Possibility of maintaining remission with topical therapy alone after withdrawal of dupilumab in Japanese patients with atopic dermatitis and their characteristics in the real world.

Psossibility and appropriate timing of discontinuation of dupilumab for atopic dermatitis (AD) remain unclear. We explored the possibility of patients, who could maintain remission with topical therapy alone after withdrawing dupilumab in the real world. Furthermore, we identified their characteristics. All adult AD patients who initiated dupilumab from June 2018 to July 2022 and were treated with dupilumab for more than 3 months at our hospital were included in this study. The observation period was from June 2018 to July 2023. In 138 patients, 58 (42.0%) discontinued dupilumab at least once. Among them, 18 (13.0%) discontinued dupilumab but reinitiated dupilumab later due to exacerbation. Only seven patients (5.1%) could maintain remission with topical therapy alone after discontinuation of dupilumab, with characteristics of lower POEM, VAS of pruritus, serum levels of TARC and LDH, and neutrophil counts at baseline, and those of longer duration of dupilumab until its discontinuation (24.0 ± 13.3 vs. 12.8 ± 7.3 months) and lower EASI and affected BSA at the discontinuation of dupilumab. In 118 patients treated with dupilumab for at least 1 year, 38 patients (32.2%) discontinued at least once. Only four patients (3.4%) could maintain remission with topical therapy alone after discontinuation of dupilumab, with characteristics of lower POEM at baseline and lower EASI at the discontinuation of dupilumab. In conclusion, maintaining remission after withdrawing dupilumab is challenging. Discontinuation of dupilumab may be considered in patients with low baseline POEM, after more than 2 years of dupilumab treatment, with a substantial decrease in EASI.

Impact of 1-year treatment with dupilumab on work productivity in Japanese patients with atopic dermatitis.

Atopic dermatitis (AD) places a burden on work productivity. Recently, dupilumab was approved for AD, but its impact on work productivity in Japanese patients has not been reported. Furthermore, data on the effect of long-term treatment with dupilumab on work productivity are limited. We investigated the work productivity and activity in Japanese patients with moderate-to-severe AD, utilizing the Japanese version of the Work Productivity and Activity Impairment (WPAI-AD-Japan) questionnaire. Furthermore, we examined the impact of dupilumab on work productivity. Adult moderate-to-severe AD patients treated with dupilumab for more than 12 months from March 2020 to June 2022 who filled out the WPAI-AD-Japan questionnaire were included. Twenty-eight adult AD patients were analysed. Absenteeism was low (mean: 5.3%), but presenteeism, work productivity loss and activity impairment were high (36.8%, 39.7%, 48.9%, respectively). Significant positive correlations were observed between work productivity loss and visual analogue scale (VAS) score of pruritus and between activity impairment and dermatology life quality index (DLQI). Dupilumab treatment significantly reduced presenteeism, work productivity loss and activity impairment at both 6 and 12 months. The extent of their amelioration was numerically higher at 12 months than at 6 months. The reduction rates in presenteeism, work productivity loss and activity impairment were positively correlated with the reduction rates in DLQI and VAS score of pruritus at 12 months. Dupilumab improved work productivity in Japanese AD patients. Long-term remission of pruritus and improved quality of life are important for comprehensive improvement of work productivity.

Dynamic Stabilization of Unit Polyion Complexes Incorporating Small Interfering RNA by Fine-Tuning of Cationic Block Length in Two-Branched Poly(ethylene glycol)-b-poly(l-lysine).

