Association of Plasma Claudin-5 with Age and Alzheimer Disease.

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.

Japanese Regulatory Considerations for Interoperability of Medical Devices.

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.

SARS-CoV-2 disrupts respiratory vascular barriers by suppressing Claudin-5 expression.

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.

A doxycycline-inducible CYP3A4-Caco-2 cell line as a model for evaluating safety of aflatoxin B1 in the human intestine.

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.

Safety evaluation of MA-T after ingestion in mice.

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.

Homoharringtonine is a transdermal granular permeation enhancer.

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.

Modifying the blood-brain barrier by targeting claudin-5: Safety and risks.

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.

Exploring compounds to be used as cosmetic agents that activate peroxisome proliferator-activated receptor alpha.

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 barrier 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 α (PPARα) 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 PPARα are expected to control skin function. Therefore, we identified PPARα 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 PPARα activator screening cell line independently constructed in our laboratory. Next, reporter assays were performed using the Gal4-chimera system to evaluate whether these extracts act as ligands for PPARα. We then analysed their effect on primary normal human epidermal keratinocyte cells by using real-time RT-PCR. Finally, we evaluated PPARα activation effect by the combination of these extracts. RESULTS: We identified 36 extracts having the effect of activating PPARα. In particular, #419, a Typha angustifolia spike extract, showed concentration-dependent transcriptional activation through PPARα-LBD and was considered to be likely to contain a compound that is a ligand of PPARα. #419 increased the expression of PPARα 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 angustifolia spike extract could be used in cosmetics that activate PPARα, which expected to improve skin function.

OBJECTIF: L'épiderme humain se forme grâce à la prolifération et à la différenciation des kératinocytes adjacents à la membrane basale. La couche externe, dite « couche cornée ¼, possède une fonction barrière qui empêche l'évaporation de l'eau, dans laquelle les lipides intercellulaires jouent un rôle important. Lorsque la barrière fonctionne normalement, l'évaporation est évitée ; mais lorsqu'elle est altérée, l'évaporation a lieu et la peau, privée d'hydratation, devient sèche et rêche. Par conséquent, il est capital de maintenir cette fonction barrière normale pour que la peau conserve son fonctionnement normal. Le récepteur alpha activé par proliférateurs de peroxysomes (PPARα) intervient surtout non seulement dans le métabolisme lipidique du foie, mais également dans l'épiderme ; il joue en effet un rôle dans l'induction de la différenciation des kératinocytes, la promotion de la production lipidique, le maintien de la fonction barrière et la suppression de l'inflammation de l'épiderme. Par conséquent, les activateurs du PPAR-α devraient être déterminants pour une bonne fonction cutanée. Nous avons donc identifié des activateurs du PPAR-α parmi des extraits de ressources naturelles dont l'utilisation chez l'homme est approuvée, et nous avons analysé les effets de ces extraits sur la fonction cutanée. MÉTHODES: Tout d'abord, des extraits de 474 ressources naturelles ont été sélectionnés à l'aide d'une lignée cellulaire de détection des activateurs du PPAR-α, construite indépendamment dans notre laboratoire. Ensuite, des tests de gènes rapporteurs ont été effectués à l'aide du système Gal4-chimera pour voir si ces extraits jouaient le rôle de ligands pour le PPAR-α. Nous avons ensuite analysé leur effet sur les cellules kératinocytaires épidermiques humaines normales primaires par RT-PCR en temps réel. Enfin, nous avons évalué l'effet d'activation du PPAR-α par l'association de ces extraits. RÉSULTATS: Nous avons identifié 36 extraits ayant pour effet d'activer le PPAR-α. En particulier, le n° 419, un extrait d'épi de Typha angustifolia, a montré une activation transcriptionnelle dépendante de la concentration par le PPAR-α-LBD et a été considéré comme susceptible de contenir un composé qui est un ligand du PPAR-α. Le n° 419 a augmenté l'expression des gènes cibles du PPAR-α et des gènes liés au fonctionnement de la peau dans les kératinocytes épidermiques humains primaires mis en culture. Enfin, l'utilisation du n° 419 en association avec neuf extraits a augmenté de plus du double l'activité du PPAR par rapport au traitement par le n° 419 seul. CONCLUSIONS: Ces résultats ont montré que la lignée cellulaire rapporteuse pourrait être utile pour découvrir des extraits de ressources naturelles et que l'extrait d'épi de Typha angustifolia identifié pourrait être utilisé dans des cosmétiques qui activent le PPAR-α, ce qui devrait améliorer la fonction cutanée.

Drug discovery and development scheme for liver-targeting bridged nucleic acid antisense oligonucleotides.

