Several fat-soluble compounds such as cholesterol and fat-soluble vitamins have important physiological activities in the body, and their excess and/or deficiency have been reported to be closely associated with the onset and progression of several conditions such as lifestyle-related diseases. It is important to clarify not only the physiological activities but also in vivo kinetics of fat-soluble compounds to understand their in vivo activity (toxicity). This review introduces our recent (reverse) translational research in a combination of basic and clinical studies to reveal the regulatory mechanisms of in vivo behaviors of fat-soluble compounds and effects of their disruption in humans.
The roles of chondroitin sulfate (CS) and dermatan sulfate (DS) have been demonstrated in various biological events such as the construction of the extracellular matrix, tissue development, and cell signaling through interactions with extracellular matrix components, morphogens, and growth factors. Human genetic diseases, including skeletal abnormalities, connective tissue diseases, and heart defects, were reported to be caused by mutations in the genes encoding glycosyltransferases, epimerases, and sulfotransferases that are responsible for the biosynthesis of CS and DS. Glycobiological approaches revealed that mutations in CS- and DS-biosynthetic enzymes led to reductions in their enzymatic activities and in the levels of CS and DS. Furthermore, CS at the surface of tumor cells plays a key role in pulmonary metastasis. A receptor for advanced glycation end-products (RAGE) was predominantly expressed in the lung, and was identified as a functional receptor for CS chains. CS and anti-RAGE antibodies inhibited the pulmonary metastasis of not only Lewis lung carcinoma but also B16 melanoma cells. Hence, RAGE and CS are potential targets of drug discovery for pulmonary metastasis and a number of other pathological conditions involving RAGE in the pathogenetic mechanism. This review provides an overview of glycobiological studies on characterized genetic disorders caused by the impaired biosynthesis of CS, as well as DS, and on the pulmonary metastasis of Lewis lung carcinoma cells involving CS and RAGE.
Brain functions are performed by highly interconnected neurons distributed across the whole brain. Imaging of the whole brain at subcellular resolution is crucial for precise understanding of the pathological and therapeutic mechanisms of neuropsychiatric disorders; however, microscopic imaging of the whole brain remains a challenge due to the trade-offs between imaging speed and spatial resolution. To overcome this, we have recently developed block-face serial microscopy tomography (FAST), which is a novel serial-section imaging system using high-speed spinning-disk confocal microscopy. FAST enables high-throughput imaging of whole mouse brains (2.4 h per brain at maximum speed) and can be applied to nonhuman primate whole brains and human postmortem brains. Whole-brain neuronal activation mapping using FAST and Arc-dVenus mice reveals differences in brain-wide activation patterns between acute and chronic stress exposure. These applications of FAST are expected to contribute to unbiased and hypothesis-free analyses for understanding the anatomical and functional relationships of the brain underlying disease and pharmacotherapy.
Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (iPS-HLCs) are expected to be applicable to large-scale in vitro hepatotoxicity screening systems. Accordingly, methods for generating HLCs from human iPS cells have been improved over the past decade. However, although human hepatocytes have zone-specific characteristics in vivo, there is currently no technique to generate zone-specific HLCs from human iPS cells. Therefore, to generate HLCs with zone-specific properties from human iPS cells, we cultured iPS-HLCs using a parenchymal or nonparenchymal cell-conditioned medium (CM). The results showed that urea production and gluconeogenesis capacity in iPS-HLCs were increased by culturing with cholangiocyte-CM, and glutamine production and drug metabolism capacity in iPS-HLCs were increased by culturing with hepatocyte-CM. It was thus clarified that iPS-HLCs acquire zone 1 hepatocyte-like properties by culturing with cholangiocyte-CM and that iPS-HLCs acquire zone 3 hepatocyte-like properties by culturing with hepatocyte-CM. In addition, we found that WNT inhibitory factor-1 secreted from cholangiocytes, and WNT7B and WNT8B secreted from hepatocytes play important roles in the zone-specific conversion of iPS-HLCs. We hope that our findings will facilitate the application of iPS-HLCs to drug discovery research.