To stabilize small interfering RNA (siRNA) in the bloodstream for systemic RNAi therapeutics, we previously fabricated ultrasmall siRNA nanocarriers that were sub-20 nm in hydrodynamic diameter, named as unit polyion complexes (uPICs), using two-branched poly(ethylene glycol)-b-poly(l-lysine) (bPEG-PLys). The blood retention time of uPICs is dramatically increased in the presence of free bPEG-PLys, suggesting dynamic stabilization of uPICs by free bPEG-PLys based on their equilibrium. Herein, we examined how the degree of polymerization of PLys (DPPLys) affected the dynamic stability of uPICs in the bloodstream during prolonged circulation. We prepared a series of bPEG-PLys with DPPLys values of 10, 13, 20, 40, and 80 for the uPIC formation and siRNA with 40 negative charges. These bPEG-PLys were then evaluated in physicochemical characterization and pharmacokinetic analyses. Structural analyses revealed that the uPIC size and association numbers were mainly determined by the molecular weights of PEG and DPPLys, respectively. Under bPEG-PLys-rich conditions, the hydrodynamic diameters of uPICs were 15-20 nm, which were comparable to that of the bPEG block (i.e., ∼18 nm). Importantly, DPPLys significantly affected the association constant of bPEG-PLys to siRNA (Ka) and blood retention of free bPEG-PLys. A smaller DPPLys resulted in a lower Ka and a longer blood retention time of free bPEG-PLys. Thus, DPPLys can control the dynamic stability of uPICs, i.e., the balance between Ka and blood concentration of free bPEG-PLys. Ultimately, the bPEG-PLys with DPPLys values of 14 and 19 prolonged the blood circulation of siRNA-loaded uPICs with relatively small amounts of free bPEG-PLys. This study revealed that the uPIC formation between siRNA and bPEG-PLys can be controlled by their charges, which may be helpful for designing PIC-based delivery systems.

Effective mRNA Protection by Poly(l-ornithine) Synergizes with Endosomal Escape Functionality of a Charge-Conversion Polymer toward Maximizing mRNA Introduction Efficiency.

For efficient delivery of messenger (m)RNA, delivery carriers need two major functions: protecting mRNA from nucleases and translocating mRNA from endolysosomes to the cytoplasm. Herein, these two complementary functionalities are integrated into a single polyplex by fine-tuning the catiomer chemical structure and incorporating the endosomal escape modality. The effect of the methylene spacer length on the catiomer side chain is evaluated by comparing poly(l-lysine) (PLL) with a tetramethylene spacer and poly(L-ornithine) (PLO) with a trimethylene spacer. Noteworthily, the nuclease stability of the mRNA/catiomer polyplexes is largely affected by the difference in one methylene group, with PLO/mRNA polyplex showing enhanced stability compared to PLL/mRNA polyplex. To introduce the endosomal escape function, the PLO/mRNA polyplex is wrapped with a charge-conversion polymer (CCP), which is negatively charged at extracellular pH but turns positive at endosomal acidic pH to disrupt the endosomal membrane. Compared to the parent PLO/mRNA polyplex, CCP facilitated the endosomal escape of the polyplex in cultured cells to improve the protein expression efficiency from mRNA by approximately 80-fold. Collectively, this system synergizes the protective effect of PLO against nucleases and the endosomal escape capability of CCP in mRNA delivery.

Treatment of primary and metastatic breast and pancreatic tumors upon intravenous delivery of a PRDM14-specific chimeric siRNA/nanocarrier complex.

PRDM14 is highly expressed in several cancers but is not detected in normal tissues. It confers cancer stem cell-like properties, including chemoresistance and distant metastasis, to cancer cells. Herein, we aimed to develop a highly effective therapy against advanced stage cancer based on intravenously delivered PRDM14-targeted siRNA. First, we examined PRDM14 expression and gene amplification in breast and pancreatic tumors and cell lines. PRDM14 was expressed in breast cancer, including the triple-negative subtype, and pancreatic cancer. PRDM14 was amplified in 23.8% of patients with PRDM14+ breast cancer. Next, we investigated the inoculated tumor growth and distant metastasis following PRDM14 depletion by administering mice with PRDM14-specific chimeric siRNA combined with a novel branched PEGylated poly-L-ornithine (PLO)-based intravenous drug delivery system, designated PRDM14 unit polyion complex (uPIC) (n = 6/group). Inhibition of PRDM14 expression with PRDM14 uPIC by systemic intravenous injection effectively reduced tumor size and metastasis in vivo, thereby improving survival. Finally, pharmacokinetic/toxicokinetic analyses were performed on PRDM14 uPIC, which was intravenously administered to rats (n = 10-15/group) and cynomolgus monkeys (n = 3-5/group), twice weekly for 4 weeks. This revealed that PRDM14 uPIC was relatively nontoxic and the siRNA exposure in serum was greater than that predicted by the administered dose ratio when delivered as a uPIC. Taken together, our study indicated that PRDM14 uPIC is highly effective in suppressing malignant features of solid cancers and does not cause severe toxicity, making it a promising therapeutic agent for cancer treatment.

