Quantitative Model Analysis and Simulation of Pharmacokinetics and Metastasis-Associated Lung Adenocarcinoma 1 RNA Knockdown Effect After Systemic Administration of Cholesterol-Conjugated DNA/RNA Heteroduplex Oligonucleotide Crossing Blood-Brain Barrier of Mice.

The cholesterol-conjugated heteroduplex oligonucleotide (Chol-HDO) is a double-stranded complex; it comprises an antisense oligonucleotide (ASO) and its complementary strand with a cholesterol ligand. Chol-HDO is a powerful tool for achieving target RNA knockdown in the brains of mice after systemic injection. Here, a quantitative model analysis was conducted to characterize the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD), non-coding RNA metastasis-associated lung adenocarcinoma 1 (Malat1) RNA, of Chol-HDO, in a time-dependent manner. The established PK model could describe regional differences in the observed brain concentration-time profiles. Incorporating the PD model enabled the unique knockdown profiles in the brain to be explained in terms of the time delay after single dosing and enhancement following repeated dosing. Moreover, sensitivity analysis of PK exposure/persistency, target RNA turnover, and knockdown potency identified key factors for the efficient and sustained target RNA knockdown in the brain. The simulation of an adequate dosing regimen quantitatively supported the benefit of Chol-HDO in terms of achieving a suitable dosing interval. This was achieved via sufficient and sustained brain exposure and subsequent strong and sustained target RNA knockdown in the brain, even after systemic injection. The present study provides new insights into drug discoveries and development strategies for HDO in patients with neurogenic disorders. SIGNIFICANCE STATEMENT: The quantitative model analysis presented here characterized the PK/PD relationship of Chol-HDO, enabled its simulation under various conditions or assumptions, and identified key factors for efficient and sustained RNA knockdown, such as PK exposure and persistency. Chol-HDO appears to be an efficient drug delivery system for the systemic administration of desired drugs to brain targets.

Expression of secretory calcium-binding phosphoprotein (scpp) genes in medaka during the formation and replacement of pharyngeal teeth.

BACKGROUND: Analyses of tooth families and tooth-forming units in medaka with regard to tooth replacement cycles and the localization of odontogenic stem cell niches in the pharyngeal dentition clearly indicate that continuous tooth replacement is maintained. The secretory calcium-binding phosphoprotein (scpp) gene cluster is involved in the formation of mineralized tissues, such as dental and bone tissues, and the genes encoding multiple SCPPs are conserved in fish, amphibians, reptiles, and mammals. In the present study, we examined the expression patterns of several scpp genes in the pharyngeal teeth of medaka to elucidate their roles during tooth formation and replacement. METHODS: Himedaka (Japanese medaka, Oryzias latipes) of both sexes (body length: 28 to 33 mm) were used in this study. Real-time quantitative reverse transcription-polymerase chain reaction (PCR) (qPCR) data were evaluated using one-way analysis of variance for multi-group comparisons, and the significance of differences was determined by Tukey's comparison test. The expression of scpp genes was examined using in situ hybridization (ISH) with a digoxigenin-labeled, single-stranded antisense probe. RESULTS: qPCR results showed that several scpp genes were strongly expressed in pharyngeal tissues. ISH analysis revealed specific expression of scpp1, scpp5, and sparc in tooth germ, and scpp5 was continually expressed in the odontoblasts of teeth attached to pedicles, but not in the osteoblasts of pedicles. In addition, many scpp genes were expressed in inner dental epithelium (ide), but not in odontoblasts, and scpp2 consistently showed epithelial-specific expression in the functional teeth. Taken together, these data indicate that specific expression of scpp2 and scpp5 may play a critical role in pharyngeal tooth formation in medaka. CONCLUSION: We characterized changes in the expression patterns of scpp genes in medaka during the formation and replacement of pharyngeal teeth.

