutics using angubindin-1 as a prototype tTJ modulator to enhance the permeation of drugs.

We applied heteroduplex oligonucleotide (HDO) technology, which uses an oligonucleotide hybridized with a complementary strand, to efficiently deliver locked nucleic acid (LNA)-based splice-switching oligonucleotides (SSOs) to the nucleus. Using an in vitro assay involving cationic lipids, we revealed that HDO technology increased the exon-skipping activity of LNA-based SSOs. To assess the effect of heteroduplex SSOs (HDSSOs) on exon-skipping activity, we designed and evaluated various HDSSOs using a series of complementary oligonucleotides with different sugar chemistries (DNA, RNA, and LNA), linkages (phosphodiester; PO and phosphorothioate; PS linkages), and lengths. HDO with different complementary oligonucleotide designs demonstrated a variety of exon-skipping activities. Next, we investigated the intracellular behavior of HDOs, which seemed to affect their efficient exon-skipping activity. We found that HDO technology increased the uptake of both SSOs and complementary oligonucleotides into the nuclei. Additionally, a series of complementary oligonucleotides showed different intracellular stabilities, and complementary oligonucleotide design appears to be one of the key factors affecting efficient exon skipping. Finally, we examined the exon-skipping activity of HDSSOs in mdx mice and found that HDSSOs exhibited higher exon-skipping activity than single-stranded LNA-based SSOs in these mice under intramuscular injections.

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.

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.

Curcumin (CUR), a polyphenol, is an attractive component of functional foods, owing to various physiological activities. However, CUR is highly hydrophobic, insoluble in water, and difficult to absorb in the body. Here, we report an amorphous CUR formulation containing the dispersant polyglycerol fatty acid ester (PGFE), demonstrating high and stable water solubility. Improved water solubility enhanced the absorbability of CUR in our amorphous formulation along with enhanced triglyceride inhibition, compared to that in a commercial formulation. Nuclear Overhauser effect spectroscopy (NOESY) analysis revealed that PGFE reduced CUR-CUR interaction, resulting in higher dispersion and improved solubility of CUR. Taylor dispersion analysis showed a lower diffusion coefficient of CUR in the highly water-soluble formulation (with PGFE) than that in the low water-soluble formulation (without PGFE), which prevents recontact and recrystallization of CUR, which is trapped by PGFE. Overall, the amorphous CUR with high solubility could be used as a promising functional food.

中枢神経系に薬物を送達する際、血液脳関門（BBB）が大きな障壁となる。BBBでは、隣接する脳微小血管内皮細胞の隙間を封印する必要があり、この封印するための仕組みとしてタイトジャンクション（TJ）が発達している。TJは複数のタンパク質から構成されているが、なかでもクローディン-5（CLDN-5）は、BBBのTJシール機能に中心的な役割を担っている。ノックアウトマウスでは、約800Daの分子が細胞間隙を介して脳内に移行していたことから、BBBのTJシール制御による脳内薬物送達技術として、CLDN-5バインダーの開発が行われてきた。本稿では、このCLDN-5を標的としたBBB制御技術の臨床応用に向けた有効性および安全性に係る論点を議論したい。

Objective: The human epidermis is formed by the proliferation and differentiation of keratinocytes adjacent to the basement membrane. The outermost layer. the stratum corneum, is equipped with a barrier function that prevents water evaporation, and intercellular lipids play an important role in this harrier function. When the barrier is functioning normally, evaporation is prevented; however, when barrier function is impaired, moisture evaporates, resulting in dry and rough skin. Therefore, maintenance of normal barrier function is critical for maintaining normal skin function. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is mainly not only involved in lipid metabolism in the liver but is also expressed in the epidermis and is involved in inducing keratinocyte differentiation, promoting lipid production, maintaining barrier function and suppressing skin inflammation. Hence, compounds that activate PPAR alpha are expected to control skin function. Therefore, we identified PPAR alpha activators from among extracts of natural resources that have been approved for use in humans and analysed the effects of these extracts on skin function. Methods: First, extracts of 474 natural resources were screened using a PPAR alpha activator screening cell line independently constructed in our laboratory. Next, reporter assays were performed using the Ga14-chimera system to evaluate whether these extracts act as ligands for PPAR alpha. We then analysed their effect on primary normal human epidermal keratinocyte cells by using real-time RT-PCR. Finally, we evaluated PPAR alpha activation effect by the combination of these extracts. Results: We identified 36 extracts having the effect of activating PPAR alpha. In particular, #419, a Typha angustifolia spike extract, showed concentration-dependent transcriptional activation through PPAR alpha-LBD and was considered to be likely to contain a compound that is a ligand of PPAR alpha. #419 increased the expression of PPAR alpha target genes and genes related to skin function in primary cultured human epidermal keratinocytes. Finally, the use of #419 in combination with nine extracts increased PPAR activity more than twice as much as #419 alone treatment. Conclusions: These results showed that the reporter cell line could be useful for discovering extracts of natural resources and that the identified Typha angustifoha spike extract could he used in cosmetics that activate PPAR alpha, which expected to improve skin function.

