In this study, we determined effects of an anionic siRNA delivery vector, siRNA ternary complex, which is constructed with biodegradable dendrigraft poly-L-lysine (DGL) and γ-polyglutamic acid (γ-PGA) on the melanoma cells and melanoma lung metastasis. The siRNA ternary complex showed high cellular uptake and silencing effect in melanoma cell line B16-F10/Luc cells. After intravenous administration of the siRNA ternary complex, high silencing effect was also observed in the lung of B16-F10/Luc melanoma metastasis model mice. Therefore, we applied vascular endothelial growth factor (VEGF)-siRNA on the siRNA ternary complex and determined the effect on the melanoma lung metastasis. The siRNA ternary complex containing VEGF-siRNA reduced VEGF protein levels significantly in in vitro and in vivo, and the complex successfully inhibited melanoma lung metastasis. This biodegradable and effective siRNA delivery vector, siRNA ternary complex, could be available for clinical trials.

In this study, we evaluated the effect of several promoters on the transfection activity and immune-induction efficiency of a plasmid DNA (pDNA)/polyethylenimine/γ-polyglutamic acid complex (pDNA ternary complex). Model pDNAs encoding firefly luciferase (Luc) were constructed with several promoters, such as simian virus 40 (SV40), eukaryotic elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and chicken beta actin hybrid (CBh) (pSV40-Luc, pEF1-Luc, pCMV-Luc, and pCBh-Luc, respectively). Four types of pDNA ternary complexes, each with approximately 145-nm particle size and −30-mV ζ-potential, were stably constructed. The pDNA ternary complex containing pSV40-Luc showed low gene expression, but the other complexes containing pEF1-Luc, pCMV-Luc, and pCBh-Luc showed high gene expression in DC2.4 cells and spleen after intravenous administration. After immunization using various pDNA encoding ovalbumin (OVA) such as pEF1-OVA, pCMV-OVA, and pCBh-OVA, only the pDNA ternary complex containing pCBh-OVA showed significant anti-OVA immunoglobulin G (IgG) induction. In conclusion, our results showed that the CBh promoter is potentially suitable for use in pDNA ternary complex-based DNA vaccination.

Typical examples of non-viral vectors are binary complexes of plasmid DNA with cationic polymers such as polyethyleneimine (PEI). However, problems such as cytotoxicity and hemagglutination, owing to their positively charged surfaces, hinder their in vivo use. Coating binary complexes with anionic polymers, such as γ-polyglutamic acid (γ-PGA), can prevent cytotoxicity and hemagglutination. However, the role of interactions between these complexes and serum components in in vivo gene transfer remains unclear. In this study, we analyzed the contribution of serum components to in vivo gene transfer using PEI/plasmid DNA binary complexes and γ-PGA/PEI/plasmid DNA ternary complexes. In binary complexes, heat-labile components in the serum greatly contribute to the hepatic and splenic gene expression of the luciferase gene. In contrast, serum albumin and salts affected the hepatic and splenic gene expression in the ternary complexes. Changes in physicochemical characteristics, such as increased particle size and decreased absolute values of ζ-potential, might be involved in the enhanced gene expression. These findings would contribute to a better understanding of in vivo non-viral gene transfer using polymers, such as PEI and γ-PGA.

Community-acquired pneumonia (CAP) is an acute pulmonary parenchymal infection acquired outside the hospital. The utility of blood cultures in inpatients with CAP to reduce mortality and length of hospital stay is controversial. This study aimed to determine the utility of blood cultures on the first day of hospitalization for CAP inpatients and its influence on mortality, length of hospital stay, and antibiotics use. We conducted a fact-finding survey on the implementation of blood culture in inpatients with CAP in Japan. A propensity score (PS)-matched analysis based on the National Database of Health Insurance Claims and Specific Health Check-ups of Japan database was conducted. Overall, 163173 patients were included in the analysis, and PS matching extracted 68104 pairs. The results of the comparison between the PS-matched blood culture group and PS-matched control group were as follows: mortality and length of hospital stay were significantly lower in the PS-matched blood culture group than in the control group. The adjusted odds ratio (OR) (95% confidence interval (CI)) for in-hospital mortality with blood culture test was 0.73 (0.68-0.79). Moreover, for days of antibiotic usage, number of antibiotics used were significantly higher in the PS-matched blood culture group than that in the control group. Our findings indicated that performing a blood culture on the first day of hospitalization for inpatients with CAP was associated with reduced mortality. To our knowledge, this is the largest epidemiological study to assess the utility of blood culture in Japanese inpatients with CAP. This testing method shows potential for application in clinical practice.

