Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 40, Issue 9
Displaying 1-39 of 39 articles from this issue
Review
  • Masayuki Kaneko, Kazunori Imaizumi, Atsushi Saito, Soshi Kanemoto, Rie ...
    2017 Volume 40 Issue 9 Pages 1337-1343
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Secretory and membrane proteins are synthesized in ribosomes, then mature in the endoplasmic reticulum (ER), but if ER function is impaired, immature defective proteins accumulate in the ER. This situation is called ER stress: in response, a defensive mechanism called the unfolded protein response (UPR) is activated in cells to reduce the defective proteins. During the UPR, the ER transmembrane sensor molecules inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and RNA-dependent protein kinase (PKR)-like ER kinase (PERK) are activated, stress signals are transduced to the outside of the ER, and various cell responses, including gene induction, occur. In ER-associated degradation (ERAD), one type of UPR, defective proteins are eventually expelled from the ER and degraded in the cytoplasm through the ubiquitin proteasome system. Since ER stress has been reported to have relationships with neurodegenerative diseases, diabetes, metabolic syndromes, and cancer, it is the focus of increased attention from the perspectives of elucidating pathogenic mechanisms, and in the development of therapeutics.

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Regular Articles
  • Momoko Kawakami, Sachiko Takenoshita-Nakaya, Yuko Takeba, Yuki Nishimu ...
    2017 Volume 40 Issue 9 Pages 1344-1351
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 14, 2017
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    CYP2D6 plays an important role in the metabolism of many drugs such as opioids and antidepressants. Polymorphisms of the CYP2D6 gene are widely observed in the Japanese population, and can affect the first-pass metabolism of orally administered drugs. Several CYP enzymes have been identified in the small intestine of Caucasians, but intestinal CYP enzymes have not been reported in the Japanese population, except for CYP3A4 and CYP2C19. In this study, we evaluated the CYP2D6 metabolic capacity by measurement of CYP2D6 mRNA and protein levels and activity in the small intestine of Japanese individuals. Normal jejunal tissues were obtained from 31 patients who had undergone pancreaticoduodenectomy, and the CYP2D6*10 variant was identified in these tissues. CYP2D6 mRNA and CYP2D6 protein levels were analyzed using real-time RT-PCR and Western blotting, respectively. Bufuralol 1′-hydroxylation, a marker of CYP2D6 activity, was analyzed using HPLC. Frequencies of the CYP2D6*1/*1, *1/*10, and *10/*10 genotypes in the jejunal tissue were 29.0% (n=9), 35.5% (n=11), and 35.5% (n=11), respectively. CYP2D6 protein and activity levels did not differ significantly between the genotypes. A positive correlation was found between CYP2D6 protein and activity levels. Furthermore, CYP2D6 protein levels and activity in the small intestine were significantly lower than those in the liver. These findings suggest that the metabolic capacity of CYP2D6 in the small intestine of the Japanese population has a relatively small effect on drug metabolism.

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  • Yan Wang, Zhi He, Xianhui Li
    2017 Volume 40 Issue 9 Pages 1352-1360
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Rapamycin (Rap) has been demonstrated to affect lipid metabolism through stimulating lipolysis, inhibiting de novo lipogenesis and reducing adiposity. In the present study, we investigated rapamycin exposure’s influence on adipose tissue browning in high-fat diet-induced fatty mice. Four-week old C57BL/6J mice were fed normal chow or high-fat diet for a period of 6 weeks and then divided into three groups: (1) Nor group: mice fed with normal chow; (2) high fat diet (HFD) group: fatty mice fed with high-fat diet; (3) Rap group: high-fat diet-fed fatty mice treated intragastrically with rapamycin at a dose of 2.5 mg/kg per day for 5 weeks. Body weights and food intakes of the mice were recorded weekly. At the end of the study, blood samples were collected for glucose, lipid and insulin evaluations. Adipose tissues were weighed and lipid contents were monitored. Moreover, real-time PCR and Western blotting were applied to detect the expression levels of beige and brown fat marker genes in white adipose tissue (WAT) and brown adipose tissue (BAT). Our data demonstrated that Rap exposure significantly ameliorated metabolic defects including hyperglycaemia, dyslipidaemia and insulin resistance in the fatty mice. Furthermore, Rap treatment led to decreased tissue weights and lipid contents both in WAT and BAT. Remarkably, expression levels of BAT marker genes including uncoupling protein-1 (UCP-1), cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDEA), PR-domain containing protein-16 (PRDM16) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) were significantly down-regulated in Rap-treated fatty mice. This report demonstrates Rap exposure is capable of inhibiting adipose tissue browning in high-fat diet-induced fatty mice, and provides evidence for deeper understanding of Rap’s influence on lipid homeostasis.

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  • Hokuto Namba, Yuki Nishimura, Norimitsu Kurata, Mariko Iwase, Takahito ...
    2017 Volume 40 Issue 9 Pages 1361-1365
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    There have been few reports concerning to the drug–drug interactions (DDIs) with OTC drugs although an increase in the use of OTC drugs in recent years. This current study was conducted to clarify the DDIs through CYP3A inhibition by oxethazaine (OXZ), an antacid available as an OTC drug. Midazolam (MDZ) was used as a probe drug for CYP3A activity. In an in vivo study, a single oral dose of OXZ (50 mg/kg) was administered to rats 30, 60, or 120 min before oral MDZ administration (15 mg/kg). Serum concentrations of MDZ were analyzed by HPLC, and its pharmacokinetic parameters were compared with a water-treated control group. The inhibitory effect of OXZ on MDZ 1′-hydroxylation (MDZ 1′-OH) activity was investigated in vitro using rat liver and intestinal microsomes. Pretreatment of OXZ 120 min before MDZ administration significantly increased the area under the serum concentration–time curve (AUC0–∞) of MDZ six-fold compared to the control group without a change in elimination half-life (t1/2). In contrast, OXZ pretreatment 30 or 60 min before MDZ administration did not show any remarkable change in MDZ pharmacokinetic parameters. The in vitro study showed that OXZ inhibited MDZ 1′-OH activity in a concentration-dependent manner both in liver and intestinal microsomes. These results suggested that OXZ increases serum MDZ concentration presumably by the inhibition of liver and/or intestinal CYP3A activity. OXZ was predicted to cause the DDIs mediated by CYP3A inhibition, although this effect depended on the dose interval.

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  • Ramakanta Lamichhane, Se-Gun Kim, Sona Kang, Kyung-Hee Lee, Prakash Ra ...
    2017 Volume 40 Issue 9 Pages 1366-1373
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 03, 2017
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    Sulfuretin is a natural flavonoid found in the plant Rhus verniciflua STOKES. The plant has been traditionally used as medicinal agent for antiviral, cathartic, diaphoretic, anti-rheumatic and sedative activities in East Asia. In this study we isolated and identified sulfuretin from R. verniciflua and investigated its anti-adipogenic activity against 3T3-L1 preadipocytes cells. We evaluated the effects of sulfuretin on the adipogenic transcription factors like peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), Fabp4, adiponectin and zinc fingerprint protein (Zfp) 521 by gene expression (real-time QPCR) and Western blot analysis. Sulfuretin treatment at Day 0 and 2 showed significant reduction of lipid production in 3T3-L1 cells in concentration dependent manner. Gene expression analysis (real-time PCR) revealed that sulfuretin inhibited the both major adipogenic factors (C/EBPα, C/EBPβ and PPARγ) and minor adipogenic factors (sterol regulatory element-binding protein (SREBP1c), adiponectin, FAS, Fabp4, Zfp423, and Ebf1). Western blot analysis showed the increased expression of β-catenin and suppression of PPARγ after sulfuretin treatment. Overall, sulfuretin is a natural flavonoid having potent anti-adipogenic activity through the suppression of major adipogenic factors C/EBPα, C/EBPβ and PPARγ, which initiate adipogenesis.