Glycoprotein NMB promotes tumor formation and malignant progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC), although one of the most common head and neck cancers, has a static or slightly decreased survival rate because of difficulties in early diagnosis, lack of effective molecular targeting therapy, and severe dysfunction after radical surgical treatments. Therefore, a novel therapeutic target is crucial to increase treatment efficacy and survival rates in these patients. Glycoprotein NMB (GPNMB), whose role in LSCC remains elusive, is a type 1 transmembrane protein involved in malignant progression of various cancers, and its high expression is thought to be a poor prognostic factor. In this study, we showed that GPNMB expression levels in LSCC samples are significantly higher than those in normal tissues, and GPNMB expression is observed mostly in growth-arrested cancer cells. Furthermore, knockdown of GPNMB reduces monolayer cellular proliferation, cellular migration, and tumorigenic growth, while GPNMB protein displays an inverse relationship with Ki-67 levels. Therefore, we conclude that GPNMB may be an attractive target for future LSCC therapy.

Asparagine residue 368 is involved in Alzheimer's disease tau strain-specific aggregation.

In tauopathies, tau forms pathogenic fibrils with distinct conformations (termed "tau strains") and acts as an aggregation "seed" templating the conversion of normal tau into isomorphic fibrils. Previous research showed that the aggregation core of tau fibril covers the C-terminal region (243-406 amino acids (aa)) and differs among the diseases. However, the mechanisms by which distinct fibrous structures are formed and inherited via templated aggregation are still unknown. Here, we sought to identify the key sequences of seed-dependent aggregation. To identify sequences for which deletion reduces tau aggregation, SH-SY5Y cells expressing a series of 10 partial deletion (Del 1-10, covering 244-400 aa) mutants of tau-CTF24 (243-441 aa) were treated with tau seeds prepared from a different tauopathy patient's brain (Alzheimer's disease, progressive supranuclear palsy, and corticobasal degeneration) or recombinant tau, and then seed-dependent tau aggregation was assessed biochemically. We found that the Del 8 mutant lacking 353-368 aa showed significantly decreased aggregation in both cellular and in vitro models. Furthermore, to identify the minimum sequence responsible for tau aggregation, we systematically repeated cellular tau aggregation assays for the delineation of shorter deletion sites and revealed that Asn-368 mutation suppressed tau aggregation triggered by an AD tau seed, but not using other tauopathy seeds. Our study suggested that 353-368 aa is a novel aggregation-responsible sequence other than PHF6 and PHF6*, and within this sequence, the Asn-368 residue plays a role in strain-specific tau aggregation in different tauopathies.

A new class of pentapeptide KISS1 receptor agonists with hypothalamic-pituitary-gonadal axis activation.

The kisspeptin (Kp, Kp-54, metastin)/KISS1R system plays crucial roles in regulating the secretion of gonadotropin-releasing hormone. Continuous administration of nonapeptide Kp analogs caused plasma testosterone depletion, whereas bolus administration caused strong plasma testosterone elevation in male rats. To develop a new class of small peptide drugs, we focused on stepwise N-terminal truncation of Kp analogs and discovered potent pentapeptide analogs. Benzoyl-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (16) exhibited high agonist activity for KISS1R and excellent metabolic stability in rat serum. A single injection of a 4-pyridyl analog (19) at the N-terminus of 16 into male Sprague Dawley rats caused a robust increase in plasma luteinizing hormone levels, but unlike continuous administration of nonapeptide Kp analogs, continuous administration of 19 maintained moderate testosterone levels in rats. These results indicated that small peptide drugs can be successfully developed for treating sex hormone deficiency.

A quantitative LC/MSMS method for determination of edoxaban, a Xa inhibitor and its pharmacokinetic application in patients after total knee arthroplasty.