Target identification (target-ID) is an important step in elucidating the mechanisms of action of bioactive small molecules. In the past few decades, a number of target-ID methods have been developed. Among these, affinity labeling has been reliably used for specific modifications, as well as for the identification of weakly interacting protein targets, membrane-associated protein targets, and target-interacting proteins under native cellular conditions, which are generally difficult to achieve by conventional pull-down methods. In general, affinity labeling utilizes chemical probes composed of a bioactive small molecule, a reactive group, and a detection unit. However, the design and synthesis of highly functionalized chemical probes is often time-consuming. To address this issue, we have recently developed some simple affinity labeling methods using small fluorogenic tags, such as 4-alkoxy-7-nitro-2,1,3-benzoxadiazole (O-NBD), 2,3-dichloromaleimide (diCMI), and 4-azidophthalimide (AzPI), and successfully achieved the specific fluorescent labeling of target proteins, even in living cells. These methods should be useful for target-ID in phenotypic drug discovery.
Oxidative stress, including reactive oxygen species (ROS) generation and resulting glutathione oxidation, have been implicated in numerous aspects of cell physiology and human pathology such as cell senescence, cell differentiation, and inflammation. Significant effort has been made to establish methods of analyzing ROS levels and glutathione oxidation within a living cell. The recent development of redox-sensitive green fluorescent protein (GFP) variants enables a robust and accurate estimation of ROS level and glutathione oxidation at subcellular resolution. We created membrane-targeted versions of glutathione and hydrogen peroxide sensors by attaching palmitoylation signals to existing sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively), and demonstrated the nonuniform distribution of these oxidative elements within cytosol. In living cells, cytosolic glutathione is highly reduced, and hydrogen peroxide is barely detected. Nevertheless, near the cytoplasmic side of intracellular vesicular membranes, significant glutathione oxidation and hydrogen peroxide were successfully probed by our sensors, clearly showing the difference between various areas within cytosol. Currently, these sensors are being applied to an intestinal inflammation model which is constituted by co-culturing intestinal epithelial cells and macrophage-like inflammatory cells derived from THP-1. This review covers the current status of studies regarding the association of oxidative stress and intestinal inflammation, with a focus on the redox regulation of intracellular glutathione.
Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are powerful molecular imaging methods for examining disease-related factors in the whole body using specific imaging probes. Recently, we tried to develop molecular imaging probes that specifically visualize pathological factors associated with cancers and infectious diseases. Although survivin is highly expressed in several cancers, its expression is undetectable in non-dividing tissues. Thus, we developed several small molecular imaging probes that target survivin. These ligands not only showed high affinity for survivin protein, but also showed consistent cellular accumulation with respect to survivin expression levels, thereby indicating the feasibility of their backbones as scaffolds for tumor-specific imaging agents that target survivin. Prion diseases are fatal neurodegenerative diseases characterized by the deposition of amyloid plaques containing abnormal prion protein aggregates (PrPSc). Thus, we developed flavonoids, acridines, and benzofurans as PrPSc-imaging probes. A styrylchromone derivative ([123I]SC-OMe) appears to be a particularly promising SPECT radioligand for monitoring prion deposit levels in living brains. Gallium-68 is a positron emitter in clinical PET applications that can be produced by a 68Ge/68Ga generator without a cyclotron. Notably, we developed new adsorbents for 68Ge by introducing N-methylglucamine groups into the Sephadex series to serve as a hydrophilic polymer matrix. We also demonstrated that generator-eluted 68Ga-citrate could be used for PET imaging of infectious mouse models. Our polysaccharide-based 68Ge/68Ga generators were shown to be prospectively cost-effective production systems for 68Ga radiopharmaceuticals. This Review describes the major findings of these three studies and the future prospect of these fields.