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  • Qing Liu, Qingrui Li, Ting Han, Tingting Hu, Xuemei Zhang, Junhua Hu, ...
    2017 Volume 40 Issue 9 Pages 1374-1380
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 24, 2017
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    Currently, all commercial available nebulized salbutamol in China is in its racemic form. It is known that only R-salbutamol (eutomer) has therapeutic effects, while S-salbutamol (distomer) may exacerbate asthma after chronic use. Therefore, it is an unmet clinical need to develop R-salbutamol as a nebulized product that is more convenient for young and old patients. In our study, a stable aerosol solution of R-salbutamol sulfate was established, and its antiasthmatic effects were confirmed. The decomposition rate and racemization effect of the R-salbutamol sulfate solution were evaluated over a pH range from 1 to 10 (except pH=7, 8) at 60°C. The aerodynamic particle size of the R-salbutamol sulfate solution and commercial RS-salbutamol sulfate solution were both tested in vitro by Next-Generation Impactor (NGI) in 5°C. Laser diffractometer was used to characterize the droplet-size distribution (DSD) of both solutions. We next conducted an in vivo animal study to document the antiasthmatic effect of R-salbutamol aerosol sulfate solution and determine the relationship to RS-salbutamol. The results showed that the R-salbutamol sulfate solution was more stable at pH 6. In vitro comparison studies indicated that there was no distribution difference between R-salbutamol sulfate solution and the commercial RS-salbutamol solution. The animal results showed that R-salbutamol was more potent than RS-salbutamol against the same dose of histamine challenge. Unlike commercial RS-salbutamol, which was acidified to a pH of 3.5 to extend bench life but may cause bronchoconstriction in asthmatic patients, the neutralized R-salbutamol solution was more suitable for clinic use.

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  • Jonghwan Jegal, Ki Wung Chung, Hae Young Chung, Eun Ju Jeong, Min Hye ...
    2017 Volume 40 Issue 9 Pages 1381-1388
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    In European folk medicine, the fruits of Juniperus communis are used in the treatment of skin-related disorders such as skin infection, itching, and psoriasis. Previously, we reported that the EtOAc fraction of J. communis (EAJC) contained tyrosinase inhibition properties in vitro non-cellular experiment. The aim of this study was to evaluate anti-melanogenic effect of standardized EAJC on a hyperpigmentation animal model. Therapeutic effects of EAJC toward skin hyperpigmentation were confirmed by both in vivo experiment and in vitro cell-based assay. Skin depigmenting effect was detected by topical treatment of EAJC for 11 d to HRM-2 melanin-possessing hairless mice. Histologic findings including significantly decreased melanin depositions could be observed in dorsal skin samples of EAJC-treated group. In addition, the EAJC (50 µg/mL) attenuated melanin production through down-regulation of tyrosinase activity and protein expression in B16 murine melanoma cells. According to the phytochemical analysis, EAJC was found to contain hypolaetin-7-O-β-D-xylopyranoside and isoscutellarein-7-O-β-D-xylopyranoside as main components. Hypolaetin-7-O-β-D-xylopyranoside was responsible for the skin-lightening effect of EAJC by reducing the number of melanocytes in dorsal skins of HRM-2 mice. The present study provided direct experimental evidence for skin-lightening effect of EAJC in UV-irradiated hairless mouse model. Therapeutic attempts with the J. communis might be useful in the management of skin pigmentation-related diseases.

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  • Shinji Oshima, Takako Enjuji, Akio Negishi, Hayato Akimoto, Kousuke Oh ...
    2017 Volume 40 Issue 9 Pages 1389-1398
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 03, 2017
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    In order to avoid adverse drug reactions (ADRs), pharmacists are reconstructing ADR-related information based on various types of data gathered from patients, and then providing this information to patients. Among the data provided to patients is the time-to-onset of ADRs after starting the medication (i.e., ADR onset timing information). However, a quantitative evaluation of the effect of onset timing information offered by pharmacists on the probability of ADRs occurring in patients receiving this information has not been reported to date. In this study, we extracted 40 ADR–drug combinations from the data in the Japanese Adverse Drug Event Report database. By applying Bayes’ theorem to these combinations, we quantitatively evaluated the usefulness of onset timing information as an ADR detection predictor. As a result, when information on days after taking medication was added, 54 ADR–drug combinations showed a likelihood ratio (LR) in excess of 2. In particular, when considering the ADR–drug combination of anaphylactic shock with levofloxacin or loxoprofen, the number of days elapsed between start of medication and the onset of the ADR was 0, which corresponded to increased likelihood ratios (LRs) of 138.7301 or 58.4516, respectively. When information from 1–7 d after starting medication was added to the combination of liver disorder and acetaminophen, the LR was 11.1775. The results of this study indicate the clinical usefulness of offering information on ADR onset timing.

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  • Caina Li, Miaomiao Yang, Guojiang Hou, Shuainan Liu, Yi Huan, Dongan Y ...
    2017 Volume 40 Issue 9 Pages 1399-1408
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 17, 2017
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    GW002 is a recombinant protein engineered by fusing the C-terminal region of human glucagon-like peptide-1 (GLP-1) to the N-terminal region of human serum albumin (HSA) with a peptide linker. This study aims to evaluate its anti-diabetic effects both in vitro and in vivo. The GLP-1 receptor-dependent luciferase reporter plasmid was transiently transfected in NIT-1 cells to calculate the half-maximal concentration (EC50) for GLP-1 receptor activation, and normal ICR mice and diabetic KKAy mice were acutely injected with GW002 (1, 3, 9 mg/kg) subcutaneously to evaluate the hypoglycemic action, while the diabetic KKAy and db/db mice were treated with GW002 once daily for 7 weeks to evaluate the effects on glucose metabolism. The results showed that GW002 activated GLP-1 receptor in NIT-1 cells with higher EC50 versus exendin-4 (46.7 vs. 7.89 nM), and single subcutaneous injection of GW002 at doses of 1, 3 and 9 mg/kg efficiently restrained the glycemia variation after oral glucose loading in ICR mice for at least 4 d, as well as reducing the non-fasting blood glucose in KKAy mice for about 2 d, while repeated injections of GW002 significantly improved abnormal glycaemia, hemoglobin (Hb)A1c levels, oral glucose intolerance and β-cell function in diabetic db/db mice. These results suggested that GW002 showed prolonged hypoglycemic action by activating its cognate receptor and provided efficient control of glucose metabolism. Thus GW002 may be a potential treatment for the management of type 2 diabetes.