Edoxaban was extracted from human plasma by simple protein precipitation with acetonitrile, followed by quantitative determination using a liquid chromatography-mass spectrometry method. The recoveries of edoxaban and the internal standard (ticlopidine) from human plasma were >85%, and the within- and between-day coefficients of variation were within 15%. The limit of quantification in human plasma was 1 ng/mL. The concentration of edoxaban in blood decreased at room temperature, but remained unchanged for 1 week at 4°C. On the other hand, the concentration in plasma at both -20 and -80°C remained unchanged for 5 months. These results indicated that blood samples should be centrifuged immediately or stored at 4°C, and that plasma samples should be stored below -20°C until analysis. This method was applied to human plasma obtained from four patients after total knee arthroplasty. Analysis of edoxaban pharmacokinetics demonstrated an absorption time lag of 4h, a maximum concentration of 110 ± 26 ng/mL and an oral clearance of 37 ± 16 L/h. The analytical methods established in this study will be suitable for determining the concentrations of edoxaban in human plasma.

The twenty-four KDa C-terminal tau fragment increases with aging in tauopathy mice: implications of prion-like properties.

The truncated tau protein is a component of the neurofibrillary tangles found in the brains with tauopathies. However, the molecular mechanisms by which the truncated tau fragment causes neurodegeneration remain unknown. Tau pathology was recently suggested to spread through intercellular propagation, and required the formation of 'prion-like' species. We herein identified a new fragment of the tau protein that consisted of four binding domains and a C-terminal tail (Tau-CTF24), but lacked the N-terminal projection domain, and found that it increased with aging in tauopathy model mice (Tg601). Tau-CTF24-like fragments were also present in human brains with tauopathies. A mass spectroscopic analysis revealed that Tau-CTF24 was cleaved behind R242. The digestion of full-length tau (Tau-FL) by calpain produced Tau-CTF24 in vitro and calpain activity increased in old Tg601. Recombinant Tau-CTF24 accelerated heparin-induced aggregation and lost the ability to promote microtubule assembly. When insoluble tau from diseased brains or aggregated recombinant tau was introduced as seeds into SH-SY5Y cells, a larger amount of insoluble tau was formed in cells overexpressing Tau-CTF24 than in those overexpressing Tau-FL. Furthermore, lysates containing the Tau-CTF24 inclusion propagated to naive tau-expressing cells more efficiently than those containing the Tau-FL inclusion. Immunoblot and confocal microscopic analyses revealed that aggregated Tau-CTF24 bound to cells more rapidly and abundantly than aggregated Tau-FL. Our results suggest that Tau-CTF24 contributes to neurodegeneration by enhancing prion-like propagation as well as deteriorating the mechanisms involved in microtubule function.

Identification of AHCY inhibitors using novel high-throughput mass spectrometry.

S-adenosylhomocysteine hydrolase (AHCY) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and l-homocysteine. This enzyme is frequently overexpressed in many tumor types and is considered to be a validated anti-tumor target. In order to enable the development of small molecule AHCY inhibitors as targeted cancer therapeutics we developed an assay based on a RapidFire high-throughput mass spectrometry detection system, which allows the direct measurement of AHCY enzymatic activity. This technique avoids many of the problems associate with the previously reported method of using a thiol-reactive fluorescence probes to measure AHCY activity. Screening of a ∼500,000 compound library using this technique identified multiple SAH competitive hits. Co-crystal structures of the hit compounds complexed with AHCY were obtained showing that the compounds indeed bind in the SAH site of the enzyme. In addition, some hit compounds increased the SAH levels in HCT116 cells and showed growth inhibition. These compounds could be promising starting points for the optimization of cancer treatments.

Discovery of GPX4 inhibitory peptides from random peptide T7 phage display and subsequent structural analysis.

The phospholipid hydroperoxidase glutathione peroxidase (GPX4) is an enzyme that reduces lipid hydroperoxides in lipid membranes. Recently, GPX4 has been investigated as a target molecule that induces iron-dependent cell death (ferroptosis) selectively in cancer cells that express mutant Ras. GPX4 inhibitors have the potential to become novel anti-cancer drugs. However, there are no druggable pockets for conventional small molecules on the molecular surface of GPX4. To generate GPX4 inhibitors, we examined the use of peptides as an alternative to small molecules. By screening peptide libraries displayed on T7 phages, and analyzing the X-ray crystal structures of the peptides, we successfully identified one peptide that binds to near Sec73 of catalytic site and two peptides that bind to another site on GPX4. To our knowledge, this is the first study reporting GPX4 inhibitory peptides and their structural information.