[A case of pancreatic actinomycosis diagnosed by EUS-FNA].

A 60-year-old male patient had alcoholic chronic pancreatitis. Three months prior, he had undergone an exchange of pancreatic duct stents. In December 201X-1, magnetic resonance imaging and computed tomography (CT) scan results showed a caput pancreatic mass and common bile duct dilatation. We considered that it was because of chronic pancreatitis and decided to follow up by imaging studies. Further, in March 201X, a CT scan result revealed worsening of the mass and bile duct dilation. We assessed the mass by endoscopic ultrasound and fine-needle aspiration. Histological findings revealed to an interstitial tissue infiltrated by several neutrophils and plasma cells and abscess-forming inflammation like sulfur granule. The mass was improved by antibiotic administration for 6 months.

Noncovalent Stabilization of Vesicular Polyion Complexes with Chemically Modified/Single-Stranded Oligonucleotides and PEG-b-guanidinylated Polypeptides for Intracavity Encapsulation of Effector Enzymes Aimed at Cooperative Gene Knockdown.

For the simultaneous delivery of antisense oligonucleotides and their effector enzymes into cells, nanosized vesicular polyion complexes (PICs) were fabricated from oppositely charged polyion pairs of oligonucleotides and poly(ethylene glycol) (PEG)-b-polypeptides. First, the polyion component structures were carefully designed to facilitate a multimolecular (or secondary) association of unit PICs for noncovalent (or chemical cross-linking-free) stabilization of vesicular PICs. Chemically modified, single-stranded oligonucleotides (SSOs) dramatically stabilized the multimolecular associates under physiological conditions, compared to control SSOs without chemical modifications and duplex oligonucleotides. In addition, a high degree of guanidino groups in the polypeptide segment was also crucial for the high stability of multimolecular associates. Dynamic light scattering and transmission electron microscopy revealed the stabilized multimolecular associates to have a 100 nm sized vesicular architecture with a narrow size distribution. The loading number of SSOs per nanovesicle was determined to be ∼2500 using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated ribonuclease H (RNase H) as an effector enzyme at ∼10 per nanovesicle through simple vortex-mixing with preformed nanovesicles. Ultimately, the RNase H-encapsulated nanovesicle efficiently delivered SSOs with RNase H into cultured cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesicles (without RNase H) or a mixture of nanovesicles with RNase H without encapsulation. These results demonstrate the great potential of noncovalently stabilized nanovesicles for the codelivery of two varying bio-macromolecule payloads for ensuring their cooperative biological activity.

Synthetic miR-143 Exhibited an Anti-Cancer Effect via the Downregulation of K-RAS Networks of Renal Cell Cancer Cells In Vitro and In Vivo.

To understand the role of RAS-signaling networks in the pathogenesis of renal cell carcisnoma, we clarified the relationship between miR-143 and RAS. The expression of miR-143 was extremely downregulated in tumor tissues from renal cell carcinoma patients compared with that in the adjacent normal tissues and Caki-1 cells. We developed a synthetic miR-143#12, and we found that the ectopic expression of it inhibited cell growth with autophagy in Caki-1 cells. Also, the expression level of c-Myc was markedly decreased, resulting in the perturbation of cancer-specific energy metabolism by negatively modulating the expression of GLUT1 and the PTBP1/PKMs axis. A partial metabolic shift from glycolysis to oxidative phosphorylation induced autophagy through increasing the intracellular level of reactive oxygen species (ROS). In an in vivo study, the potent anti-tumor activity of polyion complex (PIC)-loaded miR-143#12 (miR-143#12/PIC) was shown by systemic administration of it to Caki-1 cell-xenografted mice. Higher levels of miR-143 were found in both blood and tumor tissues after the systemic administration with miR-143#12/PIC compared to those with lipoplexes in the xenografted mice. These findings indicated that this synthetic miR-143#12 induced a marked growth inhibition by impairing K-RAS-signaling networks in vitro and in vivo.

Systemic Brain Delivery of Antisense Oligonucleotides across the Blood-Brain Barrier with a Glucose-Coated Polymeric Nanocarrier.