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  • Yuka Kato, Yuji Mukai, Anders Rane, Nobuo Inotsume, Takaki Toda
    2017 Volume 40 Issue 9 Pages 1409-1415
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Epoxyeicosatorienoic acids (EETs) are generated from arachidonic acid (AA) by CYPs. EETs comprise four regioisomers (14,15-, 11,12-, 8,9-, and 5,6-EET). EETs show potent physiological effects, including vasodilation, anti-inflammation, myocardial preconditioning, and anti-platelet aggregation effects. We recently demonstrated that telmisartan, one of angiotensin II receptor blockers, inhibits AA metabolism by CYP enzymes, including CYP2C8, CYP2C9, and CYP2J2. We conducted studies of AA metabolism using recombinant CYP enzymes to estimate the inhibition constant and the type of inhibition by telmisartan of CYP2C9 and CYP2C8. The contribution ratio (CR) of each CYP enzyme was investigated using human liver microsomes. Dixon and Lineweaver–Burk plots indicated that telmisartan is a mixed inhibitor of both CYP2C9 and CYP2C8; telmisartan did not show a time-dependent inhibition toward these CYP enzymes. Based on the CRs, both CYP2C9 and CYP2C8 are the key enzymes in the metabolism of AA in the human liver. Uptake of telmisartan in the liver by organic anion transporting polypeptide (OATP) 1B3 and the non-linear metabolism in gastrointestinal tract augment the potential of the drug to inhibit the CYP enzymes in the liver.

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  • Jihyun Youm, Hyunyong Lee, Hwan Bong Chang, Jihyun Jeon, Mi Hee Yoon, ...
    2017 Volume 40 Issue 9 Pages 1416-1422
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    DW2008 is an anhydrous ethanol extract of Justicia procumbens produced by Dong-Wha Pharmaceutical, Inc., Co. as a candidate anti-asthmatic drug. In this study, DW2008 selectively reduced T helper 2 (Th2) cytokines in mouse splenocytes and ameliorated ovalbumin-induced airway inflammation by downregulating pulmonary infiltration of differential inflammatory cells and Th2 cytokines more than a decoction or ethanol extract of J. procumbens did in a mouse asthma model. DW2008 also significantly inhibited airway hyperresponsiveness and reduced the thickness of the airway epithelium. HPLC analysis showed that the major peaks (justicidin A and B) of DW2008 were higher than those of the other extracts. Justicidin A and B significantly suppressed Th2 cytokine levels in mouse spleen cells and exhibited a protective effect in ovalbumin-induced airway inflammation. Our findings indicate that DW2008 effectively inhibits allergic airway inflammatory reactions and airway hyperresponsiveness in a mouse model of asthma, suggesting its potential as an anti-asthmatic agent.

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  • Hae Sun You, Ailyn Fadriquela, Ma Easter Joy Sajo, Johny Bajgai, Jesmi ...
    2017 Volume 40 Issue 9 Pages 1423-1431
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Acidic electrolyzed water is an innovative sanitizer having a wide-spectrum of applications in food industry, and healthcare industry but little is known on its effect and mechanism in wound healing. The study was conducted to identify the effect and mechanism of slightly acidic electrolyzed water (SAEW) on cutaneous wounds in hairless mice. SAEW (pH: 5–6.5, oxidation reduction potential: 800 mV, chlorine concentration: 25 ppm) was prepared through electrolysis of water and was applied to the wounds of hairless mice three times a day for seven days. Wound size, immune response and oxidative stress were explored and compared to conventional agents such as Betadine and alcohol. We found that SAEW-treated group showed the highest wound reduction percentage (p<0.01). Antioxidant activities such as glutathione peroxidase, catalase and myeloperoxidase activities of SAEW group surpassed the total reactive oxygen species in skin. Nuclear factor erythroid-2-related-factor-2 and aryl hydrocarbon receptor were upregulated in SAEW group. Further, SAEW recruited the production of intracellular calcium and promoted its utilization for faster healing. In line, SAEW treatment decreased pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, keratinocyte chemoattractant, and tumor necrosis factor-α] in serum. Other hallmarks of wound healing, matrixmetalloproteinases (MMP)1 and MMP9 were also upregulated. Collectively, our study indicates that SAEW is effective in wound healing of hairless mice via immune-redox modulation, and heals better/faster than conventional agents.

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  • Yuecheng Lu, Baohua Lin, Junmin Zhong
    2017 Volume 40 Issue 9 Pages 1432-1438
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 14, 2017
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    Diabetic neuropathy pain (DNP) is a common chronic complication of diabetes characterized by spontaneous pain, hyperalgesia and allodynia. Dexmedetomidine is a selective α2 adrenergic agonist that relieves sympathetic nervous tension and reduces the release of glutamate. Thus, it is possible that dexmedetomidine may relieve DNP as well. In this study, we examined the effect of dexmedetomidine on DNP in the presence or absence of the α2 adrenergic antagonist yohimbine in rats utilizing a streptozotocin (STZ)-induced diabetes as a model of DNP. To examine DNP, we examined behavior using the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests, and microglia and astrocyte activation was examined by immunofluorescence staining. The levels of pro-inflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the spinal cord were measured by enzyme-linked immunosorbent assay (ELISA). Cell apoptosis in spinal cord was examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Glutamate production in caudal lumbar was measured by HPLC. We found that STZ-treated rats had decreased pain threshold, elevated activation of microglia but not astrocytes, increased level of pro-inflammatory cytokines, increased apoptosis and glutamate production compared to control animals, and these effects were ameliorated by dexmedetomidine treatment. Pretreatment of yohimbine abolished almost all of the protective effects of dexmedetomidine except for glutamate production. In conclusion: our data confirmed that dexmedetomidine can relieve hyperalgesia in diabetic neuropathy pain, and protect spinal cord cells from apoptotic death. The mechanism may be related to dexmedetomidine-mediated inhibition of microglia activation, reduction of inflammatory reaction in the spinal cord, and suppression of glutamate production.

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  • Jiaming Liu, Jindan Wang, Yilu Shi, Wei Su, Jie Chen, Zhengzheng Zhang ...
    2017 Volume 40 Issue 9 Pages 1439-1446
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Short chain fatty acids acetate and propionate have been demonstrated protective function in the intestinal mucosa. However, their impact on gastric mucosa has not yet been elucidated. The current study aimed to investigate the potential protective effects of acetate and propionate against ethanol-induced gastric mucosal lesion and the underlying mechanism in mice. ICR mice were orally treated with acetate and propionate, respectively, 30 min prior to the establishment of gastric mucosal injury model by challenge with absolute ethanol. The gastric samples were collected for the detection of oxidative, inflammatory and apoptotic related parameters. Acetate, but not propionate, attenuated the severity of gastric mucosal damage as evidenced by the gross changes of gastric mucosa, pathological aberrations. Acetate alleviated oxidative stress as shown by the increase in glutathione (GSH) content and superoxide dismutase (SOD) activities, and the decrease of malondialdehyde (MDA) level. The elevated concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6, and the activation of nuclear factor-kappaB (NF-κB) p65 by ethanol stimulation was also reduced by acetate. Moreover, the anti-inflammatory factors, IL-4, LXA4 and IL-10, were up-regulated in acetate treated group. With respect to gastric mucosal apoptosis, acetate suppressed caspase-3 activity and BAX expression in favor of cell survival. These favorable actions were maybe associated with up-regulation of the gastric MUC5AC, the key defense factor of gastric mucosal system. These findings accentuate the gastroprotective actions of acetate in ethanol-induced gastric injury which were mediated via concerted multi-prolonged actions, including suppression of gastric oxidation, inflammation and apoptosis and promotion of MUC5AC expression.