Identification of PARP14 inhibitors using novel methods for detecting auto-ribosylation.

Poly(ADP-ribose) polymerases (PARPs) use nicotinamide adenine dinucleotide (NAD+) as a co-substrate to transfer ADP-ribose when it releases nicotinamide as the metabolized product. Enzymes of the PARP family play key roles in detecting and repairing DNA, modifying chromatin, regulating transcription, controlling energy metabolism, and inducing cell death. PARP14, the original member of the PARP family, has been reported to be associated with the development of inflammatory diseases and various cancer types, making it a potential therapeutic target. In this study, we purified the macrodomain-containing PARP14 enzyme and established an assay for detecting the auto-ribosylation activity of PARP14 using RapidFire high-throughput mass spectrometry and immunoradiometric assay using [3H]NAD+. Subsequently, we performed high-throughput screening using the assays and identified small-molecule hit compounds, which showed NAD+-competitive and PARP14-selective inhibitory activities. Co-crystal structures of PARP14 with certain hit compounds revealed that the inhibitors bind to the NAD+-binding site. Finally, we confirmed that the hit compounds interacted with intracellular PARP14 by a cell-based protein stabilization assay. Thus, we successfully identified primary candidate compounds for further investigation.

Design and synthesis of a novel series of orally active, selective somatostatin receptor 2 agonists for the treatment of type 2 diabetes.

The discovery of a novel series of ß-methyltryptophan (ß MeTrp) derivatives as selective and orally active non-peptide somatostatin receptor 2 (SSTR2) agonists for the treatment of Type 2 diabetes is described. In our previous research, Compound A, ß-MeTrp derivative with highly potent and selective SSTR2 agonistic activity IC50 (SSTR2/SSTR5)=0.3/>100 (nM), was identified asa drug candidate for treatment of Type 2 diabetes which lowers significantly plasma glucose level in Wistar fatty rats in its oral administrations. However, as serious increase in AUC and phospholipidosis (PLsis) were observed in its toxicological studies in rats, follow-up compounds were searched to avoid risk of PLsis with reference to their in vitro PLsis potentials evaluated on the basis of accumulation of phospholipids in HepG2 cells exposed to the compounds. It has been found that introduction of a carbonyl group onto the piperidine and piperazine or aniline moiety of compounds A and B reduced markedly the in vitro PLsis potentials. And further modification of the compounds and their evaluation led to a discovery of compounds 3k with lower in vitro PLsis potentials exhibiting lowering effect of hypoglycemia-induced glucagon secretion in SD rats (ED50=1.1mg/kg) and glucose excursion in meal tolerance test in Wistar fatty diabetic rats (MED=3.0mg/kg) in oral administrations. Compound 3k was selected asa new drug candidate of selective and orally active non-peptide SSTR2 agonists for treatment of Type 2 diabetes with low in vivo PLsis potential.

Usefulness of a peripherally inserted central catheter for total parenteral nutrition in patients with inflammatory bowel disease.

BACKGROUNDS AND AIMS: Peripherally inserted central catheters (PICC) have been widely used as a blood access route for total parenteral nutrition (TPN) in recent years. However, there have been few reports that evaluated the usefulness of PICC for patients with inflammatory bowel disease (IBD). In this study, we compared the clinical courses in patients with IBD who received TPN during their hospitalization by conventional central venous catheters (CVC) and PICC. PATIENTS AND METHODS: A total of 137 IBD patients were enrolled. The CVC group and the PICC group included 56 and 81 patients, respectively. The clinical courses in both groups were compared retrospectively. RESULTS: As a complication of the puncture, pneumothorax occurred in two patients (3.6%) in the CVC group, but in none (0%) in the PICC group. The PICC group had significantly higher rates of achieving the scheduled TPN without removing the catheter, lower rates of catheter-related blood stream infection (CRBSI) and longer periods without CRBSI than the CVC group. CONCLUSION: PICC might be more useful than CVC in terms of safety and the ability to deliver scheduled TPN for IBD patients.