Fullerenes, large spherical molecules composed solely of carbon atoms, have gathered much attention for practical applications that take advantage of their unique spherical structure, physical properties, and biological activities. For example, fullerene C60 can function as a photosensitizer, an antioxidant, a bioimaging agent, and as a gene or drug carrier. However, the practical use of C60 for these potential biomedical applications has been hampered by the fact that it is only sparingly soluble in water. In this review, we focus on the development of hydrophilic C60 nanoparticles, the surface of which is covered by cyclodextrin (CD), and then evaluate its biological activities. C60/CD nanoparticles were stable under physiological conditions, and even under much harsher conditions. The nanoparticles generate reactive oxygen species (ROS) under visible light irradiation. Efficient photodynamic therapy against tumor growth was achieved by the intravenous injection of C60/CD nanoparticles to tumor bearing mice, followed by photoirradiation. In addition, C60(OH)10, which is regarded as a potential candidate for use in scavenging ROS, was also prepared in the form of water soluble nanoparticles. C60(OH)10/CD nanoparticles protect the liver from injury by the suppression of oxidative stress occurring in the mitochondria, for example, by scavenging ROS such as superoxide anion radicals (O2・−), nitric oxide (NO) and peroxynitrite (ONOO−), which act as critical mediators in liver injuries. C60-based nanoparticles represent a potentially promising material for use in the treatment of cancer and oxidative stress-related diseases, and are promising as well in terms of extensive biological applications.
Intercellular lipids in the stratum corneum (SC), including ceramides (CERs), cholesterol, and fatty acids, are important for maintaining the skin barrier function. CERs in the SC have vital roles in water retention and the barrier function. A decrease in intercellular lipids, however, reduces the skin barrier function. In this study, the ability of CER precursors to increase the level of CERs in the SC and improve the skin barrier function was examined. Glucosylceramide and sphingomyelin liposomes were used as CER precursors and prepared with a thin-film method. The particle diameter and surface potential of glucosylceramide liposomes were 120.0 nm and −20 mV, while those of sphingomyelin liposomes were 153.3 nm and −11.4 mV, respectively. Transmission electron microscopy images showed that both liposomes were closed vesicles having a lamellar structure. These liposomes were applied from the SC side of a three-dimensional cultured human epidermis model, and the level of CERs in the epidermis was measured by high-performance thin-layer chromatography. In this study, the application of glucosylceramide or sphingomyelin liposomes increased the amount of CERs. In addition, the precursors of CERs were effective in improving the skin barrier function.
The skin is the largest human organ, comprising the epidermis that is composed of epithelial tissue, the dermis composed of connective tissue, and the innermost subcutaneous tissue. Generally, skin conditions are due to aging and the influence of the external environment, but empirically patients with gastrointestinal diseases are more prone to pruritus and inflammation caused by dry skin. A decrease in the skin barrier function, involving immunocompetent mast cells and oxidative stress, was noted in indomethacin-induced small intestine inflammation, dextran sodium sulfate (DSS)-induced ulcerative colitis, and azoxymethane+DSS-induced colorectal cancer. A possible correlation was found to exist between inflammatory gastrointestinal diseases and the skin, and this correlation was investigated using a rheumatoid arthritis model as representative of inflammatory diseases. Similar to previously reported results, deterioration of the skin barrier function was observed, and new information was obtained by analyzing changes in inflammatory markers in the blood and skin tissues. Understanding the underlying mechanism of decreased skin barrier function will help in establishing effective prophylaxis and treatment methods and clarify the importance of crosstalk between organs. It will also help accelerate drug development.
Severe cutaneous adverse reactions (SCARs) are important in postmarketing drug safety because SCAR patients were highest in the adverse drug reaction relief system of Japan. The SCAR symptoms of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) include high fever, severe mucosal impairment, and epidermal necrosis-induced erosions and blisters. Approximately 600 cases of SJS and 300 cases of TEN are reported annually in Japan. Many suspected drugs such as acetaminophen, lamotrigine, allopurinol, and carbamazepine have been reported. Over the last 15 years, an association between human leukocyte antigen and SJS/TEN onset has been reported with several drugs. Pathophysiological examinations in those reports revealed marked CD8-positive T cell infiltration into epidermal lesions, and the presence of cytotoxic granulysin, soluble Fas ligand, and tumor necrosis factor (TNF)-α in blister fluid. Therefore, SJS and TEN are immunological disorders that lead to epidermal necrosis and are consequently treated with the systemic administration of corticosteroids and with high-dose intravenous immunoglobulin therapy and plasma exchange in severe cases. Additionally, because the epidermal necrosis has characteristics similar to those of organ rejection after transplantation, the administration of cyclosporine, an immunosuppressant that inhibits helper T cell activation, has been attempted. Further, the administration of the TNF-α inhibitor etanercept has also been reported. This review summarizes current knowledge on the mechanisms of onset of SJS/TEN and their treatments.