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  • Mayu Takeyama, Kimie Sai, Takuya Imatoh, Katsunori Segawa, Noriyasu Hi ...
    2017 Volume 40 Issue 9 Pages 1447-1453
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Supplementary material

    The anti-receptor activator of nuclear factor kappa-B ligand (RANKL) antibody, Denosumab (DEN), was approved in April 2012 in Japan, but a Dear Healthcare Professional Letter of Rapid Safety Communication was released in September, 2012 by the regulatory authority because of the severe hypocalcemia risks. Currently, the effectiveness of this regulatory action has not been evaluated and, therefore, this study aimed to assess its impact on DEN-induced hypocalcemia using the Japanese Adverse Drug Event Report database (JADER). The case reports from April 2012 to September 2014 were collected from the JADER, which included 151642 adverse events for the primary suspected drugs. The reporting odds ratio (ROR) of hypocalcemia as a signal of the target adverse event was analyzed for DEN and zoledronic acid (ZOL, a reference drug). Changes in RORs were compared between the pre- (Pre, April 2012 to September 2012) and post- (Post 1, October 2012 to September 2013 and Post 2, October 2013 to September 2014) periods of the regulatory action. A decrease in the hypocalcemia ROR was observed for DEN in the post-periods, especially Post 2. Multivariate logistic regression analysis showed a significant decrease in hypocalcemia signal in Post 1 (p=0.0306 vs. Pre) and Post 2 (p=0.0054 vs. Pre). ZOL caused no significant changes in ROR of hypocalcemia, and none of the drugs caused ROR changes in jaw osteonecrosis (a reference adverse event). This study suggests that the regulatory action against hypocalcemia in DEN effectively decreased hypocalcemia signal. Further studies using medical information databases are needed to confirm this result.

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  • Tae Gi Kim, Ju Hee Lee, Mi Young Lee, Ka-Ul Kim, Jeong Hyun Lee, Chi H ...
    2017 Volume 40 Issue 9 Pages 1454-1462
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z′ factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z′ factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.

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  • Satoru Mitsuboshi, Hitoshi Yamada, Kazuhiko Nagai, Hideo Okajima
    2017 Volume 40 Issue 9 Pages 1463-1467
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    To determine the response of hemodialysis (HD) patients to topiroxostat after a switch from febuxostat, we evaluated the efficacy, tolerability, and serum concentration of topiroxostat in HD patients after the switch. In this 16-month prospective observational study, we assessed the serum uric acid (UA) levels, other laboratory data, and serum topiroxostat concentrations of 10 HD patients who had been receiving febuxostat at a dose of 10 mg/d for over 1 year. No statistical difference was observed between the tolerability index at baseline and 16 months after the switch to topiroxostat. Serum UA after the switch in all patients (attained serum UA levels of ≤6 mg/dL) was 5.6±1.7 mg/dL (60%) at baseline, 4.9±0.5 mg/dL (100%) at 6 months and 5.7±0.4 mg/dL (50%) at 16 months (p=0.25), respectively. In patients with baseline serum UA levels >6 mg/dL, serum UA was significantly reduced at 6 and 16 months compared with baseline. Minimum serum concentrations of serum topiroxostat were lower than the limit of quantification (<25 ng/mL). Our results indicate that a switch from febuxostat 10 mg/d to topiroxostat 40 mg/d might reduce serum UA levels, with no change in other clinical laboratory data over the long term. These effects were more frequent in patients with high serum UA levels. Furthermore, topiroxostat therapy was more cost effective than febuxostat therapy. Thus, topiroxostat therapy could be a better treatment option for HD patients who develop high serum UA levels after febuxostat 10 mg/d administration.

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  • Kazunobu Ogiwara, Susumu Ohya, Yoshiaki Suzuki, Hisao Yamamura, Yuji I ...
    2017 Volume 40 Issue 9 Pages 1468-1474
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Salt-sensitive hypertension induces renal injury via decreased blood flow in the renal artery (RA), and ion channel dysfunction in RA myocytes (RAMs) may be involved in the higher renal vascular resistance. We examined the effects of several voltage-gated K+ (KV) channel blockers on the resting tension in endothelium-denuded RA strips and delayed-rectifier K+ currents in RAMs of Dahl salt-sensitive hypertensive rats (Dahl-S) fed with low- (Dahl-LS) and high-salt diets (Dahl-HS). The tetraethylammonium (TEA)-induced contraction in RA strips were significantly larger in Dahl-HS than Dahl-LS. Correspondingly, TEA-sensitive KV currents were significantly larger in the RAMs of Dahl-HS than Dahl-LS. Among the TEA-sensitive KV channel subtypes, the expression levels of KV2.1 transcript and protein were significantly higher in the RA of Dahl-HS than Dahl-LS, while those of KV1.5, KV7.1, and KV7.4 transcripts was comparable in two groups. KV2.1 currents detected as the guangxitoxin-1E-sensitive component were larger in the RAMs of Dahl-HS than Dahl-LS. These suggest that the up-regulation of the KV2.1 channel in RAMs may be involved in the compensatory mechanisms against decreased renal blood flow in salt-sensitive hypertension.

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    Editor's pick

    Salt-sensitive hypertension induces renal injury via decreased blood flow in the renal artery. Voltage-gated, delayed-rectifier K+ (KV) channels play key roles in the regulation of vascular tone, and their dysfunction in arterial myocytes may be involved in the higher vascular resistance. In their report, Ogiwara et al. described that there is a large contribution of KV2.1 to the resting tension maintenance of renal artery in Dahl salt-sensitive hypertensive rats and suggested the up-regulation of the KV2.1 channel in renal artery might be involved in the compensatory mechanisms against decreased renal blood flow in salt-sensitive hypertension.



  • Ju-Young Park, Byung-Wook Kim, Hae Un Lee, Dong-Kug Choi, Sung-Hwa Yoo ...
    2017 Volume 40 Issue 9 Pages 1475-1482
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 08, 2017
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    A series of methyl ester of clovamide analogues, where the hydroxyl group of catechol moiety in caffeic acid and L-3,4-dihydroxyphenylalanine (L-dopa) was replaced with various functional groups, were synthesized and their inhibitory effects on nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-induced BV2 cells were tested. Among the synthesized compounds, 3,5-ditrifluoromethyl analogue 9l (IC50=2.8 µM) exhibited a potency about 26.3 times greater than that of the parent compound 9a (IC50=73.6 µM) and suppressed NO production dose-dependently without cytotoxicity. Compound 9l also inhibited iNOS expression in LPS-induced BV2 cells at 2.5, 5 and 10 µM concentrations. These results suggested that the dihydroxyl group of catechol moiety in caffeic acid unit is not essential for the suppression of NO production and that 9l has potential as a potent inhibitor of NO production.