Visual Field Improvement and Electrode Extrusion Prevention by Extended Endaural Incision With Cavity Obliteration for Cochlear Implantation in Ears After Canal Wall-Down Mastoidectomy: Experience of Four Cases.

There is no global consensus on the surgical technique of cochlear implantation (CI) in ears with an open cavity after canal wall-down (CWD) mastoidectomy. Here, we report CI surgery with an endaural incision for the ears after CWD mastoidectomy. The endaural incision was extended upward to obliterate the open cavity of the temporal fascial flap. The endaural incision was extended downward to close the open cavity inlet. After inserting the implanted electrode, the open cavity was obliterated using a temporal fascial flap, and the cavity was closed at the inlet. We performed this type of CI surgery in four ears in three patients. This extended endaural incision provided an excellent view for pedicling the temporal fascial flap with the superficial temporal artery and for open cavity closure without any serious complications. This technique allowed us to opt for CI surgery of the ears after CWD mastoidectomy.

A rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies.

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

Discovery of TAK-960: an orally available small molecule inhibitor of polo-like kinase 1 (PLK1).

Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies.

Quantitation and characterization of glucosylsphingosine in cerebrospinal fluid (CSF), plasma, and brain of monkey model with Gaucher disease.

Treatment with conduritol-ß-epoxide (CBE) in preclinical species is expected to be a powerful approach to generate animal models of Gaucher disease (GD) and Parkinson's disease associated with heterozygous mutations in Glucocerebrosidase (GBA-PD). However, it is not fully elucidated how quantitatively the change in glucosylsphingosine (GlcSph) levels in cerebrospinal fluid (CSF) correlates with that in the brain, which is expected to be clinically informative. Herein, we aimed to investigate the correlation with successfully quantified GlcSph in monkey CSF by developing highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The GlcSph in normal monkey CSF was 0.635 ± 0.177 pg/mL at baseline and increased by CBE treatment at 3 mg/kg daily for five days up to a moderate level, comparable to that in GD patients. The balance between GlcSph and galactosylsphingosine (GalSph) in the CSF matched that in the brain rather than plasma. In addition, GlcSph in the CSF was increased, accompanied by that in the brain at a dose of 3 mg/kg daily. These results indicate that GlcSph in the CSF is worth evaluating for concentration changes in the brain. Thus, this model can be useful for evaluating GBA-related diseases such as GD and GBA-PD.

Endoscopic observation of pharyngeal mucosal suture healing process after total laryngectomy: case series.

BACKGROUND: Pharyngocutaneous fistula (PCF), a major complication of total laryngectomy, is caused by pharyngeal repair failure. OBJECTIVE: Assess the usefulness of endoscopic observation of the pharyngeal suture's healing process for the early detection of PCF development. METHODS: Pharyngeal mucosal sutures were endoscopically observed postoperatively in patients who underwent total laryngectomy with primary closure. RESULTS: Postoperatively, a white coat adhered to the pharyngeal mucosal suture of all patients. In most cases, the white coat gradually receded, which was considered to be a normal healing process. Thickening of the white coat and/or dehiscence of surgical wound were interpreted as 'poor healing conditions'. Three cases were judged to have developed poor healing conditions of the pharyngeal mucosal suture and one patient developed PCF. The other two patients did not develop PCF, possibly due to early detection of 'poor healing condition' and conservative approach, such as discontinuation of oral intake. CONCLUSIONS: Postoperative poor healing conditions of the pharyngeal mucosal suture may be precursors to PCF development. Endoscopic observation enables early detection of these conditions and may enable the prevention of PCF.