In the initial process of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects respiratory epithelial cells and then transfers to other organs the blood vessels. It is believed that SARS-CoV-2 can pass the vascular wall by altering the endothelial barrier using an unknown mechanism. In this study, we investigated the effect of SARS-CoV-2 on the endothelial barrier using an airway-on-a-chip that mimics respiratory organs and found that SARS-CoV-2 produced from infected epithelial cells disrupts the barrier by decreasing Claudin-5 (CLDN5), a tight junction protein, and disrupting vascular endothelial cadherin–mediated adherens junctions. Consistently, the gene and protein expression levels of CLDN5 in the lungs of a patient with COVID-19 were decreased. CLDN5 overexpression or Fluvastatin treatment rescued the SARS-CoV-2–induced respiratory endothelial barrier disruption. We concluded that the down-regulation of CLDN5 expression is a pivotal mechanism for SARS-CoV-2–induced endothelial barrier disruption in respiratory organs and that inducing CLDN5 expression is a therapeutic strategy against COVID-19.

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.

MA-T (Matching Transformation System®) is a proprietary chemical mixture for on-demand production of aqueous chlorine dioxide that is used for the treatment of oral malodor. MA-T is also an effective disinfectant against at least 39 pathological microorganisms, including severe acute respiratory syndrome coronavirus 2, and therefore may be useful as a disinfectant mouthwash to prevent the spread of infection. Accidental ingestion is the putative worst hazard scenario associated with mouthwash use; therefore, here we investigated the safety of MA-T ingestion in mice. Mice were provided drinking water containing 0-3000 μg/ml MA-T for 7 days followed by non-spiked drinking water for an additional 14 days. At day 7, mice ingesting 1000 or 3000 μg/ml MA-T showed significantly decreased body weight and significantly increased liver, kidney, and heart tissue injury biomarkers compared with control. However, at 14 days after stopping MA-T ingestion, body weight and tissue injury biomarkers had returned to normal. Histological analysis revealed that MA-T-induced injuries in liver, kidney, spleen, stomach, duodenum, colon, and rectum had also recovered at 14 days after stopping MA-T ingestion; however, mild vascular endothelial injuries remained in heart, jejunum, and ileum in the worst-case scenario. Taken together, MA-T may be potentially safety for further development as a disinfectant mouthwash by risk management, such as placing a caution of the label and adding a distinctive flavor.

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.

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.

With the rapid technological innovations of the Internet of Things (IoT), the situation surrounding medical devices and medical systems has been changing. Interoperable medical devices—medical devices capable of interoperating in a clinically significant way with other medical devices—have been developed, and interoperable medical systems consisting of two or more interconnected interoperable medical devices are being used in clinical settings. However, general points that need to be considered to ensure safe and effective interoperability have yet to be fully established in Japan. A research project (FY2019–FY2021) to discuss issues associated with ensuring safe and effective interoperability was commissioned by the Japan Agency for Medical Research and Development. A pivotal aspect identified in that project is how to manage the sharing of data and information among interoperable medical devices from different manufacturers. Characteristics and timestamps of data and information need to be exchanged between interoperable medical devices. Risks associated with interoperable devices should be managed in a manner appropriate to the characteristics and the intended use of the interoperable medical devices. In this review, we summarize the aspects of data and information that this study group judged were important to consider for ensuring safety and effective interoperability.

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.

The blood-brain barrier (BBB), which is composed of endothelial cells, pericytes, astrocytes, and neurons, separates the brain extracellular fluid from the circulating blood, and maintains the homeostasis of the central nervous system (CNS). The BBB endothelial cells have well-developed tight junctions (TJs) and express specific polarized transport systems to tightly control the paracellular movements of solutes, ions, and water. There are two types of TJs: bicellular TJs (bTJs), which is a structure at the contact of two cells, and tricellular TJs (tTJs), which is a structure at the contact of three cells. Claudin-5 and angulin-1 are important components of bTJs and tTJs in the brain, respectively. Here, we review TJ-modulating bioprobes that enable drug delivery to the brain across the BBB, focusing on claudin-5 and angulin-1.

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.