On-demand lectures have become common in Japan. We are now developing a video re-editing tool for the purpose of utilizing the lecture videos used in such lectures. This tool is designed to shorten the playback time of lecture videos as much as possible without reducing the lecture content and to create digest videos by picking out the important parts from multiple lecture videos. In developing the tool, we conducted a survey of lecture videos actually used in on-demand lectures. We also confirmed that the time required for video playback can be reduced by about 30% by removing silent time. In addition, we conducted a basic investigation to see if it is possible to detect the parts of the lecture that the lecturer considers particularly important by using voice emotion recognition AI.

We previously found that a nanoparticle constructed with an antigen, benzalkonium chloride (BK) and γ-polyglutamic acid (γ-PGA) showed high Th1 and Th2-type immune induction after subcutaneous administration. For prophylaxis of respiratory infections, however, mucosal immunity should be induced. In this study, we investigated the effect of pulmonary administration of a nanoparticle comprising ovalbumin (OVA) as a model antigen, BK, and γ-PGA on induction of mucosal immunity in the lungs and serum. The complex was strongly taken up by RAW264.7 and DC2.4cells. After pulmonary administration, lung retention was longer for the OVA/BK/γ-PGA complex than for OVA alone. OVA-specific serum immunoglobulin (Ig)G was highly induced by the complex. High IgG and IgA levels were also induced in the bronchoalveolar lavage fluid, and in vivo toxicities were not observed. In conclusion, we effectively and safely induced mucosal immunity by pulmonary administration of an OVA/BK/γ-PGA complex.

We previously found that a complex comprising plasmid DNA (pDNA), polyethylenimine (PEI), and γ-polyglutamic acid (γ-PGA) had high transgene efficiency without cytotoxicity in vitro and in vivo. However, messenger RNA (mRNA) remains an attractive alternative to pDNA. In this study, we developed a safe and effective delivery system for mRNA to prevent its degradation and efficiently deliver it into target cells. Various cationic and anionic complexes were produced containing PEI, γ-PGA, and an mRNA encoding firefly luciferase. Their physicochemical properties and cytotoxicities were analyzed and the in vitro and in vivo protein expression were determined. The cationic mRNA/PEI complex showed high in vitro protein expression with strong cytotoxicity. The anionic complex was constructed as mRNA/PEI8/γ-PGA12 complex with a theoretical charge ratio of 1:8:12 based on the phosphate groups of the mRNA, nitrogen groups of PEI, and carboxylate groups of γ-PGA. It was stable and showed high in vitro protein expression without cytotoxicity. After intravenous administration of mRNA/PEI8/γ-PGA12 complex to mice, high protein expression was observed in the spleen and liver and slight expression was observed in the lung over 24 h. Thus, the newly constructed mRNA/PEI8/γ-PGA12 complex provides a safe and effective strategy for the delivery of mRNA.

We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and β-tricalcium phosphate (β-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 μg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 μg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

