Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 40 , Issue 6
Showing 1-29 articles out of 29 articles from the selected issue
Review
  • Yasuo Endo, Hiroyuki Kumamoto, Masanori Nakamura, Shunji Sugawara, Ter ...
    2017 Volume 40 Issue 6 Pages 739-750
    Published: June 01, 2017
    Released: June 01, 2017
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    Bisphosphonates (BPs), with a non-hydrolysable P-C-P structure, are cytotoxic analogues of pyrophosphate, bind strongly to bone, are taken into osteoclasts during bone-resorption and exhibit long-acting anti-bone-resorptive effects. Among the BPs, nitrogen-containing BPs (N-BPs) have far stronger anti-bone-resorptive effects than non-N-BPs. In addition to their pyrogenic and digestive-organ-injuring side effects, BP-related osteonecrosis of jaws (BRONJ), mostly caused by N-BPs, has been a serious concern since 2003. The mechanism underlying BRONJ has proved difficult to unravel, and there are no solid strategies for treating and/or preventing BRONJ. Our mouse experiments have yielded the following results. (a) N-BPs, but not non-N-BPs, exhibit direct inflammatory and/or necrotic effects on soft tissues. (b) These effects are augmented by lipopolysaccharide, a bacterial-cell-wall component. (c) N-BPs are transported into cells via phosphate transporters. (d) The non-N-BPs etidronate (Eti) and clodronate (Clo) competitively inhibit this transportation (potencies, Clo>Eti) and reduce and/or prevent the N-BP-induced inflammation and/or necrosis. (e) Eti, but not Clo, can expel N-BPs that have accumulated within bones. (f) Eti and Clo each have an analgesic effect (potencies, Clo>Eti) via inhibition of phosphate transporters involved in pain transmission. From these findings, we propose that phosphate-transporter-mediated and inflammation/infection-promoted mechanisms underlie BRONJ. To treat and/or prevent BRONJ, we propose (i) Eti as a substitution drug for N-BPs and (ii) Clo as a combination drug with N-BPs while retaining their anti-bone-resorptive effects. Our clinical trials support this role for Eti (we cannot perform such trials using Clo because Clo is not clinically approved in Japan).

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Regular Articles
  • Nattapong Puthdee, Wunchana Seubwai, Kulthida Vaeteewoottacharn, Thida ...
    2017 Volume 40 Issue 6 Pages 751-757
    Published: June 01, 2017
    Released: June 01, 2017
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    Berberine is a natural compound found in several herbs. Anticancer activity of berberine was reported in several cancers, however, little is known regarding the effects of berberine against cholangiocarcinoma (CCA). In this study, the growth inhibitory effects of berberine on CCA cell lines and its molecular mechanisms were explored. Cell growth and cell cycle distribution were examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. The expression levels of cell cycle regulatory proteins were determined by Western blot analysis. Berberine significantly inhibited growth of CCA cell lines in a dose and time dependent fashion. The inhibition was largely attributed to cell cycle arrest at the G1 phase through the reduction of cyclin D1, and cyclin E. Moreover, berberine could reduce the expression and activation of signal transducers and activator of transcription 3 (STAT3) and probably nuclear factor-kappaB (NF-κB) via suppression of extracellular signal-regulated kinase (ERK) 1/2 action. These results highlight the potential of berberine to be a multi-target agent for CCA treatment.

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  • Graciela Pinto Vitorino, María Cecilia Becerra, Gustavo Daniel Barrera ...
    2017 Volume 40 Issue 6 Pages 758-764
    Published: June 01, 2017
    Released: June 01, 2017
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    The effects of different combinations of ciprofloxacin (CIP) and norfloxacin (NOR) against Escherichia coli and Staphylococcus aureus were studied using checkerboard, fractional inhibitory concentration (FIC) and time–kill analysis methods. Results obtained by the checkerboard method showed that the more effectives combinations against Escherichia coli were 0.0009 µg/mL CIP+0.0312 µg/mL NOR and 0.0037 µg/mL CIP+0.0075 µg/mL NOR with a FIC index of 0.62. For Staphylococcus aureus, the combination of 0.0625 µg/mL CIP+0.2500 µg/mL NOR showed a synergistic effect, with a FIC index of 0.50. The results of the time–kill method demonstrated either indifference or additivity of the combinations 0.0009 µg/mL CIP+0.0312 µg/mL NOR, 0.0018 µg/mL CIP+0.0312 µg/mL NOR, 0.0037 µg/mL CIP+0.0075 µg/mL NOR and 0.0037 µg/mL CIP+0.0156 µg/mL NOR at 24 h against E. coli. The combination 0.0037 µg/mL CIP+0.0312 µg/mL NOR showed synergistic activity. All the analyzed combinations evidenced bactericidal effects at 4 h. The combinations 0.0625 µg/mL CIP+0.2500 µg/mL NOR and 0.0625 µg/mL CIP+0.0625 µg/mL NOR showed indifference or additivity against S. aureus. None of them generated bactericidal effect at 4 h. Moreover, this last equimolecular combination (equivalent to 1/4 minimum inhibitory concentration (MIC) CIP+1/16 MIC NOR) generated higher reduction of nitro blue tetrazolium than drugs alone. By another way, combinations not equimolecular of CIP and NOR assayed, generated less levels of reactive oxygen species (ROS) than the components alone.