Itching, or pruritus, can be defined as an unpleasant sensation that evokes the desire to scratch. Pruritus is most commonly associated with a primary skin disorder such as atopic dermatitis (AD), psoriasis, etc., and can have a major impact on the quality of life of those patients. Itch-induced scratching can further damage the skin barrier, leading to a worsening of symptoms. For that reason, it is important to manage pruritus. Topical glucocorticoids are commonly the first-line therapy in the management of AD and psoriasis patients. We found that topical glucocorticoids induce pruritus in mice under certain conditions. Topical glucocorticoids may induce pruritus in a mouse model of allergic contact dermatitis via inhibition of prostaglandin (PG)D2 production in antigen-mediated activated mast cells in the skin. Additionally, topical glucocorticoids do not induce pruritus in healthy skin. These results indicate the importance of controlling skin inflammation to a healthy level by applying sufficient quantities of glucocorticoids to avoid glucocorticoid-induced pruritus. However, topical “steroid phobia” is common in Japan, and most patients apply inadequate amounts of topical glucocorticoids for this reason. This may cause glucocorticoid-induced pruritus in patients by prolonging the skin inflammation. We conducted a survey regarding community pharmacists' instructions on the application quantity of topical glucocorticoids and found that most community pharmacists have experienced inappropriate instructions concerning this point.
To investigate medication adherence to oral antihyperglycemic agents and its associated factors in Japanese type 2 diabetic patients, a questionnaire survey was conducted in 983 adult patients receiving once-daily (QD) or twice-daily (BID) dipeptidyl peptidase-4 inhibitors (DPP-4 inhibitor) or BID biguanides (BG) as monotherapy at 502 pharmacies in Japan. The percentage of patients with good adherence (the proportion of days in which patients took all pills as prescribed in the past 7 days ≥80%) was high (≥90%) in any dosing regimen with no significant difference among the groups. The following factors were identified as associating with good adherence: the longer duration of type 2 diabetes (≥1 year) (p=0.002), “Feeling your disease gets worse if you don't take medications” (p=0.031), “Not forgetting to bring along your medicine when you leave home” (p=0.007), “Feeling anxiety on taking medications for long period of time” (p=0.042), “Neither feeling nor not feeling anxiety on taking medications for a long period of time” (p=0.004), “Never run out of your medicine because you get a refill on time” (p=0.035), and the lower MMAS-4 score (p<0.001). Subgroup analyses revealed that adherence of younger patients (<65 years) with BG (BID) was lower than those with DPP-4 inhibitor (QD) (p=0.021). Additionally, around 60% of patients currently prescribed with QD preferred QD regimen, and ≥80% patients prescribed with BID equally preferred once-weekly or QD regimen, suggesting a large discrepancy exists between their preference and the actual regimen in patients on BID.
The purpose of this study is to evaluate the collaboration system between Muscat Pharmacy and community general support center by verifying the results of tests for early detection of dementia, and the early support response for the participants with suspected dementia by the center. From December 2016 to November 2018, we conducted measurement sessions using a touch panel-type test, MSP-1100 at 23 events that included health promotion classes and local community events directed by community general support center. At a later date, a visit survey was administered by the center to the participants with suspected dementia those who received a score lower than 12, and their individual records of early support responses and follow-ups were evaluated and analyzed. During the period, 597 participants had measurements taken with the touch panel-type test. Among them, 89 (14.9% of total) participants received a score lower than 12. The contents of the support by the center for the low-scoring participants were classified into nine categories. Forty of the 89 low-scoring participants were judged to have no problems. Others were cooperatively followed up with medical consultation recommendation, receiving home care management, and/or having their information provided physicians by the Muscat pharmacy and the center. The results of our study indicate that the cooperation between community pharmacy and community general support centers could allow for the early detection of dementia and follow-ups for suspected dementia.