Proton beam therapy in multimodal treatment for locally advanced squamous cell carcinoma of the nasal cavity and paranasal sinus.

BACKGROUND: To evaluate proton beam therapy (PBT) in multimodal treatment for locally advanced squamous cell carcinoma of the nasal cavity and paranasal sinus (NPSCC). METHODS: The cases in this study included T3 and T4 NPSCC without distant metastases that were treated at our center using PBT between July 2003 and December 2020. These cases were classified into 3 groups based on resectability and treatment strategy: surgery followed by postoperative PBT (group A); those indicated to be resectable, but the patient refused surgery and received radical PBT (group B); and those declared unresectable based on the extent of the tumor and treated with radical PBT (group C). RESULTS: The study included 37 cases, with 10, 9 and 18 in groups A, B and C, respectively. The median follow-up period in surviving patients was 4.4 years (range 1.0-12.3 years). The 4-year overall survival (OS), progression-free survival (PFS), and local control (LC) rates were 58%, 43% and 58% for all patients; 90%, 70% and 80% in group A, 89%, 78% and 89% in group B; and 24%, 11% and 24% in group C. There were significant differences in OS (p = 0.0028) and PFS (p = 0.009) between groups A and C; and in OS (p = 0.0027), PFS (p = 0.0045) and LC (p = 0.0075) between groups B and C. CONCLUSIONS: PBT gave favorable outcomes in multimodal treatment for resectable locally advanced NPSCC, including surgery followed by postoperative PBT and radical PBT with concurrent chemotherapy. The prognosis for unresectable NPSCC was extremely poor, and reconsideration of treatment strategies, such as more active use of induction chemotherapy, may improve outcomes.

Effect of Average Relative Humidity on Epistaxis.

Background Epistaxis is a very common symptom. The occurrence of epistaxis may be affected by dry environments, but there are some differences among previous reports and this view is controversial. Objective We investigated the relationship between the number of epistaxes and daily average relative humidity. Methods Data on patients with epistaxis between March 2011 and February 2021 were collected from two hospitals. The daily average relative humidity was examined, and the change in the number of patients with epistaxis due to humidity was investigated using a generalized linear mixed model. Results A total of 4184 cases of epistaxis were identified. The number of epistaxis cases per day was significantly associated with the daily average relative humidity (p < 0.001). One percent increment in average relative humidity decreases the number of epistaxis cases per day by 1.1%. Conclusion A negative correlation was found to exist between daily average relative humidity and occurrences of epistaxis.

Elevation of C-reactive protein during concurrent chemoradiotherapy is a poor predictive factor for head and neck cancer.

OBJECTIVE: The prognostic role of pretreatment C-reactive protein (CRP) has been reported for head and neck cancer. However, little is known about the relationship between the changes in CRP levels during treatment and prognosis. This study aimed to investigate the correlation between CRP elevation during concurrent chemoradiotherapy (CCRT) and survival outcomes. METHODS: The medical records of patients with oropharyngeal, hypopharyngeal, and laryngeal cancer treated with CCRT at the University of Tsukuba Hospital and National Hospital Organization Mito Medical Center from April 2014 to December 2019 were retrospectively reviewed. Patients were divided into normal (<0.3 mg/dl) and elevated (≥0.3 mg/dl) CRP groups according to the CRP level after the first cycle of cisplatin. The primary endpoint was progression-free survival (PFS). RESULTS: A total of 74 patients were enrolled, of whom 36 (49%) showed elevated CRP levels after the first cycle of cisplatin. The 3-year PFS was 83.3% and 61.0% in the normal and elevated CRP groups, respectively, showing significant differences between the two groups. CONCLUSION: Elevated CRP levels after the first cycle of cisplatin is an objective predictive marker for survival in patient with head and neck squamous cell carcinoma treated with CCRT.