Ethenzamide (ETZ), an antipyretic analgesic categorized as a non-steroidal anti-inflammatory drug (NSAID), is widely used as an OTC drug in combination with other NSAIDs. However, its site of action and mechanism underlying its analgesic action have not yet been fully elucidated. In this study, we performed in vitro pharmacological assays to identify the mechanism underlying the analgesic action of ETZ, and also conducted the rat formalin test to investigate its analgesic effect and site of action. Of the 85 receptors, ion channels, transporters and enzymes tested, we found that ETZ binds to the 5-hydroxytryptamine (5HT)_2B receptor in concentration-dependent manner with modest inhibitory effects on monoamine oxidase-A and transient potential vanilloid 1 channel. The 5HT_2B receptor antagonist activity of ETZ was also confirmed in a cellular functional assay. Furthermore, the drug exerted no inhibitory effects on cycrooxygenase-1 and -2. In the rat formalin test, oral administration of ETZ significantly reduced the nociceptive responses of the second phase and also the number of c-Fos-expressing cells in the spinal dorsal horn, in a dose-dependent manner. Moreover, intrathecal administration of ETZ significantly reduced the nociceptive responses. These results suggest that the analgesic effect of ETZ is exerted at least in the spinal cord, and the effect would be attributed to multiple mechanisms of action including 5HT_2B receptor blockade.

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.

The objective of this study was to evaluate the influence of cancer cachexia on pain control in cancer patients receiving a transdermal fentanyl patch (FP) and to investigate whether dry skin was a factor related to cancer cachexia and uncontrolled pain. We retrospectively reviewed the medical records of 77 patients receiving FP treatment for the first time, who were classified into cancer cachexia and non-cancer-cachexia groups, according to European Palliative Care Research Collaborative criteria. On day 7 after FP administration, the mean FP dose and morphine equivalent dose (MED) in the cancer cachexia group were significantly higher than in the non-cancer-cachexia group. Additionally, in the cancer cachexia group, there was a significantly larger degree of variation in pain intensity over 7 d than in the non-cachexia group. In patients who were switched from FP to morphine injection, the mean pain intensity and MED on day 3 after morphine injection were significantly lower than those immediately before morphine injection. Subsequently, to investigate whether dry skin was involved in poor pain control in the cancer cachexia group, transepidermal water loss (TEWL) was compared between 15 additional patients classified into cancer cachexia and non-cancer cachexia groups; the mean TEWL in the cancer cachexia group was found to be significantly lower. Our data suggest that cancer cachexia may be a risk factor for poor pain control in patients receiving FP treatment, and that uncontrolled pain in FP treatment may be caused by the inhibition of fentanyl transdermal absorption due to dry skin.

Bone loss and bone-related disease are associated with the deregulation of osteoclast function, and therefore agents that affect osteoclastogenesis have attracted attention. The purpose of the present study was to discover modified kavalactone analogs as potential anti-osteoclastogenic agents. We assessed the effect of 26 analogs on osteoclast differentiation in vitro. The most potent compound, (E)-6-(2-fluorostyryl)-4-methoxy-2H-pyran-2-one (22), suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenic differentiation of RAW264 cells with IC₅₀ values of 4.3 µM. A partial structure–activity relationship study revealed the importance of fluorine and its position within the 5,6-dehydrokawain skeleton. The results of a pit formation assay suggested that compound 22 prevents osteoclastic bone resorption by inhibiting osteoclastogenesis. Moreover, compound 22 downregulated mRNA expression levels of RANKL-induced nuclear factor of activated T cells c1 (NFATc1) and osteoclastogenesis-related genes. These results suggest that (E)-6-(2-fluorostyryl)-4-methoxy-2H-pyran-2-one scaffold could lead to the identification of new anti-resorptive agents.