Antisense oligonucleotides (ASOs) containing bridged nucleic acids (BNAs) have been proven to be very powerful. However, ensuring a reliable discovery and translational development scheme for this class of ASOs with wider therapeutic windows remains a fundamental challenge. We here demonstrate the robustness of our scheme in the context of the selection of ASOs having two different BNA chemistries (2,'4'-BNA/locked nucleic acid [LNA] and amido-bridged nucleic acid [AmNA]) targeting human proprotein convertase subtilisin/kexin type 9 (PCSK9). The scheme features a two-step process, including (1) a unique and sensitive in vitro screening approach, called Ca2+ enrichment of medium (CEM) transfection, and (2) a ligand-targeted drug delivery approach to better reach target tissues, averting unintended accumulation of ASOs. Using CEM screening, we identified a candidate ASO that shows >70% cholesterol-lowering action in monkeys. An N-acetylgalactosamine (GalNAc) ligand then was appended to the candidate ASO to further broaden the therapeutic margin by altering the molecule's pharmacokinetics. The GalNAc conjugate, HsPCSK9-1811-LNA, was found to be at least ten times more potent in non-human primates (compared with the unconjugated counterpart), with reduced nephrotoxicity in rats. Overall, we successfully showed that our drug development scheme is better suited for selecting clinically relevant BNA-based ASOs, especially for the treatment of liver-associated diseases.

Claudin-5: A Pharmacological Target to Modify the Permeability of the Blood-Brain Barrier.

Claudin-5 is the dominant tight junction protein in brain endothelial cells and exclusively limits the paracellular permeability of molecules larger than 400 Da across the blood-brain barrier (BBB). Its pathological impairment or sustained down-regulation has been shown to lead to the progression of psychiatric and neurological disorders, whereas its expression under physiological conditions prevents the passage of drugs across the BBB. While claudin-5 enhancers could potentially act as vascular stabilizers to treat neurological diseases, claudin-5 inhibitors could function as delivery systems to enhance the brain uptake of hydrophilic small-molecular-weight drugs. Therefore, the effects of claudin-5 manipulation on modulating the BBB in different neurological diseases requires further examination. To manipulate claudin-5 expression levels and function, several claudin-5 modulating molecules have been developed. In this review, we first describe the molecular, cellular and pathological aspects of claudin-5 to highlight the mechanisms of claudin-5 enhancers/inhibitors. We then discuss recently developed claudin-5 enhancers/inhibitors and new methods to discover these molecules.

Safety and efficacy of an anti-claudin-5 monoclonal antibody to increase blood-brain barrier permeability for drug delivery to the brain in a non-human primate.

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.

Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids.

Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (Tm) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same Tm values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower Tm values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.

A Method to Prepare a Bioprobe for Regulatory Science of the Drug Delivery System to the Brain: An Angulin Binder to Modulate Tricellular Tight Junction-Seal.

Epithelium acts as a barrier separating the interior and exterior of the body, and the epithelial and endothelial cells form tight junctions (TJs) by sealing the paracellular space. The blood-brain barrier (BBB) endothelial cells have well-developed TJs and express specific polarized transport systems to tightly control paracellular movements of solutes, ions, and water. Thus, more than 98% of small-molecular-weight drugs cannot pass the BBB. The tricellular TJ (tTJ) is a structure at contacts of three cells. Angulin-1, also known as lipolysis-stimulated lipoprotein receptor (LSR), is one of angulin family and is abundantly expressed in brain endothelial cells, which plays an important role in barrier function of the BBB. The C-terminal domain of a receptor-binding component of Clostridium perfringens iota-toxin (Ib421-664), also named as angubindin-1, binds to its receptors angulin-1 and angulin-3. This angubindin-1 modulates the tTJ barrier and is able to deliver a 16-mer gapmer antisense oligonucleotide (5.3 kDa) without adverse effects. Thus, angulin binders, such as angubindin-1, are useful tools for studying the safety assessment of tTJ-targeted drug delivery and BBB permeability modulation. Here, we provide a protocol for the expression and purification of recombinant angubindin-1 protein as angulin binders, an analysis method for angubindin-1 binding affinity, and a procedure for assessing the effect of modulating tight junction integrity.

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.

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.

Tight Junction Modulating Bioprobes for Drug Delivery System to the Brain: A Review.

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.

The Roadmap to Approval under Japan's Two-Track Regulatory System: Comparing Six Regenerative Medical Products.

The Japanese conditional and time-limited (CTL) approval framework, a tiered system for regenerative medical products, was initiated in 2014. Here, we compare the dossiers of six regenerative medical products with either direct full approval or CTL approval, and we discuss the regulatory considerations for obtaining approval under the CTL approval framework.

New Japanese Regulatory Frameworks for Post-Marketing Management of Pharmaceutical Products.

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.

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.

Structural Basis for PPARα Activation by 1H-pyrazolo-[3,4-b]pyridine Derivatives.

Small-molecule agonism of peroxisome proliferator-activated receptor α (PPARα), a ligand-activated transcriptional factor involved in regulating fatty acid metabolism, is an important approach for treating dyslipidemia. Here, we determined the structures of the ligand-binding domain (LBD) of PPARα in complex with 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives, which were recently identified as PPARα-selective activators with markedly different structures from those of the well-known PPARα agonists fibrates. The crystal structures of the complexes showed that they form a canonical hydrogen-bond network involving helix 12 in the LBD, which is thought to be essential for PPARα activation, as also observed for fibrates. However, the phenyl side chain of the compounds occupies a small cavity between Ile272 and Ile354, which is rarely accessed by fibrates. This unique feature may be essential for subtype selectivity and combine with the well-characterized binding mode of fibrates to improve activity. These findings demonstrate the advantage of using 1H-pyrazolo-[3,4-b]pyridine as a skeleton of PPARα agonists and provide insight into the design of molecules for treating dyslipidemia.