We developed a biocompatible splenic vector for a DNA vaccine against melanoma. The splenic vector is a ternary complex composed of plasmid DNA (pDNA), biodegradable dendrigraft poly-L-lysine (DGL), and γ-polyglutamic acid (γ-PGA), the selective uptake of which by the spleen has already been demonstrated. The ternary complex containing pDNA encoding luciferase (pCMV-Luc) exhibited stronger luciferase activity for RAW264.7 mouse macrophage-like cells than naked pCMV-Luc. Although the ternary complex exhibited strong luciferase activity in the spleen after its tail vein injection, luciferase activity in the liver and spleen was significantly decreased by a pretreatment with clodronate liposomes, which depleted macrophages in the liver and spleen. These results indicate that the ternary complex is mainly transfected in macrophages and is a suitable formulation for DNA vaccination. We applied the ternary complex to a pUb-M melanoma DNA vaccine. The ternary complex containing pUb-M suppressed the growth of melanoma and lung metastasis by B16-F10 mouse melanoma cells. We also examined the acute and liver toxicities of the pUb-M ternary complex at an excess pDNA dose in mice. All mice survived the injection of the excess amount of the ternary complex. Liver toxicity was negligible in mice injected with the excess amount of the ternary complex. In conclusion, we herein confirmed that the ternary complex was mainly transfected into macrophages in the spleen after its tail vein injection. We also showed the prevention of melanoma metastasis by the DNA vaccine and the safety of the ternary complex.

Melanoma is a highly malignant skin cancer that frequently metastasizes to the lung, bone, and brain at an early phase. Therefore, noninvasive detection of metastasized melanoma could be beneficial to determine suitable therapeutic strategies. We previously reported a biocompatible ternary anionic complex composed of plasmid DNA (pDNA), polyethyleneimine (PEI), and γ-polyglutamic acid (γ-PGA) based on an electrostatic interaction, which was highly taken up by melanoma cells (B16-F10), even if it was negatively charged. Here, we developed a radiolabeled γ-PGA complex by using indium-111 (111In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 (125I)-labeled PEI instead of native PEI, and evaluated its effectiveness as a melanoma-targeted imaging probe. This ternary complex was synthesized at a theoretical charge ratio; carboxyl groups of 111In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125I-PEI : carboxyl groups of γ-PGA was 1 : 8 : 16, and the size and zeta potential were approximately 29 nm and -33 mV, respectively. This complex was taken up by B16-F10 cells with time. Furthermore, a biodistribution study, using normal mice, demonstrated its accumulation in the liver, spleen, and lung, where macrophage cells are abundant. Almost the same level of radioactivity derived from both 111In and 125I was observed in these organs at an early phase after probe injection. Compared with the normal mice, significantly higher lung-to-blood ratios of radioactivity were observed in the B16-F10-lung metastatic cancer model. In conclusion, the radiolabeled γ-PGA complex would hold potentialities for nuclear medical imaging of lung metastatic melanoma.

In a previous study, we constructed a lung-targeting lipopolyplex containing polyethyleneimine (PEI), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and N-lauroylsarcosine (LS). The lipopolyplex exhibited an extremely high gene expression in the lung after intravenous administration. Here, we optimized the lipopolyplex and used it to deliver a TGF-β1 shRNA to treat refractory pulmonary fibrosis. We constructed several lipopolyplexes with pDNA, various cationic polymers, cationic lipids, and LS to select the most effective formulation. Then, the pDNA encoding shRNA against mouse TGF-β1 was encapsulated in the lipopolyplex and injected into mice with bleomycin-induced pulmonary fibrosis. After optimizing the lipopolyplex, dendrigraft poly-L-lysine (DGL) and DOTMA were selected as the appropriate cationic polymer and lipid, respectively. The lipopolyplex was constructed with a pDNA, DGL, DOTMA, and LS charge ratio of 1:2:2:4 showed the highest gene expression. After intravenous administration of the lipopolyplex, the highest gene expression was observed in the lung. In the in vitro experiment, the lipopolyplex delivered pDNA into the cells via endocytosis. As a result, the lipopolyplex containing pDNA encoding TGF-β1 shRNA significantly decreased hydroxyproline in the pulmonary fibrosis model mice. We have successfully inhibited pulmonary fibrosis using a novel lung-targeting lipopolyplex.