We previously showed that adhesive aggregates were formed when levofloxacin hydrate tablets and lansoprazole orally disintegrating (OD) tablets were suspended in water in the clinical context. In this study, we have clarified the factors causing aggregate formation, focusing on the role of pharmaceutical additives and electrostatic interaction. Co-suspension of enteric-coated proton pump inhibitor (PPI) esomeprazole magnesium hydrate with levofloxacin resulted in aggregate formation, whereas the non-enteric-coated PPI vonoprazan fumarate did not. A comparison of pharmaceutical additive in the two PPIs highlighted polysorbate 80 and methacrylic acid copolymer LD as candidates causing aggregation. When these pharmaceutical additives were added to levofloxacin, only methacrylic acid copolymer LD induced aggregate formation. Since levofloxacin is zwitterionic, we examined another zwitterionic ingredient, ampicillin sodium, and found that it also formed aggregates with methacrylic acid copolymer LD, while benzylpenicillin sodium, which is not zwitterionic, did not form aggregates. When we next examined a series of zwitterionic quinolone antimicrobial drugs, we found that ofloxacin, which is highly soluble, formed aggregates with lansoprazole OD tablets, whereas poorly soluble quinolone antimicrobial drugs did not form aggregates. Further, although cefepime hydrochloride and cephalexin did not form aggregates with methacrylic acid copolymer LD in tap water, aggregates were formed when a suspension of cefepime hydrochloride or cephalexin with methacrylic acid copolymer LD was adjusted to pH 7.0. Our results indicate that electrostatic interaction between zwitterionic ingredients and methacrylic acid copolymer LD can result in aggregate formation under conditions where the drug and methacrylic acid copolymer LD are both sufficiently soluble.

Sesamin is a major lignan in sesame seeds, and a recent meta-analysis of controlled trials indicated that sesamin intake decreases blood pressure. The antihypertensive effect of sesamin has been suggested to be due to sesamin-mediated suppression of 20-hydroxyeicosatetraenoic acid production catalyzed by CYP4F2. However, the detailed mechanism underlying inhibition of CYP4F2 function by sesamin remains unclear. In this study, the effects of sesamin on catalytic activity of CYP4F2 were investigated in vitro. Sesamin inhibited luciferin-4F2/3 O-dealkylase activity of recombinant human CYP4F2 with an IC₅₀ value of 0.381 µM. When preincubated in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) for 20 min, sesamin potentiated the inhibition of CYP4F2 activity. Moreover, kinetic analysis of the inactivation revealed that sesamin showed a preincubation time- and concentration-dependent inhibition of CYP4F2 activity yielding a maximal inactivation rate constant (k_inact) value of 0.354 min⁻¹ and half-maximal inhibitory concentration (K_I) value of 1.12 µM. The inactivation of CYP4F2 by sesamin required NADPH. These results indicated that sesamin is a mechanism-based inactivator of human CYP4F2.

5-Aminosalicylic acid (5-ASA) is used as first line therapy for symptom remission and maintenance of inflammatory bowel disease (IBD). Because 5-ASA is well absorbed from the small intestine when orally administered, several 5-ASA formulations for selective delivery to the colon have been developed and used in clinical practice. However, its delivery efficiency to local inflamed colonic sites remains low. Intestinal H⁺-coupled oligopeptide transporter 1 (PEPT1) expression in the colon is low, whereas its expression is induced in the colon under chronic inflammation conditions, such as IBD. Therefore, we considered that PEPT1 would be a target transporter to improve 5-ASA delivery efficiency to local colonic lesions. We evaluated the transport characteristics of dipeptide-like 5-ASA derivatives, which were coupling glycine (Gly), lysine, glutamic acid (Glu), valine (Val) and tyrosine to amino or carboxyl group of 5-ASA, in Caco-2 cells. [³H]Glycylsarcosine (Gly-Sar) uptake into Caco-2 cells was inhibited by all 5-ASA derivatives. In addition, 5-ASA derivatives (Gly-ASA, Glu-ASA and Val-ASA), which were coupled by glycine, glutamic acid and valine to amino group of 5-ASA, were taken up in a pH- and concentration-dependent manner and their uptake was inhibited by excess Gly-Sar. Two-electrode voltage-clamp experiment using human PEPT1 expressing Xenopus oocytes showed that Gly-ASA, Glu-ASA and Val-ASA induced marked currents at pH 6.0. Taken together, these results showed that these 5-ASA derivatives are transportable substrates for PEPT1.