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  • Ki Mo Kim, A-Rang Im, Sanghyun Lee, Sungwook Chae
    2017 Volume 40 Issue 6 Pages 765-773
    Published: June 01, 2017
    Released: June 01, 2017
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    The leaves of Petasites japonicus are used for their anti-allergic properties in traditional Korean, Japanese, and Chinese medicine. This study aimed to identify bioactive compounds isolated from P. japonicus leaves. All compounds were assessed for their ability of transcriptional activation, induction of phase 2 enzymes and heat shock proteins (HSPs), as well as protection against the UVB-induced apoptotic cell death. Bioactive compounds were isolated from P. japonicus leaves. All compounds were evaluated for their protective effect using human dermal fibroblasts (HDF) and human epidermal keratinocyte cells (HEKC) treated with UVB radiation. Four flavonoids were isolated from the leaves of P. japonicus and identified as kaempferol-3-O-(6″-acetyl)-β-D-glucoside (1), quercetin-3-O-(6″-acetyl)-β-D-glucoside (2), kaempferol-3-O-β-D-glucoside (3), and quercetin-3-O-β-D-glucoside (4). These compounds activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heat-shock response transcription elements (HSE) that resulted in the induction of heme oxygenase-1 (HO-1) and HSP70, respectively. Activation of these pathways provided protection to the skin cells against UVB radiation. The isolated compounds activated the Nrf2 and HSE pathways and could protect against UVB-induced apoptosis.

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  • Nina Xue, Chen Wei, Lihong Zhang, Hongying Liu, Xiaojuan Wang, Lili Wa ...
    2017 Volume 40 Issue 6 Pages 774-781
    Published: June 01, 2017
    Released: June 01, 2017
    [Advance publication] Released: March 04, 2017
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    Stimulatory G protein α-subunit (Gsα) mediated cAMP signal is required for elevated hepatic glucose production (HGP) in diabetic patients. However, it remains obscure of the exact characteristics of hepatic Gsα-cAMP signal axis (including Gsα, glucagon receptor, β2-adrenergic receptor, cAMP, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in insulin resistance (IR) and type 2 diabetes mellitus (T2DM). In current study, we investigated the changing characteristics of hepatic Gsα-cAMP signal axis and blood glucose in high-sugar-high-fat (HSHF)-diet-induced IR Wistar rats and db/db diabetic mice. As expected, the HSHF-diet rats were characterized by hyperinsulinemia, hyperglycemia and impaired glucose tolerance. According to a threshold (1.7) of homeostasis model assessment ratio (HOMA-R), the process of IR in HSHF-diet rats could be divided into slight and high IR stages, with the week-23 as the cut-off point. In early slight IR stage, key molecules expressions of hepatic Gsα-cAMP signal axis in HSHF-diet rats were up-regulated with significantly elevated fasting blood glucose (FBG) from 18 to 23 weeks. Unexpectedly, in high IR stage, hepatic Gsα-cAMP signal axis was recovered comparatively to that of normal chow-diet rats, and no significant differences in FBG levels were found. However, in diabetic db/db mice, up-regulation of hepatic Gsα-cAMP signal axis was responsible for its severely increased fasting hyperglycaemia. Our data revealed a positive correlation between hepatic Gsα-cAMP signal axis and FBG in slight IR stage of HSHF-diet rats and diabetic db/db mice. The current finding thus suggested hepatic Gsα-cAMP signal axis plays a central role in regulating of FBG during the developing and development of T2DM.

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  • Ayako Hara, Kazuaki Matsumoto, Yuta Yokoyama, Junko Kizu
    2017 Volume 40 Issue 6 Pages 782-788
    Published: June 01, 2017
    Released: June 01, 2017
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    Hepatitis B caused by chemotherapy- and immunosuppression-associated hepatitis B virus reactivation is likely to become fulminant, and a high mortality rate has been reported. In this study, using the Japanese adverse drug event report database (JADER), factorial analysis of patients who developed hepatitis B as an adverse event was performed. The number of reported cases of hepatitis B during the survey period was 781 and 185 of them (24%) died. Rituximab and prednisolone were administered to many cases (233, 216 cases, respectively), and the reporting odds ratios were high (65.35, 13.40, respectively), suggesting their strong association with the development of hepatitis B. Regarding the onset time, rituximab-induced hepatitis B developed within one year after administration in 83%, being a high frequency. Prednisolone-induced hepatitis B developed even after one year in 36%. Since prednisolone is used to treat rheumatoid arthritis at a dose ≤10 mg/d, the patients were divided based on the prednisolone dose into the groups treated at >10 and ≤10 mg/d, and the onset time was investigated in each group. The median onset time was 113 and 330 d, respectively, showing a significant difference. On time-to-event analysis using the Weibull distribution, rituximab was classified as the early failure type, and prednisolone and methotrexate for rheumatoid arthritis were classified as the wear out failure type. These findings are important information which may lead to early discovery of and taking actions against hepatitis B being helpful for providing appropriate medical care.

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  • Hayato Akimoto, Shinji Oshima, Kousuke Ohara, Akio Negishi, Hanako Hir ...
    2017 Volume 40 Issue 6 Pages 789-796
    Published: June 01, 2017
    Released: June 01, 2017
    [Advance publication] Released: March 04, 2017
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    Depressive disorders cause large socioeconomic effects influencing not only the patients themselves but also their family and broader community as well. To better understand the physiologic factors underlying depression, in this study, we performed metabolomics analysis, an omics technique that comprehensively analyzes small molecule metabolites in biological samples. Specifically, we utilized high-resolution magic-angle spinning-1H-NMR (HRMAS-1H-NMR) spectroscopy to comprehensively analyze the changes in metabolites in the hippocampal tissue of rats exposed to chronic stress (CS) via multi-step principal component analysis (multi-step PCA). The rats subjected to CS exhibited obvious depression-like behaviors. High correlations were observed between the first principal component (PC1) score in the score plot obtained using multi-step PCA and measurements from depression-like behavioral testing (body weight, sucrose preference test, and open field test). Alanine, glutamate, glutamine, and aspartate levels in the hippocampal tissue were significantly lower, whereas N-acetylaspartate, myo-inositol, and creatine were significantly higher in the CS group compared to the control (non-CS) group. As alanine, glutamate, and glutamine are known to be involved in energy metabolism, especially in the tricarboxylic acid cycle, chronic exogenous stress may have induced abnormalities in energy metabolism in the brains of the rats. The results suggest that N-acetylaspartate and creatine levels may have increased in order to complement the loss of energy-producing activity resulting from the development of the depression-like disorder. Multi-step PCA therefore allowed an exploration of the degree of depression-like symptoms as represented by changes in intrinsic metabolites.