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  • Erina Anzai, Kensuke Hirata, Misato Shibazaki, Chiaki Yamada, Mariko M ...
    2017 Volume 40 Issue 9 Pages 1483-1489
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Supplementary material

    Epithelial-to-mesenchymal transition (EMT) is an important process during embryonic development and tumor progression by which adherent epithelial cells acquire mesenchymal properties. Forkhead box protein A1 (FOXA1) is a transcriptional regulator preferentially expressed in epithelial breast cancer cells, and its expression is lost in mesenchymal breast cancer cells. However, the implication of this biased expression of FOXA1 in breast cancer is not fully understood. In this study, we analyzed the involvement of FOXA1 in EMT progression in breast cancer, and found that stable expression of FOXA1 in the mesenchymal breast cancer MDA-MB-231 cells strongly induced the epithelial marker E-cadherin at the mRNA and protein levels. Furthermore, stable expression of FOXA1 was found to reduce the mRNA and protein expression of Slug, a repressor of E-cadherin expression. FOXA1 knockdown in the epithelial breast cancer MCF7 cells reduced E-cadherin protein expression without decreasing its mRNA expression. In addition, FOXA1 knockdown in MCF7 cells up-regulated Slug mRNA and protein expression. Notably, similar to FOXA1 knockdown, stable expression of Slug in MCF7 cells reduced E-cadherin protein expression without decreasing its mRNA expression. Taken together, these results suggest that although FOXA1 can induce E-cadherin mRNA expression, it preferentially promotes E-cadherin expression at the protein level by suppressing Slug expression in epithelial breast cancer, and that the balance of this FOXA1-Slug axis regulates EMT progression.

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  • Xiaojing Zhang, Huihui Hu, Jingxiong Luo, Huixing Deng, Pei Yu, Zaijun ...
    2017 Volume 40 Issue 9 Pages 1490-1498
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 22, 2017
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    In this study, we investigated the cardioprotective mechanisms of action of DT-010, a novel danshensu-tetramethylpyrazine conjugate. DT-010 significantly preserved cell viability and suppressed cell apoptosis in H9c2 cells injured by tert-butylhydroperoxide (t-BHP), iodoacetic acid (IAA) and hypoxia-reoxygenation. In addition, DT-010 pre-treatment reduced the intracellular level of free radicals including superoxide anion (·O2), hydroxyl radical (·OH) and peroxynitrite anion (ONOO) after t-BHP exposure. Moreover, DT-010 up-regulated the protein expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and nuclear factor-E2-related factor 2 (Nrf2) as well as mitochondrial transcription factor A (Tfam) and heme oxygenase-1 (HO-1) in H9c2 cells. DT-010 also triggered Nrf2 nuclear translocation. In a rat myocardial ischemia-reperfusion model, DT-010 significantly alleviated myocardial infarction. The results indicated that DT-010 may be a promising candidate for the treatment of cardiovascular diseases, particularly myocardial ischemia and reperfusion injury.

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  • Mayako Uchida, Yasuo Mori, Tsutomu Nakamura, Koji Kato, Kenjiro Kameza ...
    2017 Volume 40 Issue 9 Pages 1499-1505
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    In the present study, the antiemetic effect of palonosetron, not combined with dexamethasone and aprepitant, on chemotherapy-induced nausea and vomiting was evaluated in patients with malignant lymphoma receiving first-line rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy, and was compared to that of granisetron. A total of 74 patients with non-Hodgkin lymphoma were included in this study (April 2007 to December 2015). Palonosetron (0.75 mg) or granisetron (3 mg) was intravenously administered before R-CHOP therapy. The proportions of patients with complete response (CR) during the overall (0–120 h after the start of R-CHOP therapy), acute (0–24 h) and delayed (24–120 h) phases were evaluated. CR was defined as no vomiting and no use of antiemetic rescue medication. A total of 32 and 42 patients were treated with palonosetron and granisetron, respectively. The CR rate in the palonosetron group was significantly higher than that in the granisetron group during the delayed phase (90.6 and 61.9%, respectively; p=0.007). Logistic regression analysis showed that use of palonosetron improved the CR rate during the delayed phase, compared to use of granisetron. Female sex, age less than 60 years, no habitual alcohol intake, and Eastern Cooperative Oncology Group performance status (ECOG-PS) score of 1 were significant risk factors associated with non-CR. The findings of this study suggested the superiority of palonosetron to granisetron, without accompanying dexamethasone and aprepitant, for chemotherapy-induced nausea and vomiting in patients with malignant lymphoma.

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  • Nipaporn Muangchan, Suwattanee Kooptiwut, Sompol Tapechum, Pravit Akar ...
    2017 Volume 40 Issue 9 Pages 1506-1514
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Gastric motility disturbance is commonly found in long-standing hyperglycemia. Both delayed and rapid gastric emptying has been reported in diabetes. However, very few studies have followed the changes in gastric emptying during disease progression in diabetes because of technical limitations. 13C-Acetic acid breath test is a validated method which is non-invasive and can be used repeatedly or serially to evaluate gastric emptying changes in animal. We investigated the gastric emptying changes in different stages of diabetes using 13C-acetic acid breath test, as well as its related mechanisms involving interstitial cells of Cajal (ICCs), and stem cell factor (SCF) in streptozotocin-induced diabetic rats. The results showed that gastric emptying was accelerated at the early stage (12 weeks of diabetes) whereas intramuscular ICCs (ICC-IM) networks were not different from normal group. At long-term stage (28 weeks of diabetes), gastric emptying had returned to normal pattern with no delayed. ICC-IM networks were decreased in the diabetic group compared to 12th weeks, and were lower than in the normal group at the same time point. SCF levels were constantly high in the diabetic group than in the normal group. This result indicated that 13C-acetic acid breath test is useful to track the alteration in gastric emptying during disease progression. The change of gastric emptying was not found to be significantly associated with ICC-IM. Elevated SCF may help to preserve ICC-IM, especially in the early phase of diabetes.

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  • Hitomi Ozawa, Atsushi Imaizumi, Yoshihiko Sumi, Tadashi Hashimoto, Mas ...
    2017 Volume 40 Issue 9 Pages 1515-1524
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Curcumin, a polyphenol derived from the rhizome of the naturally occurring plant Curcuma longa, has various pharmacological actions such as antioxidant and anti-inflammatory effects. In this paper, we evaluated the role of its internal metabolite, curcumin β-D-glucuronide (curcumin monoglucuronide, CMG), by investigating curcumin kinetics and metabolism in the blood. Firstly, we orally administered highly bioavailable curcumin to rats to elucidate its kinetics, and observed not only the free-form of curcumin, but also, curcumin in a conjugated form, within the portal vein. We confirmed that curcumin is conjugated when it passes through the intestinal wall. CMG, one of the metabolites, was then orally administered to rats. Despite its high aqueous solubility compared to free-form curcumin, it was not well absorbed. In addition, CMG was injected intravenously into rats in order to assess its metabolic behavior in the blood. Interestingly, high levels of free-form curcumin, thought to be sufficiently high to be pharmacologically active, were observed. The in vivo antitumor effects of CMG following intravenous injection were then evaluated in tumor-bearing mice with the HCT116 human colon cancer cell line. The tumor volume within the CMG group was significantly less than that of the control group. Moreover, there was no significant loss of body weight in the CMG group compared to the control group. These results suggest that CMG could be used as an anticancer agent without the serious side effects that most anticancer agents have.