Current antisense oligonucleotide (ASO) therapies for the treatment of central nervous system (CNS) disorders are performed through invasive administration, thereby placing a major burden on patients. To alleviate this burden, we herein report systemic ASO delivery to the brain by crossing the blood-brain barrier using glycemic control as an external trigger. Glucose-coated polymeric nanocarriers, which can be bound by glucose transporter-1 expressed on the brain capillary endothelial cells, are designed for stable encapsulation of ASOs, with a particle size of about 45 nm and an adequate glucose-ligand density. The optimized nanocarrier efficiently accumulates in the brain tissue 1 h after intravenous administration and exhibits significant knockdown of a target long non-coding RNA in various brain regions, including the cerebral cortex and hippocampus. These results demonstrate that the glucose-modified polymeric nanocarriers enable noninvasive ASO administration to the brain for the treatment of CNS disorders.

Self-Assembly of siRNA/PEG- b-Catiomer at Integer Molar Ratio into 100 nm-Sized Vesicular Polyion Complexes (siRNAsomes) for RNAi and Codelivery of Cargo Macromolecules.

Vesicular polyion complexes (PICs) were fabricated through self-assembly of rigid cylindrical molecules, small interfering RNAs (siRNAs), with flexible block catiomers of poly(ethylene glycol) (2 kDa) and cationic polyaspartamide derivative (70 units) bearing a 5-aminopentyl side chain. 100 nm-sized siRNA-assembled vesicular PICs, termed siRNAsomes, were fabricated in specific mixing ranges between siRNA and block catiomer. The siRNAsome membrane was revealed to consist of PIC units fulfilling a simple molar ratio (1:2 or 2:3) of block catiomer and siRNA. These ratios correspond to the minimal integer molar ratio to maximally compensate the charge imbalance of PIC, because the numbers of charges per block catiomer and siRNA are +70 and -40, respectively. Accordingly, the ζ-potentials of siRNAsomes prepared at 1:2 and 2:3 were negative and positive, respectively. Cross-section transmission electron microscopic observation clarified that the membrane thicknesses of 1:2 and 2:3 siRNAsomes were 11.0 and 17.2 nm, respectively. Considering that a calculated long-axial length of siRNA is 5.9 nm, these thickness values correspond to the membrane models of two (11.8 nm) and three (17.7 nm) tandemly aligned siRNAs associating with one and two block catiomers, respectively. For biological application, siRNAsomes were stabilized through membrane-cross-linking with glutaraldehyde. The positively charged and cross-linked siRNAsome facilitated siRNA internalization into cultured cancer cells, eliciting significant gene silencing with negligible cytotoxicity. The siRNAsome stably encapsulated dextran as a model cargo macromolecule in the cavity by simple vortex mixing. Confocal laser scanning microscopic observation displayed that both of the payloads were internalized together into cultured cells. These results demonstrate the potential of siRNAsomes as a versatile platform for codelivery of siRNA with other cargo macromolecules.

Tunable nonenzymatic degradability of N-substituted polyaspartamide main chain by amine protonation and alkyl spacer length in side chains for enhanced messenger RNA transfection efficiency.

Degradability of polycations under physiological conditions is an attractive feature for their use in biomedical applications, such as the delivery of nucleic acids. This study aims to design polycations with tunable nonenzymatic degradability. A series of cationic N-substituted polyaspartamides were prepared to possess primary amine via various lengths of alkyl spacers in side chains. The degradation rate of each polyaspartamide derivative was determined by size exclusion chromatography under different pH conditions. The N-substituted polyaspartamide containing a 2-aminoethyl moiety in the side chain (PAsp(AE)) showed considerable degradability under physiological conditions (pH 7.4, 37 °C). In contrast, the N-substituted polyaspartamides bearing a longer alkyl spacer in the side chain, i.e. the 3-aminopropyl (PAsp(AP)) and 4-aminobutyl moieties (PAsp(AB)), more strongly suppressed degradation. Further, a positive correlation was observed between the degradation rate of N-substituted polyaspartamides and a deprotonation degree of primary amines in their side chains. Therefore, we conclude that the deprotonated primary amine in the side chain of N-substituted polyaspartamides can induce the degradation of the main chain through the activation of amide nitrogen in the side chain. When N-substituted polyaspartamides were utilized as a messenger RNA (mRNA) delivery vehicle via formation of polyion complexes (PICs), degradable PAsp(AE) elicited significantly higher mRNA expression efficiency in cultured cells compared to PAsp(AP) and PAsp(AB). The higher efficiency of PAsp(AE) might be due to the facilitated destabilization of PICs within the cells, directed toward mRNA release. Additionally, degradation of PAsp(AE) considerably reduced its cytotoxicity. Thus, our study highlights a useful design of well-defined cationic poly(amino acid)s with tunable nonenzymatic degradability.