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  • Mi Hee Jang, Kwang-Youn Kim, Phil Hyun Song, Su Youn Baek, Hye Lim Seo ...
    2017 Volume 40 Issue 6 Pages 797-806
    Published: June 01, 2017
    Released: June 01, 2017
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    Moutan Cortex, the root bark of Paeonia suffruticosa ANDREWS in Ranunculaceae, has widely demonstrated analgesic, anti-spasmodic, and anti-inflammatory effects in various cancer and immune cell lines. Oxidative stress is associated with development of several diseases, including liver disease. We prepared the water extract of Moutan Cortex (MCE) to investigate the cytoprotective activities and its mechanism. MCE protected hepatocytes from arachidonic acid (AA)+iron induced oxidative stress, as indicated by reactive oxygen species (ROS) production and cell viability analysis. MCE also suppressed mitochondrial dysfunction in AA+iron-treated human hepatocyte-derived hepatocellular carcinoma cell line, HepG2 cells. In addition, MCE treatment induces AMP-activated protein kinase (AMPK) and liver kinase B1 phosphorylation, which play a role in inhibition of oxidative stress induced cell death. Moreover, one of the MCE compounds, chlorogenic acid, exerted protective effects against oxidative stress and apoptosis. Taken together, MCE protected hepatocytes against AA+iron-induced oxidative stress through AMPK activation, and may be a candidate for the treatment of liver disease.

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  • Keisuke Obara, Daisuke Chino, Yoshio Tanaka
    2017 Volume 40 Issue 6 Pages 807-814
    Published: June 01, 2017
    Released: June 01, 2017
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    Distigmine is a cholinesterase (ChE) inhibitor used for the treatment of detrusor underactivity in Japan. Distigmine’s pharmacological effects are known to be long-lasting, but the duration of its effect on urinary bladder contractile function has not been fully elucidated. The present study aimed to determine these effects in relation to the plasma concentrations of distigmine and its inhibition of ChE activities in blood, plasma, and bladder tissue. Intravesical pressures were recorded in anesthetized guinea-pigs for 12 h after the intravenous administration of saline or distigmine (0.01–0.1 mg/kg). Plasma distigmine concentrations were measured by liquid chromatograph-tandem mass spectrometry (LC-MS/MS), while ChE activities were assayed using 5,5′-dithiobis(2-nitrobenzoic acid). Distigmine (0.1 mg/kg) significantly increased the maximum intravesical pressure at micturition reflex for 12 h post-administration. In contrast, plasma distigmine was only detectable for 6 h post-administration in these animals and a one-compartment model calculated an elimination half-life of 0.7 h. However, bladder and blood acetylcholinesterase activities were significantly inhibited for 12 h after distigmine administration, although plasma ChE activities were not affected. The pharmacodynamic effects of distigmine thus persisted after its elimination from the circulation, indicating that it may bind to bladder acetylcholinesterase, producing sustained enzyme inhibition and enhancement of bladder contractility.

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  • Shaimaa Ibrahim, Tatsuaki Tagami, Tetsuya Ozeki
    2017 Volume 40 Issue 6 Pages 815-823
    Published: June 01, 2017
    Released: June 01, 2017
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    The trans platinum–chloroquine diphosphate dichloride (PtCQ) is a new type of antimalarial drug used to fight parasites resistant to traditional drugs. PtCQ is synthesized by mixing platinum and chloroquine diphosphate (CQ). This study examines two efficient methods for forming a nanodrug, PtCQ-loaded liposomes, for use as a potential antimalarial drug-delivery system: the thin drug–lipid film method to incorporate the drug into a liposomal membrane, and a remote-loading method to load the drug into the interior of a cationic liposome. The membranes accordingly comprised PEGylated neutral or cationic liposomes. PtCQ was efficiently loaded into PEGylated neutral and cationic liposomes using the thin drug–lipid film method (encapsulation efficiency, EE: 76.1±6.7% for neutral liposomes, 1 : 14 drug-to-lipid weight ratio; 70.4±9.8% for cationic liposomes, 1 : 14 drug-to-lipid weight ratio). More PtCQ was loaded into PEGylated neutral liposomes using the remote-loading method than by the thin drug–lipid film method and the EE was maximum (96.1±4.5% for neutral liposomes, 1 : 7 (w/w)). PtCQ was encapsulated in PEGylated cationic liposomes comprising various amounts of cationic lipids (0–20 mol%; EE: 96.9–92.3%) using the remote-loading method. PEGylated neutral liposomes and cationic liposomes exhibited minimum leakage of PtCQ after two months’ storage at 4°C, and further exhibited little release under in vitro culture conditions at 37°C for 72 h. These results provide a useful framework for the design of future liposome-based in vivo drug delivery systems targeting the malaria parasite.