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  • Maiko Akutagawa, Kazuki Ide, Yohei Kawasaki, Mie Yamanaka, Ryo Iketani ...
    2017 Volume 40 Issue 9 Pages 1525-1529
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 09, 2017
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    To compare the rate of treatment discontinuation due to adverse events for telaprevir-based triple (T/PR) and pegylated interferon-alfa-2b and ribavirin (PR) therapy for the treatment of hepatitis C virus (HCV) infection in patients over the age of 65 years, in Japan. Retrospective analysis of the health data of patients over the age of 65 years treated for a HCV infection genotype 1 using T/PR or PR therapy, from 38 prefectures in Japan. The primary outcome was the rate of treatment discontinuation due to adverse events for T/PR and PR. The secondary outcome was to evaluate the prevalence and type of adverse events during the treatment period that resulted in treatment discontinuation for both therapies. For comparison, the T/PR and PR populations were matched using the propensity score method, and adjusted odds ratios (ORs) for treatment discontinuation calculated by multivariate logistic regression analysis. The study group included 1330 patients, 328 in the T/PR group and 1002 in the PR group. The rate of treatment discontinuation due to adverse events in the matched population was lower for T/PR (19.82%) than PR (35.98%) therapy, (adjusted OR, 0.418; 95% confidence interval, 0.292–0.599; p<0.01). Malaise was the principal cause of treatment discontinuation in both groups (T/PR, 30.77%, and PR, 42.37%). Using real-world health data of elderly individuals in Japan, we identified a lower rate of treatment discontinuation for T/PR than PR. Our outcomes provide information for a segment of the population that is generally excluded for clinical trials.

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  • Kazuhiro Yamamoto, Hiroaki Shichiri, Takahiro Ishida, Kenta Kaku, Tats ...
    2017 Volume 40 Issue 9 Pages 1530-1536
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Hand–foot skin reaction is recognized as one of the most common adverse events related to multiple tyrosine kinase inhibitors, but an effective prevention method has not been identified. The chief aim of this study was to find a mechanism-based preventive method for the skin toxicity induced by sorafenib using vitamin C derivatives. The effects of ascorbyl-2-phosphate magnesium (P-VC-Mg) on the molecular and pathological changes induced by sorafenib were investigated in human keratinocyte HaCaT cells. The cell growth inhibition and apoptotic effects of sorafenib were attenuated by P-VC-Mg. Moreover, P-VC-Mg inhibited the decrease of signal transducer and activator of transcription 3 (STAT3) phosphorylation and the expression of apoptosis suppressors treated by sorafenib. HaCaT cells transfected with the STAT3 dominant-negative form (STAT3DN) and STAT3 small interfering RNA (siRNA) combined with P-VC-Mg did not exhibit the attenuation of cell growth inhibition. Interestingly, after exposure to sorafenib in a three dimensional (3D) skin model assay, the basal layer was significantly thickened and the granular and spinous layers became thinner. In contrast, after exposure to sorafenib with P-VC-Mg, the thickness of the basal, granular, and spinous layers was similar to that of the control image. These findings suggest that P-VC-Mg attenuates sorafenib-induced apoptosis and pathological changes in human keratinocyte cells and in the 3D skin model mediated by the maintenance of STAT3 activity.

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  • Lianghong Kuang, Xiongbin Cao, Zuneng Lu
    2017 Volume 40 Issue 9 Pages 1537-1543
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
    Advance online publication: June 28, 2017
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    Baicalein, a typical flavonoid compound, has neuroprotective properties in several neurological disorders. Autophagy plays a central role in maintaining the cellular homeostasis, and is involved in the pathogenesis of Parkinson’s disease (PD). Recently, baicalein has been reported to induce autophagy. Therefore, the current study was designed to investigate whether baicalein could protect against rotenone-induced neurotoxicity via induction of autophagy both in SH-SY5Y cells and in a mouse model. A chronic PD mouse model was established by continuous intragastric administration of rotenone for 12 weeks. Baicalein was administrated from 7 to 12 week. Our results showed that baicalein prevented rotenone-induced behavioral deficits, dopaminergic neuronal loss, apoptosis and mitochondrial dysfunction. Furthermore, baicalein restored rotenone-impaired autophagy, and blocking the baicalein-induced autophagy using 3-methyladenine inhibited the neuroprotective effects of bacalein. Baicalein increased cell viability and restored mitochondrial function in SH-SY5Y cells. The beneficial effect of baicalein was abrogated by 3-methyladenine treatment. Furthermore, rapamycin increased autopahgy and reduced the rotenone-induced neurotoxicity in SH-SY5Y cells. Collectively, these results suggest that baicalein could prevent rotenone-induced neurotoxicity via restoring autophagy.

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Notes
  • Mina Lee, Sun-Yup Shim, Sang Hyun Sung
    2017 Volume 40 Issue 9 Pages 1544-1550
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Supplementary material

    Alnus japonica (Betulaceae) is a broad-leaved tree easily found in damp regions within the mountains of Korea and Japan. Four triterpenoids (14) from the fruits of A. japonica, including the newly isolated 3β-hydroxy-lanost-9(11),23(24)-dien-25,26-diol (3), inhibited the lipopolysaccharide (LPS)-induced expression of the chemotactic cytokine interleukin (IL)-8 and nitric oxide (NO) production in HT-29 colon epithelial cells and RAW264.7 macrophages, respectively. Among these triterpenoids, compound 4, which showed the most potent inhibitory activity, effectively down-regulated LPS-induced protein expression of inducible nitric oxide synthase (iNOS) in RAW264.7 cells and in HT-29 cells. Also, compound 4 concentration-dependently inhibited the levels of LPS-induced pro-inflammatory cytokines, IL-1β and IL-6 in macrophage cells. These triterpenoids isolated from A. japonica fruits are thought to contribute to the anti-inflammatory activities of macrophage and colon epithelial cells, which are important for regulating the colon immune system. They are expected to be potential candidates for therapeutic agents against inflammatory bowel disease.

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  • Iyuki Namekata, Shogo Hamaguchi, Naoko Iida-Tanaka, Taichi Kusakabe, K ...
    2017 Volume 40 Issue 9 Pages 1551-1555
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    We investigated the effect on mitochondrial Ca2+ of SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX) which reduces mitochondrial Ca2+ overload during myocardial ischemia, in digitonin-permeabilized H9c2 cells expressing the mitochondrial-targeted Ca2+ indicator, yellow cameleon 3.1. The elevation of mitochondrial Ca2+ concentration caused by an increase in extramitochondrial Ca2+ concentration was inhibited by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) or ruthenium red, but enhanced by CGP-37157, a mitochondrial NCX inhibitor. SEA0400 had no effect on mitochondrial Ca2+ under normal and ischemic conditions. Thus, the mitochondria-protective effects of SEA0400 could be explained by inhibition of plasmalemmal NCX but not mitochondrial NCX.