Tuned Density of Anti-Tissue Factor Antibody Fragment onto siRNA-Loaded Polyion Complex Micelles for Optimizing Targetability into Pancreatic Cancer Cells.

Antibody fragment (Fab')-installed polyion complex (PIC) micelles were constructed to improve targetability of small interfering RNA (siRNA) delivery to pancreatic cancer cells. To this end, we synthesized a block copolymer of azide-functionalized poly(ethylene glycol) and poly(l-lysine) and prepared PIC micelles with siRNA. Then, a dibenzylcyclooctyne (DBCO)-modified antihuman tissue factor (TF) Fab' was conjugated to azido groups on the micellar surface. A fluorescence correlation spectroscopic analysis revealed that 1, 2, or 3 molecule(s) of Fab'(s) were installed onto one micellar nanoparticle according to the feeding ratio of Fab' (or DBCO) to micelle (or azide). The resulting micelles exhibited ∼40 nm in hydrodynamic diameter, similar to that of the parent micelles before Fab' conjugation. Flow cytometric analysis showed that three molecules of Fab'-installed PIC micelles (3(Fab')-micelles) had the highest binding affinity to cultured pancreatic cancer BxPC3 cells, which are known to overexpress TF on their surface. The 3(Fab')-micelles also exhibited the most efficient gene silencing activity against polo-like kinase 1 mRNA in the cultured cancer cells. Furthermore, the 3(Fab')-micelles exhibited high penetrability and the highest cellular internalization amounts in BxPC3 spheroids compared with one or two molecule(s) of Fab'-installed PIC micelles. These results demonstrate the potential of anti-TF Fab'-installed PIC micelles for active targeting of stroma-rich pancreatic tumors.

Evaluation of IgG4+ Plasma Cell Infiltration in Patients with Systemic Plasmacytosis and Other Plasma Cell-infiltrating Skin Diseases.

Systemic plasmacytosis is a rare skin disorder characterized by marked infiltration of plasma cells in the dermis. IgG4-related disease is pathologically characterized by lymphoplasmacytic infiltration rich in IgG4+ plasma cells, storiform fibrosis, and obliterative phlebitis, accompanied by elevated levels of serum IgG4. Reports of cases of systemic plasmacytosis with abundant infiltration of IgG4+ plasma cells has led to discussion about the relationship between systemic plasmacytosis and IgG4-related disease. This study examined IgG4+/IgG+ plasma cell ratios in 4 patients with systemic plasmacytosis and 12 patients with other skin diseases that show marked infiltration of plasma cells. Furthermore, we examined whether these cases met one of the pathological diagnostic criteria for IgG4-related disease (i.e. IgG4+/IgG plasma cells ratio of over 40%). Only one out of 4 patients with systemic plasmacytosis met the criterion. These results suggest that systemic plasmacytosis and IgG4-related disease are distinct diseases.

Influence of RNA Strand Rigidity on Polyion Complex Formation with Block Catiomers.

Polyion complexes (b-PICs) are prepared by mixing single- or double-stranded oligo RNA (aniomer) with poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) (block catiomer) to clarify the effect of aniomer chain rigidity on association behaviors at varying concentrations. Here, a 21-mer single-stranded RNA (ssRNA) (persistence length: 1.0 nm) and a 21-mer double-stranded RNA (small interfering RNA, siRNA) (persistence length: 62 nm) are compared. Both oligo RNAs form a minimal charge-neutralized ionomer pair with a single PEG-PLL chain, termed unit b-PIC (uPIC), at low concentrations (<≈ 0.01 mg mL(-1)). Above the critical association concentration (≈ 0.01 mg mL(-1)), ssRNA b-PICs form secondary associates, PIC micelles, with sizes up to 30-70 nm, while no such multimolecular assembly is observed for siRNA b-PICs. The entropy gain associated with the formation of a segregated PIC phase in the multimolecular PIC micelles may not be large enough for rigid siRNA strands to compensate with appreciably high steric repulsion derived from PEG chains. Chain rigidity appears to be a critical parameter in polyion complex association.