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    Editor’s picks

    In their report, Ibrahim et al. described that generating platinum chloroquine diphosphate dichloride (PtCQ)-loaded polyethylene glycol (PEG)-modified (PEGylated) cationic liposomes exhibiting high drug encapsulation and high drug retention. PtCQ was encapsulated in PEGylated cationic liposomes comprising various amounts of cationic lipids using the remote-loading method. PEGylated neutral liposomes and cationic liposomes exhibited minimum leakage of PtCQ after two months’ storage at 4˚C, and further exhibited little release under in vitro culture conditions at 37˚C for 72 hours. These results provide a useful framework for the design of future liposome-based in vivo drug delivery systems targeting the drug-resistant malaria parasite.

  • Yugo Chisaki, Shoki Aoji, Yoshitaka Yano
    2017 Volume 40 Issue 6 Pages 824-829
    Published: June 01, 2017
    Released: June 01, 2017
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    In general, the risk of adverse drug reactions (ADRs) is higher in elderly patients than in younger patients. In this study, we performed a comprehensive assessment of the risks of possible drug–ADR combinations in elderly patients using the Japanese Adverse Drug Event Report (JADER) database of the Pharmaceutical and Medical Devices Agency (PMDA, Japan) using the reporting odds ratio (ROR) as an index. Data recorded from April 2004 to September 2015 in the JADER database were downloaded from the PMDA website. The patients were classified into younger (≤69 years old) and elderly (≥70 years old) groups. The ROR and 95% confidence interval (CI) were calculated for all combinations of drugs and ADRs for which there were three or more reports in the database, focusing particularly on the combinations where more than 100 cases had been reported in elderly and younger patients. The most frequently reported drug–ADR combination was methotrexate with interstitial lung disease (646 cases). The combination with the highest ROR was methotrexate with lymphoproliferative disorder (ROR: 484.6, 95% CI: 334.1–702.9). In total, 27 drug–ADR combinations were found to have high risk in elderly patients. In conclusion, the findings of this comprehensive assessment of drug–ADR combinations using the JADER database will be valuable for updating the ADR risks for elderly patients in clinical setting.

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  • Takehiro Noda, Tomoyuki Okuda, Kousuke Ban, Ryota Mizuno, Tatsuaki Tag ...
    2017 Volume 40 Issue 6 Pages 830-836
    Published: June 01, 2017
    Released: June 01, 2017
    [Advance publication] Released: January 17, 2017
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    In the development of a drug for intra-articular administration, a sustained-release formulation is desirable since it is difficult to sustain the effects of conventional injections due to fast drug leakage from the joint cavity. In this study, we prepared sustained release gel formulations for intra-articular administration containing indocyanine green (ICG) as a model drug to follow its fate after intra-articular administration in rats with in-vivo imaging system (IVIS). ICG administered as an aqueous solution leaked from the joint cavity in a short time and was excreted out of the body within a day. On the other hand, ICG in the sustained-release formulations was retained and released in the joint cavity for a week. Next, we prepared a sustained-release formulation with hyaluronic acid (HA) as the gel base containing a pain-relief drug (Drug A). We had administered it and other formulations into the rat knee where we injected bradykinin to evaluate their walking distance after 1 and 3 d. The effect of an aqueous solution of Drug A disappeared on day 3. The HA gel formulation without Drug A was more effective than the aqueous solution. The HA gel formulation with Drug A was the most effective; the walking distance was about 85% of the baseline on day 3. This study showed that the gel formulations were effective to sustain the release of a drug in the knee joint, and that the combination of a pain-relief drug with HA gel was effective to improve the mobility of the acute pain model rats.

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  • Risa Takayanagi, Kaori Fujito, Koji Kimura, Yasuhiko Yamada
    2017 Volume 40 Issue 6 Pages 837-843
    Published: June 01, 2017
    Released: June 01, 2017
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    Clinical efficacy and adverse effects of the β-blocking agents, carvedilol, bisoprolol, and metoprolol were analyzed theoretically, and then compared quantitatively, for the purpose of determining their proper use for chronic heart failure. Initially, we evaluated occupancy binding to the β1 and β2 receptors (Фssβ1 and Фssβ2) by these drugs. Thereafter, we examined the relationship between Фssβ1 values and left ventricular ejection fraction (LVEF) increase rate to determine efficacy. The result showed that the efficacy with carvedilol could be attained with a lower Фssβ1 value than the others. Therefore, we constructed a model under the assumption that β-blocking agents exert both indirect action of LVEF increase through the β1 receptor and direct action on ryanodine receptor 2. Using the model, it was suggested that these drugs have no differences in regard to the efficacy, while it was clarified theoretically that only carvedilol produces an effect that directly involves ryanodine receptor 2 at clinical doses. We also investigated decreases in heart rate and forced expiratory volume in 1 s as adverse effects of β-blocking agents using a ternary complex model. It was indicated that carvedilol is less likely to induce a heart rate decrease. Meanwhile, it was also suggested that the risk of an asthmatic attack was higher for carvedilol at clinical doses. Our results are considered useful for selection of a proper β-blocking agent and its administration at a reasonable dose for successful heart failure therapy.