Resveratrol (Res) is a natural active antioxidant that is effective in relieving inflammatory bowel disease (IBD). However, the specific mechanism for its function is unknown. In our study, dextran sodium sulfate (DSS)-induced mouse IBD disease model was constructed. All mice were randomly divided into three groups. The treatment effects of resveratrol on IBD were evaluated by observing the body weight, fecal traits, colon/spleen gross appearance, tissue hematoxylin–eosin (H&E)/immunohistochemistry (IHC) and inflammatory factors. The expression of small ubiquitin-like modifier protein 1 (SUMO1) and its Wnt/β-catenin pathway-related genes was analyzed by IHC, Western blot, Real-time PCR (RT-PCR) and Immunofluorescence. The outcome indicated that resveratrol treatment significantly relieved the symptoms of IBD. The expression level of anti-inflammatory cytokines was increased while that of pro-inflammatory cytokines was decreased in both colon and spleen tissues of resveratrol-treated mice. SUMO1 expression and Wnt/β-catenin pathway were suppressed in colon and spleen tissues of IBD mice treated with resveratrol. In addition, we provided evidence that resveratrol inhibited SUMO1 and β-catenin expression and their nuclear localization in human colonic epithelial cell line (FHC). Moreover, we found that SUMO1 and β-catenin had higher expression levels in colorectal cancer patients than in health and colitis patients. In conclusions, resveratrol alleviates DSS-induced IBD by modulating SUMO1 through Wnt/β-catenin pathway.

Edoxaban is used to prevent and treat stroke or systemic embolism such as venous thromboembolism. Although bleeding is the most common complication of anticoagulants, only a few studies have addressed the safety of direct oral anticoagulants in East Asian patients. In this study, we investigated the risk factors for bleeding in Japanese patients receiving edoxaban. A retrospective review of the records of 198 patients who received 30 mg/d edoxaban in our hospital between April 2015 and March 2017 was performed. Subsequently, these patients were followed up to 1 year. Seven (3.5%) and 22 (11.1%) patients developed major bleeding and clinically relevant bleeding, respectively. In the univariate Cox regression analyses, low baseline hemoglobin levels (p = 0.002) and low baseline creatinine clearance (p = 0.020) were significantly associated with major bleeding. Multivariate Cox regression analysis revealed that a low baseline hemoglobin level was a significant risk factor for major bleeding and clinically relevant bleeding [hazard ratio 1.67 per 1 g/dL decrease (95% confidence interval 1.14–2.56, p = 0.008) and hazard ratio 1.31 per 1 g/dL decrease (95% confidence interval 1.06–1.62, p = 0.013), respectively]. Baseline hemoglobin level in quartiles also showed a quartile-dependent decrease in major bleeding and clinically relevant bleeding event. These results suggest that low baseline hemoglobin level is a significant risk factor for both major bleeding and clinically relevant bleeding in Japanese patients receiving edoxaban. Thus, these patients should be carefully monitored.

It is therapeutically important to elucidate the factors involved in the radiation resistance of tumors. We previously showed that ATP is released from mouse melanoma B16 cells in response to γ-irradiation, but the role of adenosine, a metabolite of ATP, is still unclear. Here, we show that the adenosine A2B receptor is involved in DNA damage repair and radioresistance in mouse melanoma B16 cells. The DNA damage response after γ-irradiation was attenuated by pretreatment with A2B receptor antagonists, such as PSB603, while it was enhanced by pretreatment with A2B receptor agonists, such as BAY60-6583. γ-Irradiation decreased the cell survival rate, and pretreatment with PSB603 further reduced the survival rate. On the other hand, pretreatment with BAY60-6583 increased the cell survival rate after irradiation. The DNA damage response and the cell survival rate after γ-irradiation were both decreased in A2B-knockdown cells. In vivo experiments in mice confirmed that tumor growth was suppressed and delayed in the irradiated group pretreated with PSB603, compared with the irradiation-alone group. Our results indicate that adenosine A2B receptor contributes to radioresistance, and could be a new target for the development of agents to increase the efficacy of radiotherapy.