Naldemedine (NAL), a peripherally acting μ-opioid receptor antagonist, is effective for opioid-induced constipation (OIC). However, diarrhea is the most common adverse event. We investigated the incidence of NAL-induced diarrhea in patients who started NAL at Nagasaki University Hospital between June 2017 and March 2019. Predictors of NAL-induced diarrhea were analyzed using a multivariate logistic regression model. Two hundred and forty-two patients were included in the present study, and NAL-induced diarrhea was observed in 17.8% (43 patients). The results of multiple logistic regression analyses identified the administration of opioid analgesics for 8 d or longer before the initiation of NAL (odds ratio (OR): 2.20, 95% confidence interval (95% CI): 1.04-4.64, p = 0.039), the combination of a laxative (OR: 2.22, 95%CI: 1.03-4.81, p = 0.042), and the combination of CYP3A4 inhibitors (strong/moderate) (OR: 2.80, 95%CI: 1.02-7.67, p = 0.045) as risk factors. Therefore, the development of diarrhea needs to be considered in patients with these risk factors. Furthermore, diarrhea may be controlled by the initiation of NAL within 7 d of opioid analgesics and, where possible, the discontinuation of or change in the combination of moderate or strong CYP3A4 inhibitors.

Docetaxel and paclitaxel (PTX) are key drugs in human epidermal growth factor receptor type 2 (HER2) positive metastatic/recurrent breast cancer. However, there is little information on the recommended doses for liver dysfunction. We performed trastuzumab/pertuzumab/PTX combination therapy (50％ dose of PTX) for patients with HER2-positive breast cancer who had Child-Pugh C liver dysfunction due to multiple liver metastases. After the chemotherapy, the patient’s liver function improved rapidly, so the dose of PTX was increased to 75％. However, since febrile neutropenia (FN) occurred, PTX was continued at a dose of 50％ thereafter. As a result, the patient was able to continue chemotherapy without FN. This case will help us consider the administration method of PTX in the case of severe liver dysfunction.

Background Radiation-induced heart disease has been reported, but the underlying mechanisms remain unclear. Mesenchymal stem cells (MSCs), also residing in the heart, are highly susceptible to radiation. We examined the hypothesis that the altered secretion of extracellular vesicles (EVs) from MSCs is the trigger of radiation-induced heart disease. Methods By exposing human placental tissue-derived MSCs to 5 Gy γ-rays, we then isolated EVs from the culture medium 48 h later and evaluated the changes in quantity and quality of EVs from MSCs after radiation exposure. The biological effects of EVs from irradiated MSCs on HUVECs and H9c2 cells were also examined. Results Although the amount and size distribution of EVs did not differ between the nonirradiated and irradiated MSCs, miRNA sequences indicated many upregulated or downregulated miRNAs in irradiated MSCs EVs. In vitro experiments using HUVEC and H9c2 cells showed that irradiated MSC-EVs decreased cell proliferation ( P < 0.01), but increased cell apoptosis and DNA damage. Moreover, irradiated MSC-EVs impaired the HUVEC tube formation and induced calcium overload in H9c2 cells. Conclusions EVs released from irradiated MSCs show altered miRNA profiles and harmful effects on heart cells, which provides new insight into the mechanism of radiation-related heart disease risks.

The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and γ-polyglutamic acid (γ-PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer compared with luminal or epidermal growth factor receptor 2 subtypes, thus effective therapeutic options for TNBC are yet to be developed. Nowadays, oncogenic long noncoding RNAs (lncRNAs) are applied to cancer management as a new class of therapeutic targets. We previously showed that thymopoietin antisense transcript 1 ( TMPO-AS1 ) is a proliferation-associated lncRNA that contributes to hormone-dependent breast cancer progression by stabilizing estrogen receptor-α mRNA. We here showed that TMPO-AS1 is abundantly expressed in basal-like breast cancer subtype based on the transcriptomic data in The Cancer Genome Atlas as well as in TNBC cell lines and patient-derived cells. Small interfering RNA-based loss-of-function analyses showed that TMPO-AS1 knockdown substantially represses the proliferation and migration of TNBC cells. Expression microarray analysis showed that TMPO-AS1 alters gene signatures related to transforming growth factor-β signaling in addition to proliferative E2F signaling pathways. TMPO-AS1 -targeted siRNA treatment through engineered drug delivery systems using cancer-targeted polyion complex micelle or nanoball technology significantly impaired the in vivo growth of primary and metastatic TNBC xenograft tumors. Our findings suggest that TMPO-AS1 plays a key role in TNBC pathophysiology and could be a potential therapeutic target for TNBC.