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  • Katsuyo Saito, Tomoo Funayama, Yuichiro Yokota, Takashi Murakami, Yasu ...
    2017 Volume 40 Issue 6 Pages 844-851
    Published: June 01, 2017
    Released: June 01, 2017
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    Epigenetic processes, in addition to genetic abnormalities, play a critical role in refractory malignant diseases and cause the unresponsiveness to various chemotherapeutic regimens and radiotherapy. Herein we demonstrate that histone deacetylase inhibitors (HDACis) can be used to sensitize malignant melanoma B16F10 cells to carbon ion irradiation. The cells were first treated with HDACis (romidepsin [FK228, depsipeptide], trichostatin A [TSA], valproic acid [VPA], and suberanilohydroxamic acid [SAHA, vorinostat]) and were then exposed to two types of radiation (carbon ions and gamma-rays). We found that HDACis enhanced the radiation-induced apoptosis and suppression of clonogenicity that was induced by irradiation, having a greater effect with carbon ion irradiation than with gamma-rays. Carbon ion irradiation and the HDACi treatment induced G2/M and G0/G1 cell cycle arrest, respectively. Thus, it is considered that HDACi treatment enhanced the killing effects of carbon ion irradiation against melanoma cells by inducing the arrest of G1 phase cells, which are sensitive to radiation due to a lack of DNA homologous recombination repair. Based on these findings, we propose that pretreatment with HDACis as radiosensitizers to induce G1 arrest combined with carbon ion irradiation may have clinical efficacy against refractory cancer.

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  • Kenichi Ishibashi, Yoshihiro Takeda, Eriko Nakatani, Kana Sugawara, Ry ...
    2017 Volume 40 Issue 6 Pages 852-859
    Published: June 01, 2017
    Released: June 01, 2017
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    Type 2 diabetic Tsumura, Suzuki, obese, diabetes (TSOD) mice gradually gain weight as compared to corresponding Tsumura, Suzuki, non-obesity (TSNO) control mice, and develop insulin resistance. Although development of type 2 diabetes mellitus is associated with dysfunction of adipocytes, little is known about the properties of adipocytes from TSOD mice. Therefore, we attempted to remove intracorporeal factors and elucidate inherent properties of adipocytes of TSOD mice using adipocytes differentiated from mouse embryonic fibroblasts (MEFs) in vitro. Here, we show that MEFs of TSOD have low potency for differentiation into adipocytes. The percentage of Oil red O-stained cells and levels of adipogenic markers in cells differentiated from MEFs of TSOD are lower than those in cells differentiated from MEFs of TSNO. We further show that treatment with an agonist of peroxisome proliferator-activated receptor-γ (PPARγ) (rosiglitazone) at an early stage of differentiation increases the percentage of Oil red O-stained cells in TSOD-MEFs differentiated into adipocytes. Moreover, the lipid droplet size in those adipocytes is larger than that in the adipocytes differentiated from MEFs of TSNO. Although persistent treatment of MEFs of TSOD with rosiglitazone during differentiation increases the percentage of Oil red O-stained cells, the lipid droplet size in adipocytes treated as such does not reach the size of those treated in early stage only. Thus, activation of PPARγ by its agonist at an early stage of differentiation compensates for the low potency toward adipogenic differentiation of, and accelerates formation of enlarged lipid droplets in adipocytes derived from, MEFs of TSOD mice.

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  • Masumi Takemoto, Hiroaki Takemoto, Haruka Kouno, Tomomi Soeda, Takamic ...
    2017 Volume 40 Issue 6 Pages 860-866
    Published: June 01, 2017
    Released: June 01, 2017
    [Advance publication] Released: April 01, 2017
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    The polyphenolic compound theaflavin, the main red pigment in black tea, possesses many beneficial properties, such as fat-reducing and glucose-lowering capabilities. To produce theaflavin-containing fermentation water on a large scale, we have developed a simple, inexpensive, and selective enzymatic biotransformation method to obtain sufficient levels from fresh green tea leaves. Subsequent administration of theaflavin-containing fermentation water to obese mice on a high-fat diet inhibited body weight gain, decreased casual blood glucose and fasting blood glucose levels, and lowered mesenteric and total fat composition. To note, there were no significant differences observed in food consumption between the experimental and control (water without theaflavin) mice groups. Next, we investigated the effect of this water on blood glucose levels in healthy humans and found that it significantly inhibited blood glucose levels. Thus, we showed that theaflavin-containing fermentation water can be efficiently generated from fresh green tea leaves and demonstrated its significantly potent effects in vivo.

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  • Yuri Ikeuchi-Takahashi, Ayaka Kobayashi, Hiraku Onishi
    2017 Volume 40 Issue 6 Pages 867-877
    Published: June 01, 2017
    Released: June 01, 2017
    [Advance publication] Released: April 01, 2017
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    Topical drug application has the advantage of avoiding systemic side effects. We attempted to develop a long-acting matrix-type tablet containing indomethacin (IM) with low physical stimulus and potent mucoadhesive force to treat pain caused by oral aphtha. A mixture of polyethylene glycol (PEG) and hard fat was used as the tablet base. Ethylcellulose was added to the base in an attempt to control drug release. Tablets with PEG as a base were also prepared for comparison. Polyvinyl alcohols (PVAs) with various degrees of saponification were added to increase the mucoadhesive force. From the optical microscopic observations, formulations using PEG and hard fat exhibit PEG/hard fat dispersions caused by the stabilizing effects of PVA. Although the tablets using PEG and hard fat showed sufficient adhesiveness and sustained drug release, those using PEG as the base did not. Drug release was controlled by the amount of hard fat and the saponification degree of PVA. The drug release rate was most increased in a tablet containing PVA with an intermediate degree of saponification, PEG and hard fat. From differential scanning calorimetry and powder X-ray diffraction, IM was considered to exist in the molecular phase. From the results of buccal administration of tablets to rats, highest tissue concentrations were observed in the tablet containing PVA with the intermediate degree of saponification using PEG and hard fat, and the plasma concentrations were sufficiently low in comparison.