CD81 is a highly conserved four-transmembrane protein in mammals and widely expressed on many tissues. It belongs to the tetraspanin family and forms complexes with various cell surface membrane proteins. It also functions in cell migration and B-cell activation, which is induced by CD81 complexing with CD19, CD21 and the B-cell receptor. Thus, CD81 is thought to play a key role in regulating cell function and fate. However, little is known about the degradation mechanism of CD81. Here we found that CD81 on the plasma membrane is degraded by the lysosome pathway via endocytosis. The expression levels of CD81 in HEK293T cells treated with a proteasome inhibitor (lactacystin) and lysosome inhibitors (chloroquine and bafilomycin A1) were analyzed by flow cytometry. The expression of CD81 on the cell surface was increased by the lysosome inhibitors, but not lactacystin. A pulldown assay revealed that CD81 was conjugated with a K63- and K29-linked poly-ubiquitin chain before its degradation, and the poly-ubiquitination site was Lys8 at the N-terminal intracellular domain of CD81. Furthermore, mutant CD81, in which Lys8 was substituted with alanine (Ala), extended the CD81 half-life compared with wildtype. CD81 was mainly localized on the plasma membrane in normal cells, but also co-localized with lysosomal LAMP1 and early endosomal EEA1 in chloroquine-treated cells. Furthermore, a clathrin-mediated endocytosis inhibitor, chlorpromazine, stabilized CD81 expression on the cell surface. Hence, we demonstrated that CD81 is internalized by clathrin-mediated endocytosis and subsequently degraded via a lysosome pathway requiring the K63- and K29-linked poly-ubiquitination of CD81.

Acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) is the most popular analgesic/antipyretic agent in the world. APAP has been regarded as a safer drug compared with non-steroidal anti-inflammatory drugs (NSAIDs) particularly in terms of lower risks of renal dysfunction, gastrointestinal injury, and asthma/bronchospasm induction, even in high-risk patients such as the elderly, children, and pregnant women. On the other hand, the recent increasing use of APAP has raised concerns about its toxicity. In this article, we review recent pharmacological and toxicological findings about APAP from basic, clinical, and epidemiological studies, including spontaneous drug adverse events reporting system, especially focusing on drug-induced asthma and pre-and post-natal closure of ductus arteriosus. Hepatotoxicity is the greatest fault of APAP and the most frequent cause of drug-induced acute liver failure in Western countries. However, its precise mechanism remains unclear and no effective cure beyond N-acetylcysteine has been developed. Recent animal and cellular studies have demonstrated that some cellular events, such as c-jun N-terminal kinase (JNK) pathway activation, endoplasmic reticulum (ER) stress, and mitochondrial oxidative stress may play important roles in the development of hepatitis. Herein, the molecular mechanisms of APAP hepatotoxicity are summarized. We also discuss the not-so-familiar “dark side” of APAP as an otherwise safe analgesic/antipyretic drug.

Thiamine hydrochloride has been suggested as a natural, safe yet effective alternative for chemical insect repellents. However, there is a demand for a reassessment of the minimum required dose that is sufficient to perform a topical repellency on the human skin. Therefore, the purpose of the current work is to establish a dose–response curve from which the effective dose (ED) is calculated. A series of increasing concentrations of thiamine hydrochloride were applied to the forearm of adult volunteers, the number of bites was counted and the percent repellency calculated accordingly. Data of percent repellency were converted to probit values which were plotted against log doses. A linear relation was obtained from the dose–response curve with an r² = 0.958. Statistical validation of the equation was tested through linear regression analysis, where the slope and intercept were found significant from zero. No significant difference was shown between observed and expected responses (p > 0.05). ED 50 and 99.9% were computed from the linear equation and found to be 4.57 and 344 mg, respectively. This finding can be supported by future works in which a proper formulation of thiamine hydrochloride in the respective doses would be presented. One can get prolonged safe protection against insect bites.