Tazobactam/piperacillin (TAZ/PIPC) is a useful antimicrobial agent with broad antibacterial activity. Hypokalemia is considered a rare side effect of TAZ/PIPC; however, it may occur more often than previously thought. In this study, hypokalemia frequency and risk factors were examined in 420 patients treated with TAZ/PIPC. Our results demonstrated that the hypokalemia incidence was 24.8% (grade 1-2: 18.3%, grade 3-4: 6.4%). In addition, multivariate analysis revealed that age [odds ratio 1.057, 95% confidence interval 1.024-1.090, cutoff value 80.5 years] is a risk factor. Although the “Daily dosage/creatinine clearance” was not significant in multivariate analysis, univariate analysis indicated it be to be significant, with a cutoff value of 294.9 mg/mL/min. Furthermore, a “body mass index of 19.7 kg/m2 or higher”, “serum potassium level before administration of 3.95 mEq/L or more”, and “no empirical treatment for administration purposes” appeared to prevent the hypokalemia development. Overall, the hypokalemia incidence rate in TAZ/PIPC-administered patients was as high as 20%, with patients aged >80.5 years considered a high-risk group. Thus, careful monitoring of potassium levels in patients treated with TAZ/PIPC, particularly those aged >81 years, is warranted.
Paclitaxel (PTX)-associated acute pain syndrome (P-APS) is characterized by disabling but transient arthralgia and myalgia in up to 80% of patients administered with PTX. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely administered to patients with cancer who have pain or fever, and are mainly used to manage P-APS. In this study, we investigated how P-APS appear in the patients who were administered NSAIDs prior to PTX injection. The incidence or severity and duration of P-APS in patients previously administered NSAIDs were compared to those of patients who were not administered NSAIDs. The relationship between previously administered NSAIDs and rescue administration for the relief of P-APS was also evaluated. It was revealed that the incidence and duration of P-APS were 72% and 4.67±2.30 d, respectively, in the control group and 84% and 6.19±3.30 d, respectively, in the NSAIDs group. There was no significant difference in the incidence and duration and the severity of P-APS between the two groups. Patients who were previously administered NSAIDs tended to obtain less pain relief from NSAIDs administered as rescue medications, and needed other medication. Univariate and multivariate analysis revealed no correlation between previously administered NSAIDs or patient characteristics and the incidence of P-APS. In this study, it was found that clinical condition that needs NSAIDs and previously administered NSAIDs prior to PTX injection do not affect the incidence, severity, and duration of P-APS. These results will help in educating patients about their medications and will contribute to the management of P-APS.
Paclitaxel (PTX)-associated acute pain syndrome (P-APS) is characterized by disabling but transient arthralgia and myalgia in up to 80% of patients administered with PTX. In this study, it was revealed that clinical condition that needs non-steroidal anti-inflammatory drugs (NSAIDs) and previously administered NSAIDs prior to PTX injection do not affect the incidence, severity, and duration of P-APS. These results will help in educating patients about their medications and will contribute to the management of P-APS.
It has been reported that the risk of acute kidney injury (AKI) is higher during treatment with vancomycin and piperacillin/tazobactam compared to use of vancomycin and cefepim or meropenem. We investigated the risk of AKI in patients receiving vancomycin and piperacillin/tazobactam versus those receiving vancomycin and meropenem or doripenem. The subjects were patients over 18 years old who received either vancomycin and piperacillin/tazobactam (V+P/T therapy) or vancomycin and carbapenems (meropenem or doripenem) (V+C therapy) for at least 48 h between 1 May 2013 and 28 February 2019. The primary endpoint was the incidence of AKI in patients receiving V+P/T or V+C therapy, while the secondary outcome was the timing of AKI in each group. The incidence of AKI was 33.3% (9/27) in patients receiving V+P/T therapy versus 9.1% (5/55) in those receiving V+C therapy, and its incidence was significantly higher with the former regimen (χ2=5.90, p=0.015). Multiple logistic regression analysis confirmed that V+P/T therapy was associated with an increased risk of AKI compared to V+C therapy (adjusted odds ratio: 5.05, 95% confidence interval: 1.46-17.5, p=0.01). The time to onset of AKI after initiation of treatment was not significantly different between patients receiving V+T/P or V+C therapy [median (interquartile range): 4 d (2-6 d) versus 7 d (3-10 d); p=0.282]. V+P/T therapy was associated with a significantly higher incidence of AKI than alternative regimens, suggesting that it should be avoided. When broad spectrum antibacterial therapy is required, V+C therapy should be considered instead.