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  • Keisuke Tanamachi, Keisuke Nishino, Natsuki Mori, Toshihiro Suzuki, Se ...
    2017 Volume 40 Issue 6 Pages 878-887
    Published: June 01, 2017
    Released: June 01, 2017
    [Advance publication] Released: March 24, 2017
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    Melanoma is highly malignant, and generally exhibits radioresistance, responding poorly to radiation therapy. We previously reported that activation of P2X7, P2Y6, and P2Y12 receptors is involved in the DNA damage response after γ-irradiation of human lung adenocarcinoma A549 cells. However, it is not clear whether these receptors are also involved in the case of melanoma cells, although P2X7 receptor is highly expressed in various cancers, including melanoma. Here, we show that P2X7 receptor antagonist enhances radiation-induced cytotoxicity in B16 melanoma cells in vitro and in vivo. We confirmed that these cells express P2X7 receptor mRNA and exhibit P2X7 receptor-mediated activities, such as ATP-induced pore formation and cytotoxicity. We further examined the radiosensitizing effect of P2X7 receptor antagonist Brilliant Blue G (BBG) in vitro by colony formation assay of B16 cells. γ-Irradiation dose-dependently reduced cell survival, and pretreatment with BBG enhanced the radiation-induced cytotoxicity. BBG pretreatment also decreased the number of DNA repair foci in nuclei, supporting involvement of P2X7 receptor in the DNA damage response. Finally, we investigated the radiosensitizing effect of BBG on B16 melanoma cells inoculated into the hind footpad of C57BL/6 mice. Neither 1 Gy γ-irradiation alone nor BBG alone suppressed the increase of tumor volume, but the combination of irradiation and BBG significantly suppressed tumor growth. Our results suggest that P2X7 receptor antagonist BBG has a radiosensitizing effect in melanoma in vitro and in vivo. BBG, which is used as a food coloring agent, appears to be a promising candidate as a radiosensitizer.

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  • Kohei Matsuo, Kimihiko Matsusue, Daisuke Aibara, Soichi Takiguchi, Fra ...
    2017 Volume 40 Issue 6 Pages 888-893
    Published: June 01, 2017
    Released: June 01, 2017
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    The fat-specific protein 27 (Fsp27) gene belongs to the cell death-inducing DNA fragmentation factor 45-like effector family. Fsp27 is highly expressed in adipose tissue as well as the fatty liver of ob/ob mice. Fsp27 is directly regulated by the peroxisome proliferator-activated receptor γ (PPARγ) in livers of genetically obese leptin deficient ob/ob mice. In the present study, Fsp27 was markedly induced by 24 h fasting in genetically normal mouse livers and repressed by refeeding a high sucrose diet. In contrast with the liver, Fsp27 expression was decreased in adipose tissue by fasting and increased by refeeding. Interestingly, fasting-induced Fsp27 liver expression was independent of PPARγ. Moreover, Fsp27 expression was induced in the insulin-depleted livers of streptozotocin-treated mice. Finally, Fsp27 expression was repressed by direct injection of glucose or insulin in fasting mice. These results suggest that insulin represses Fsp27 expression in the fasting liver.

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  • Shoyo Shibata, Yusuke Wayama, Ai Tsuyuki, Maiko Matsushita, Koji Chiba ...
    2017 Volume 40 Issue 6 Pages 894-901
    Published: June 01, 2017
    Released: June 01, 2017
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    The number of elderly patients with hematologic malignancies has been steadily increasing with the aging of society. However, little research has been conducted to evaluate the prescription status of drugs for such diseases in Japan. Therefore, the aims of this study were to identify the patient population currently being prescribed drugs for hematologic malignancies in Japan and the direction of drug development. To examine the prescription pattern of drugs for the treatment of hematological malignancies in Japan from 2010–2014, we used the IMS Japan Pharmaceutical Market database and the Japanese Society of Hematology Clinical Practice Guidelines, and for drug development status, we used ClinicalTrials.gov and the University Hospital Medical Information Network Clinical Trials Registry. We found a significant upward trend in prescriptions for molecular-targeted agents, which are typically prescribed over the long term, and a significant downward trend in chemotherapeutic agents, which are usually prescribed for the short term. We also found that recent drug development in hematological malignancies has focused on molecular-targeted agents. These results suggest that drug development should be directed toward anti-tumor agents in hematological malignancies that can help maintain and improve patients’ QOL.

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  • Keiko Unno, Hiroshi Yamada, Kazuaki Iguchi, Hitoshi Ishida, Yasunori I ...
    2017 Volume 40 Issue 6 Pages 902-909
    Published: June 01, 2017
    Released: June 01, 2017
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    Theanine, an amino acid in tea, has significant anti-stress effects on animals and humans. However, the effect of theanine was blocked by caffeine and gallate-type catechins, which are the main components in tea. We examined the anti-stress effect of green tea with lowered caffeine, low-caffeine green tea, on humans. The study design was a single-blind group comparison and participants (n=20) were randomly assigned to low-caffeine or placebo tea groups. These teas (≥500 mL/d), which were eluted with room temperature water, were taken from 1 week prior to pharmacy practice and continued for 10 d in the practice period. The participants ingested theanine (ca. 15 mg/d) in low-caffeine green tea. To assess the anxiety of participants, the state-trait anxiety inventory test was used before pharmacy practice. The subjective stress of students was significantly lower in the low-caffeine-group than in the placebo-group during pharmacy practice. The level of salivary α-amylase activity, a stress marker, increased significantly after daily pharmacy practice in the placebo-group but not in the low-caffeine-group. These results suggested that the ingestion of low-caffeine green tea suppressed the excessive stress response of students. This study was registered at the University Hospital Medical Information Network (ID No. UMIN14942).