Colony stimulating factor 1 (CSF1) receptor (CSF1R) is a receptor protein-tyrosine kinase specifically expressed in monocyte-lineage cells, such as monocytes and macrophages. In this study, we characterized the pharmacological properties of an azetidine compound, JTE-952 ((2S)-3-{[2-({3-[4-(4-cyclopropylbenzyloxy)-3-methoxyphenyl]azetidine-1-yl}carbonyl)pyridin-4-yl]methoxy}propane-1,2-diol), which is a novel CSF1R tyrosine kinase inhibitor. JTE-952 potently inhibited human CSF1R kinase activity, with a half maximal inhibitory concentration of 11.1 nmol/L, and inhibited the phosphorylation of CSF1R in human macrophages and the CSF1-induced proliferation of human macrophages. It also inhibited human tropomyosin-related kinase A activity, but only at concentrations 200-fold higher than that required to inhibit the activity of CSF1R in inducing the proliferation of human macrophages. JTE-952 displayed no marked inhibitory activity against other kinases. JTE-952 potently inhibited lipopolysaccharide-induced proinflammatory cytokine production by human macrophages and in whole blood. JTE-952 (≥3 mg/kg given orally) also significantly attenuated the CSF1-induced priming of lipopolysaccharide-induced tumor necrosis factor-alpha production in mice and arthritis severity in a mouse model of collagen-induced arthritis. Taken together, these results indicate that JTE-952 is an orally available compound with potent and specific inhibitory activity against CSF1R, both in vitro and in vivo. JTE-952 is a potentially clinically useful agent for various human inflammatory diseases, including rheumatoid arthritis.

Oxaliplatin is a first-line clinical drug in cancer treatment and its side effects of peripheral neuropathic pain have also attracted much attention. Neuroinflammation induced by oxidative stress-mediated activation of nuclear factor-kappa B (NF-κB) plays an important role in the course. Current studies have shown that curcumin has various biological activities like antioxidant, anti-inflammatory, antitumor and so on, while few studies were conducted about its role in oxaliplatin-induced peripheral neuropathic pain. The aim of this study is to verify the mechanism of curcumin alleviating oxaliplatin-induced peripheral neuropathic pain. Intraperitoneal injection with oxaliplatin (4 mg/kg body weight) was given to the rats twice a week and last for four weeks to establish the model rats. Gavage administration of curcumin (12.5, 25, and 50 mg/kg body weight, respectively) was conducted for consecutive 28 d to explore the effects and potential mechanism. Our results showed that curcumin administration could increase mechanical withdrawal threshold and decrease the paw-withdrawal times of cold allodynia significantly; meanwhile, motor nerve conduction velocity (MNCV) and sense nerve conduction velocity (SNCV) were both increased and the injured neurons of the spinal cord were repaired. In addition, curcumin administration increased superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and reduced malondialdehyde (MDA). Moreover, the curcumin operation inhibited the activated of NF-κB and level of inflammatory factors like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). In conclusion, these findings suggested that curcumin could alleviate oxaliplatin-induced peripheral neuropathic pain; the mechanism might be inhibiting oxidative stress-mediated activation of NF-κB and mitigating neuroinflammation.

The Hippo intracellular signaling pathway plays a pivotal role in cell fate determination. Although previous studies have identified many components of the Hippo pathway, the whole picture of the Hippo network is just beginning to be delineated. Recent discoveries in the past decade have shed light on a newly discovered signaling network where the Hippo pathway interplays with several types of non-coding RNAs, including microRNAs, long non-coding RNAs and circular RNAs, to mediate diverse biological processes. Those non-coding RNAs communicate with each other to maintain cellular homeostasis. In this review article, we summarize the current and emerging understanding of the roles of non-coding RNAs in the regulation of and by the Hippo pathway.

The aim of the present study was to investigate the “chronotoxicity” of streptomycin (SM) in relation to its circadian periodicity. Male ICR mice were injected intraperitoneally with SM (780 mg/kg, one shot) one of six time points throughout the day. Mortality was monitored until 14 d after the injection and clearly differed depending on the timing of the injection (i.e., mice were more sensitive to injection during the dark phase). Moreover, when mice were administered with non-lethal doses of SM (550 mg/kg, every 24 h for 3 d, in the light phase or dark phase), the levels of nephrotoxicity indicators (blood urea nitrogen and renal levels of malondialdehyde and cyclooxygenase-2) were significantly increased by the injection in the dark phase, but not in the light phase. These results suggested that SM showed clear chronotoxicity. Our current data indicated that chronotoxicology may provide valuable information on the importance of injection timings for evaluations of toxicity and undesirable side effects.