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  • Yuki Asai, Yukiko Sakakibara, Haruka Onouchi, Masayuki Nadai, Miki Kat ...
    2017 Volume 40 Issue 9 Pages 1556-1560
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    β-Estradiol is conjugated by uridine 5′-diphosphate-glucuronosyltransferase (UGT) 1A to 3-glucuronide in the human liver. UGT1A has been found in the brain; therefore, UGT1A may be involved in β-estradiol 3-glucuronidation in the brain. In the present study, we aimed to characterize the β-estradiol 3-glucuronidation reaction in the rat brain. β-Estradiol 3-glucuronidation was detected in eight rat brain regions (cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, striatum, and thalamus). β-Estradiol 3-glucuronidation in the cerebellum was fitted to the Hill equation (S50=8.0 µM, n=1.1). In inhibition experiments, β-estradiol 3-glucuronidation was inhibited to 73.6% in the cerebellum by 50 µM bilirubin, whereas it was reduced to 20.5% with 5 µM bilirubin in the liver. Unlike in the liver, Ugt1a1 may not be the main isoform catalyzing this glucuronidation in the brain. Serotonin and acetaminophen at 10 mM inhibited glucuronidation to 1.17 and 25.5%, respectively, in the cerebellum. In induction experiments, the administration of β-naphthoflavone, carbamazepine, and phenobarbital did not increase β-estradiol 3-glucuronidation in the brain except for phenobarbital in the striatum. In addition, β-estradiol 3-glucuronidation was not correlated with serotonin or acetaminophen glucuronidation in the brain, suggesting that Ugt1a6 and Ugt1a7 are not major isoforms of β-estradiol 3-glucuronidation in the rat brain. In the present study, although we were unable to identify the isoform responsible for β-estradiol 3-glucuronidation, we confirmed that β-estradiol could be metabolized to glucuronide in the brain under a different metabolic profile from that in the liver.

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  • Masayuki Tsujimoto, Chie Agawa, Shinya Ueda, Takayoshi Yamane, Haruna ...
    2017 Volume 40 Issue 9 Pages 1561-1565
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Human intestinal absorption and drug metabolism vary to a large extent among individuals. For example, CYP3A4 activity has large individual variation that cannot be attributed to only genetic differences. Various flavonoids in vegetables, such as kaempferol and quercetin, possess inhibitory effects, and some vegetable and fruit juices have also been found to inhibit CYP3A4 activity. Therefore, differences in daily intake of flavonoid-containing vegetables may induce individual variation in intestinal bioavailability. To identify a vegetable that strongly inhibits CYP3A4, we investigated the effects of juices, prepared from individual vegetables, on CYP3A4 activity using recombinant CYP3A4 and LS180 cells in this study. Nine vegetable juices (cabbage, Japanese radish, onion, tomato, eggplant, carrot, Chinese cabbage, green pepper, and lettuce), were prepared and recombinant CYP3A4 and LS180 cells were used for evaluation of CYP3A4 activity. Metabolism to 6β-hydroxytestosterone by recombinant CYP3A4 was strongly inhibited by cabbage, onion, and green pepper juices, and cabbage and green pepper juices significantly inhibited CYP3A4 activity in a preincubation time-dependent manner. In addition, CYP3A4 activity in LS180 cells was significantly inhibited by cabbage and onion juices. In conclusion, this study showed that juices prepared from some individual vegetables could significantly inhibit CYP3A4 activity. Therefore, variation in the daily intake of vegetables such as cabbage and onion may be one of the factors responsible for individual differences in intestinal bioavailability.

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  • Kazunari Iwao, Rushiana Tokie Kawai, Masako Oda, Hiroshi Saitoh
    2017 Volume 40 Issue 9 Pages 1566-1571
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    The objective of this study was to evaluate the interactions between various drugs and aojiru (green juice), a popular health food in Japan, using a simple centrifugation method. The mixture of drug solution and aojiru suspension was gently shaken and centrifuged. The drug concentration in the supernatant fluid was then determined by HPLC. The concentration of rhodamine 123 (Rho-123), a model compound, in the supernatant fluid significantly decreased after mixing with aojiru, indicating extensive binding of Rho-123 to the insoluble components of aojiru. When administered into rat small intestinal loops together with aojiru, the plasma Rho-123 concentrations became much smaller than those when administered alone. This result strongly suggested that a strong interaction observed in vitro was well reflected in modulated absorption. Among seven drugs tested, chlorpromazine and imipramine exerted binding properties to aojiru similar to or greater than Rho-123. As a small part of both Rho-123 and imipramine was released when the aojiru precipitate was resuspended, their binding to aojiru was considered to be tight. The binding of diltiazem, fexofenadine, glibenclamide, metformin, and norfloxacin to aojiru was much weaker or almost negligible compared with that of chlorpromazine and imipramine. The present results suggest that aojiru can decrease the intestinal absorption of some clinically relevant drugs through tight binding in the small intestine and that the present centrifugation method is useful for predicting in vivo interactions between drugs and aojiru.

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  • Masateru Miyake, Miki Fujishima, Daisuke Nakai
    2017 Volume 40 Issue 9 Pages 1572-1575
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    We investigate the inhibitory effect of marketed drugs for treatment of inflammatory bowel disease (IBD) such as ulcerative colitis (UC) and Crohn’s disease (CD) on the uptake transporters of peptide transporter 1 (PEPT1), which are up-regulated under the inflamed condition. The uptake transport of glycylsarcosine, a typical substrate for PEPT1, was reduced to 60% only by 5-aminosalicylate at the clinically relevant concentration among tested marketed drugs in PEPT1 transfected HEK293 cell lines. These findings suggest that the inhibition of PEPT1, which were up-regulated in inflamed or non-inflamed site on UC and CD patients, contribute to the clinical effect of commercially available drugs for IBD patients through the inhibition of uptake of antigenic proinflammatory oligopeptides such as formyl-methionine (Met)-leucine (Leu)-phenylalanine (Phe) via PEPT1.

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  • Yoshimi Okamoto-Uchida, Ryosuke Nakamura, Kimie Sai, Takuya Imatoh, Ka ...
    2017 Volume 40 Issue 9 Pages 1576-1580
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous adverse drug reactions. Recent studies have revealed that the prevalence of SJS/TEN is associated with genetic backgrounds, such as polymorphisms in human leukocyte antigens (HLAs). However, non-genetic factors contributing to the etiology of SJS/TEN are largely unknown. This study aimed to assess the involvement of concurrent infection on the pathological states of SJS/TEN, examining the severity of cutaneous symptoms and ocular involvement as well as the time to onset in drug-induced SJS/TEN patients. We recruited 257 Japanese SJS/TEN patients from June 2006 to September 2013 through a nationwide case collection network and participating hospitals and reviewed the clinical information including patient backgrounds, primary disease and medication status. Association between infection and pathological states of SJS/TEN was assessed using univariate and multivariate analyses. The concurrent infectious group of SJS/TEN patients showed a significantly higher rate of exhibiting severer dermatological and ophthalmological phenotypes and an earlier onset of SJS/TEN than the non-infectious group. Our results suggest that the infection could be a risk factor to cause severer symptoms and earlier onset of SJS/TEN.