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  • Noriaki Nagai, Akina Ueno, Tadatoshi Tanino, Mikako Oka, Yoshimasa Ito
    2017 Volume 40 Issue 6 Pages 910-915
    Published: June 01, 2017
    Released: June 01, 2017
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    In a study to find ways to prevent the side effects of indomethacin (IMC), we previously reported that magnesium ion (Mg2+) can prevent the onset of IMC-induced gastric mucosa in adjuvant-induced arthritis (AA) rats, a model for rheumatoid arthritis (RA). In this study we investigated whether the co-administration of IMC and Mg2+ prevents the formation and aggravation of intestinal ulcerogenic lesions in AA rats. The single oral administration of an excessive dose of IMC (40 mg/kg) induces hemorrhagic lesions and nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) in the jejunal and ileal mucosa of AA rats, and the extent of the lesions, as well as iNOS and NO levels in AA rats are higher than in normal rats. On the other hand, the co-administration of 200 mg/kg Mg2+ attenuates intestinal ulceration and the elevation in the iNOS and NO levels in AA rats. Further, hemorrhagic lesioning and enhanced iNOS and NO levels in AA rats also result from the repetitive oral administration of 3 mg/kg IMC (therapeutic dose) for 42 d (once a day), and these changes are also prevented by the co-administration of 200 mg/kg Mg2+. In conclusion, the co-administration of Mg2+ suppresses the ulcerogenic response to IMC in the jejunal and ileal mucosa of AA rats, probably by preventing the elevation of iNOS and NO levels in the region.

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Notes
  • Takuji Machida, Akina Onoguchi, Kenji Iizuka, Sayuri Ishibashi, Mikiko ...
    2017 Volume 40 Issue 6 Pages 916-921
    Published: June 01, 2017
    Released: June 01, 2017
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    We previously reported that docosahexaenoic acid (DHA) inhibits an increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMCs) through a mechanism involving mainly voltage-dependent Ca2+ channels; however, the effect of DHA on voltage-independent pathways, such as store-operated and receptor-operated Ca2+ entry, and Ca2+ entry through Na+/Ca2+ exchanger (NCX), has not been clarified. In the present study, we investigated the effect of DHA treatment on the expression of transient receptor potential canonical (TRPC) channels, capacitative Ca2+ entry, and Ca2+ entry through NCX in rat cultured VSMCs stimulated with 5-hydroxytryptamine (5-HT). RT-PCR analysis detected TRPC1, TRPC4, and TRPC6 mRNA in cultured VSMCs. DHA treatment for 2 d slightly but significantly decreased TRPC1, but not TRPC4 and TRPC6, mRNA expression. Sarpogrelate, a selective serotonin 5-HT2A receptor inhibitor, completely inhibited the 5-HT-induced increase in [Ca2+]i in cultured VSMCs. Ca2+ influx by adding extracellular Ca2+ (1.3 mM) to the Ca2+-free condition in the presence of 5-HT was partially but significantly inhibited by sarpogrelate. DHA treatment for 2 d had no effect on Ca2+ influx when extracellular Ca2+ was added to the Ca2+-free condition in the presence of either 5-HT alone or 5-HT with sarpogrelate. KB-R7943, a selective inhibitor of reverse mode NCX, significantly suppressed the 5-HT-induced increase of [Ca2+]i. Furthermore, DHA treatment for 2 d significantly decreased NCX1 mRNA expression. These results suggest that DHA seems to have little effect on capacitative Ca2+ entry. Through decreasing NCX1 expression, DHA may suppress the 5-HT-induced increase in [Ca2+]i.

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  • Mayuko Soma, Ainari Konda, Satoko Fujieda, Yoichi Sasaki, Mitsuaki Kei ...
    2017 Volume 40 Issue 6 Pages 922-925
    Published: June 01, 2017
    Released: June 01, 2017
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    Ritodrine, a drug for the treatment of threatened premature labor, is a highly selective beta-2 agonist with the major metabolites of sulfate and glucuronide conjugates. This study investigated the continuous evaluation of the concentration of ritodrine conjugates in relation to the clinical course in twin pregnancy. The subjects were 9 twin-pregnancy mothers who delivered after receiving ritodrine treatment between April 2012 and December 2013. Serum ritodrine sulfate and glucuronide conjugates were deconjugated using their specific enzymes. Ritodrine concentration was measured by liquid chromatography-tandem mass spectrometry. The continuous infusion rate of ritodrine was 2.66±0.67 (0.8–3.54) µg/min/kg, and the average concentration of unchanged ritodrine was 118.8±33.2 (63.8–194.0) ng/mL. During the study period between week 32 and week 36 of gestation, the average ratio of unchanged ritodrine concentration and sulfate ritodrine conjugate concentration for weeks 32, 33, 34, 35, and 36 were 1.7, 1.9, 1.5, 1.7, and 1.7 not significant (N.S.), respectively. The average ratio of unchanged ritodrine concentration and glucuronide ritodrine conjugate concentration were 1.8, 2.2, 1.9, 1.8, and 2.1 (N.S.), respectively. No statistical difference was identified in the ratios of unchanged ritodrine concentration and sulfate or glucuronide ritodrine conjugate concentrations. Large individual differences were shown in the concentration of sulfate and glucuronide during the gestational period. No change in the ratio of the formation of ritodrine metabolites was identified as the gestational age progressed.