In this practical research, we assessed the effectiveness of “Protocol-based Pharmacotherapy Management” (PBPM) by community pharmacists and the Nagasaki University Hospital with the cooperation of the Nagasaki Pharmaceutical Association and Nagasaki City Pharmaceutical Association. This study was implemented from July 1 to December 15, 2018. We targeted outpatients prescribed anticancer drugs (the combination drug tegafur/gimeracil/oteracil (S-1) or capecitabine) from Nagasaki University Hospital. Community pharmacists confirmed the presence of adverse reactions in patients by follow-up phone calls throughout the medication period or drug withdrawal period, and reported patients’ situation to their attending doctors according to the protocol. Telephone support by community pharmacists was provided to 17 and seven patients for S-1 and capecitabine, respectively. The number of telephone support times was 45 (2.9 times per patient) and 20 (2.7 times per patient) for S-1 and capecitabine, respectively. There were several instances of adverse skin reaction and whole-body weariness, and hand-foot syndrome was confirmed with a particularly high frequency. Community pharmacists followed compliance with recommended methods of skin care and the usage of prescribed medications. The telephone support for patients included “extension of the drug withdrawal period for S-1” and “reduction of the dosage of capecitabine”, which contributed to the safe discontinuation of drug treatment. This report is a valuable study that indicates PBPM between community pharmacists, hospital pharmacists and medical doctors through cooperation between key hospitals and regional pharmaceutical associations effectively achieved seamless medical cooperation.

Purpose: To evaluate acute corneal epithelial toxicity induced by benzalkonium chloride (BAC) homologs with different alkyl chain lengths using an in vivo electrophysiological method. Methods: BAC homologs with C12, C14, and C16 alkyl chain lengths were used at concentrations of 0.0025%, 0.005%, and 0.01%, respectively. Cytotoxicity of BAC homologs on the normal rabbit corneal epithelial cells was examined by using a WST-1 assay. Corneal transepithelial electrical resistance (TER) was measured in living Japanese white rabbits by 2 Ag/AgCl electrodes placed in the anterior aqueous chamber and on the cornea. TER changes were then evaluated after a 60-second exposure to these BAC homologs. Morphological changes in corneal epithelium after exposure to the BAC homologs were examined using scanning electron microscopy. The antimicrobial activity of BAC homologs against Escherichia coli was also assessed. Results: All BAC homologs caused cytotoxicity and corneal barrier dysfunction in a concentration-dependent manner. However, the degree of corneal toxicity differed among the BAC homologs. Based on cytotoxicity and TER measurement, C14-BAC caused the greatest corneal impairment followed in order of severity by mixed BAC/C16-BAC and C12-BAC. Scanning electron microscopy images indicated an intact corneal epithelium after exposure to 0.005% C12-BAC, whereas 0.005% C14-BAC damaged the epithelium. There were no remarkable differences noted in the antimicrobial activity among the BAC homologs. Conclusions: Acute corneal epithelial toxicity induced by BAC homologs depends on the alkyl chain length. Thus, the use of C12-BAC instead of commercially available BAC is potentially safer for patients undergoing ophthalmological pharmacotherapy.

【目的】ナルデメジンは消化管のµオピオイド受容体に拮抗してオピオイド誘発性便秘（opioid-induced constipation: OIC）を改善するが，副作用として下痢が知られている．ナルデメジン導入後の下痢発現の予測因子についてオピオイド鎮痛薬投与期間に着目して解析した．【方法】2017年6月1日から2019年3月31日の期間に長崎大学病院においてナルデメジンをはじめて導入した患者を対象に後方視的調査を行った．【結果】調査対象は132名であり，下痢は33名（25.0％）に発現した．多変量ロジスティック回帰分析の結果，ナルデメジン導入前のオピオイド鎮痛薬投与日数8日以上は下痢発現と有意に関連した（オッズ比：3.76, 95％信頼区間：1.53-9.20, p=0.004）．【考察】OICに対してナルデメジンを使用する場合，オピオイド開始後7日以内に使用することで，下痢の発現を回避できる可能性が示唆された．