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  • Yoshihiko Hirotani, Junta Fukamachi, Rina Ueyama, Yoko Urashima, Kenji ...
    2017 Volume 40 Issue 9 Pages 1581-1585
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Obesity-induced inflammation contributes to the development of metabolic disorders such as insulin resistance, type 2 diabetes, fatty liver disease, and cardiovascular disease. In this study, we investigated whether the combination of eicosapentaenoic acid (EPA) and capsaicin could protect against high-fat diet (HFD)-induced obesity and related metabolic disorders. The experiments were performed using male C57BL/6J mice that were fed one of the following diets for 10 weeks: standard chow (5.3% fat content) (normal group), a HFD (32.0% fat content) (HFD group), or a HFD supplemented with either 4% (w/w) EPA (EPA group) or a combination of 4% (w/w) EPA and 0.01% (w/w) capsaicin (EPA+Cap group). Our results indicated that the body, fat and liver tissue weights and levels of serum glucose, insulin, total cholesterol, triglyceride, high-density lipoprotein-cholesterol, aspartate aminotransferase, and alanine aminotransferase were significantly higher in HFD group mice than in normal group mice (p<0.05 in all cases). However, the body and fat tissue weights and serum glucose levels and homeostasis model assessment of insulin resistance were significantly lower in EPA+Cap group mice group than in HFD and EPA group mice (p<0.05 in all cases). Thus, our study suggests that the combination of EPA and capsaicin might be beneficial for delaying the progression of obesity-related metabolic dysregulation and subsequent complications.

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  • Nagayoshi Asano, Justin Michael Wiseman, Fumio Tsuji, Kouichi Kawazu
    2017 Volume 40 Issue 9 Pages 1586-1589
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Meibomian gland dysfunction (MGD) is the leading cause of dry eye, and although it affects approximately 4% of the population, treatment options remain limited. Topical azithromycin is one of the most promising pharmacological agents because of its multiple mechanisms of action and long sustainability. Azithromycin is frequently used as an off-label medication in the U.S. However, although azithromycin is presumed to act directly on meibomian gland cells, the mechanisms of action that contribute to its clinical efficacy remain unclear because no studies using a pharmacokinetic approach have been performed. Therefore, we aimed to clarify whether topical azithromycin reaches the meibomian glands sufficiently to generate a biological effect. We measured azithromycin concentrations in rabbit meibomian glands collected using a recently developed method. Moreover, we also visualized the azithromycin micro-distribution using desorption electrospray ionization (DESI) imaging. Azithromycin concentration in the meibomian glands reached only 0.8 µg/g tissue following a single application of a 1% azithromycin ophthalmic solution and was 1000-fold lower than the concentration in conjunctival epithelium. Similarly, no signal was observed in the meibomian glands on DESI images. Our results clearly demonstrated that topical azithromycin had limited access to the meibomian glands and was predominantly distributed in ocular surface tissues such as the palpebral conjunctiva and lid margins. These findings provide new insight into the clinical responses to topical azithromycin therapy and will aid in the further development of effective drugs with more suitable pharmacokinetic properties.

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  • Shiori Fukaya, Hiroki Yoshioka, Tadahiro Okano, Akito Nagatsu, Nobuhik ...
    2017 Volume 40 Issue 9 Pages 1590-1594
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    A wide range of medications are routinely used to maintain and improve human health. Hence, it is essential that we understand and predict adverse effects caused by the combined use of multiple medications. In the present study, we investigated whether the combination of carbon tetrachloride (CCl4) and acetaminophen (APAP) had a detrimental effect on the liver. Mice injected with APAP (100 mg/kg) showed no significant changes in hepatic injury markers (alanine aminotransferase and aspartate aminotransferase), histopathological findings, pro-inflammatory cytokine levels, or hepatic oxidative stress. In contrast, a single injection of CCl4 (15 mg/kg) led to a significant increase in hepatic injury, in addition to an increase in pro-inflammatory cytokine levels and oxidative stress. Co-administration of APAP and CCl4 resulted in exacerbation of these hepatic injuries. Our results suggest that a non-toxic dose of APAP has the potential to increase CCl4-induced liver damage and oxidative stress.

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  • Hideki Hiraoka, Kengo Nakahara, Yuki Kaneko, Shiori Akiyama, Kosaku Ok ...
    2017 Volume 40 Issue 9 Pages 1595-1598
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Methylmercury (MeHg) results in cell death through endoplasmic reticulum (ER) stress. Previously, we reported that MeHg induces S-mercuration at cysteine 383 or 386 in protein disulfide isomerase (PDI), and this modification induces the loss of enzymatic activity. Because PDI is a key enzyme for the maturation of nascent protein harboring a disulfide bond, the disruption in PDI function by MeHg results in ER stress via the accumulation of misfolded proteins. However, the effects of MeHg on unfolded protein response (UPR) sensors and their signaling remain unclear. In the present study, we show that UPR is regulated by MeHg. We found that MeHg specifically attenuated inositol-requiring enzyme 1α (IRE1α)–x-box binding protein 1 (XBP1) branch, but not the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcriptional factor 6 (ATF6) branches. Treatment with GSK2606414, a specific PERK inhibitor, significantly inhibited MeHg-induced cell death. These findings suggest that MeHg exquisitely regulates UPR signaling involved in cell death.

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  • Shin-ichi Akanuma, Hirokazu Shimada, Yoshiyuki Kubo, Ken-ichi Hosoya
    2017 Volume 40 Issue 9 Pages 1599-1603
    Published: September 01, 2017
    Released on J-STAGE: September 01, 2017
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    Supplementary material

    Spermine is the end-product in the polyamine biosynthetic pathway, and its excess accumulation induces neuroexcitatory responses and neurotoxicity. The purpose of this study was to elucidate the involvement of transport systems at the brain barriers in the clearance of spermine. In vivo rat spermine elimination from brain parenchyma across the blood–brain barrier (BBB) and blood–cerebrospinal fluid (CSF) barrier (BCSFB) was assessed by intracerebral and intracerebroventricular administration techniques, respectively. To characterize spermine transport at the BCSFB, a transport study using rat choroid plexus was performed. After the intracerebral microinjection of [3H]spermine, no time-dependent decrease in [3H]spermine in the ipsilateral cerebrum was observed, suggesting the low contribution of the BBB to spermine clearance from the brain. In contrast, the [3H]spermine concentration in the CSF after intracerebroventricular administration was time-dependently decreased with an elimination rate constant of 0.352 min−1, and the elimination clearance of [3H]spermine was 6.6-fold greater than that of [14C]D-mannitol, reflecting bulk flow of the CSF. This [3H]spermine elimination was attenuated by co-administration of unlabeled excess spermine, indicating carrier-mediated elimination of spermine from the CSF. [3H]Spermine transport into the choroid plexus was strongly inhibited by unlabeled spermine, other polyamines (spermidine and putrescine), and organic cation transporter substrates such as corticosterone and 1-methyl-4-phenylpyridinium. However, other substrates/inhibitors for organic cation transporters (decynium-22 and tetraethylammonium) had little effect. Consequently, our study indicates that transporting molecules at the BCSFB, distinct from typical organic cation transporters, are involved in spermine clearance from the CSF.

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