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  • Masahiro Iwaki, Hiroaki Shimada, Yuri Irino, Manami Take, Sachiko Egas ...
    2017 Volume 40 Issue 6 Pages 926-931
    Published: June 01, 2017
    Released: June 01, 2017
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    Combination therapy of non-steroidal anti-inflammatory drugs (NSAIDs) and methotrexate (MTX) sometimes triggers adverse effects, such as liver injury, renal failure, gastrointestinal disorders, and myelosuppression, owing to the reduction of MTX clearance. Previous reports have suggested that NSAIDs inhibit renal MTX uptake via organic anion transporters (OATs) and reduced folate transporter (RFC)-1 and efflux via multidrug resistance-associated proteins (MRPs). Recently, our laboratory found inhibitory effects of NSAIDs-glucuronide (NSAIDs-Glu), a major metabolite of NSAIDs, on MRP-mediated MTX transport as a new site of interaction between MTX and NSAIDs. However, it remains unclear that whether NSAIDs-Glu inhibit renal uptake of MTX. Therefore, the present study aimed to evaluate inhibitory effects of several NSAIDs-Glu (diclofenac, R- and S-ibuprofen, R- and S-flurbiprofen, and R- and S-naproxen) on human OAT1 and OAT3-mediated MTX transport. In this study, [3H]MTX uptake was observed by using human OAT1 and OAT3-overexpressing HEK293 cells in the presence or absence of NSAIDs-Glu. All examined NSAIDs-Glu exhibited concentration-dependent inhibitory effects on MTX uptake via OAT1 and OAT3. Our results indicated that NSAIDs-Glu are more potent (5- to 15-fold) inhibitors of OAT3 than OAT1. Moreover, stereoselective inhibitory effects of NSAIDs-Glu on OATs-mediated MTX uptake were not observed, unlike on MRPs-mediated transport. These findings suggest that inhibition of OAT1 and OAT3-mediated renal uptake of MTX by plasma NSAIDs-Glu may be one of the competitive sites underlying complex drug interaction between MTX and NSAIDs.

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  • Cho Rong Kim, Soo Jung Choi, Jae Kyeom Kim, Chan Kyu Park, Min Chul Gi ...
    2017 Volume 40 Issue 6 Pages 932-935
    Published: June 01, 2017
    Released: June 01, 2017
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    We previously reported that the extract of Cinnamomum loureirii (C. loureirii) significantly inhibited acetylcholinesterase (AChE), and identified 2,4-bis(1,1-dimethylethyl)phenol (BP) from C. loureirii as a potential AChE inhibitor. The present study, therefore was undertaken to demonstrate the effects of BP from C. loureirii on learning and memory impairment in trimethyltin (TMT)-treated ICR mice. Y-maze and passive avoidance tests were used to test cognitive ability. Further, changes in biochemical parameters in the brain tissue were also assessed in response to TMT injection and BP intervention. BP pre-administration (20, 40 mg/kg/d) in mice significantly protected cognitive dysfunction induced by TMT (p<0.05). Moreover, BP reduced AChE activity and lipid peroxidation but increased acetylcholine levels in the brain. In conclusion, we suggested that BP protected against TMT-induced cognitive dysfunction, and might be a potential agent for alleviating symptoms of neurodegenerative disorders, such as Alzheimer’s disease, via modulating cholinergic functions.

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  • Ting Liu, Renping Wang, Haojie Xu, Yunlong Song, Yunpeng Qi
    2017 Volume 40 Issue 6 Pages 936-940
    Published: June 01, 2017
    Released: June 01, 2017
    [Advance publication] Released: March 17, 2017
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    Inflammatory bowel disease (IBD) is a refractory illness with remarkably increasing incidence rate all over the world. However, no desirable treatment scheme is available. Therefore, research and development of new drugs for treating IBD are urgently needed. Histone deacetylase 6 (HDAC6) is considered to be a pro-inflammatory factor, thus the inhibitors specifically-targeting HDAC6 may find their way in IBD treatment. In this study, we evaluated the anti-inflammatory activity of a novel potent and selective HDAC6 inhibitor, LTB2, in dextran sulfate sodium (DSS)-induced colitis mouse model. It was found that LTB2 treatment significantly alleviated DSS-induced colitis in mice, as evidenced by body weight, colon length, histological examination, and the disease activity index (DAI) scores of rectal bleeding and diarrhea. More importantly, it showed a better protective effect on the DSS-induced colitis mice than the commonly used mesalazine in the clinic. Our results demonstrated that selective HDAC6 inhibitors may have a good prospect for IBD treatment.

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  • Kanako Shiota, Susumu Hama, Toru Yoshitomi, Yukio Nagasaki, Kentaro Ko ...
    2017 Volume 40 Issue 6 Pages 941-944
    Published: June 01, 2017
    Released: June 01, 2017
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    UV rays induce melanin production in the skin, which, from a cosmetic point of view, is problematic. Reactive oxygen species (ROS) generated in the skin upon UV irradiation are thought to be responsible for melanin production. Thus, effective antioxidants are recognized as useful tools for prevention of UV-induced melanin production. Redox nanoparticles (RNPs) containing nitroxide radicals as free radical scavengers were previously developed, and shown to be effective ROS scavengers in the body. RNPs are therefore expected to be useful for effective protection against UV-induced melanin production. However, as the sizes of RNPs are typically larger than the intercellular spaces of the skin, transdermal penetration is difficult. We recently demonstrated effective transdermal delivery and accumulation of nanoparticles in the epidermal layer via faint electric treatment, i.e., iontophoresis, suggesting that iontophoresis of RNPs may be a useful strategy for prevention of UV-induced melanin production in the skin. Herein, we performed iontophoresis of RNPs on the dorsal skin of hairless mice that produce melanin in response to light exposure. RNPs accumulated in the epidermal layer upon application of iontophoresis. Further, the combination of RNPs with iontophoresis decreased UV-induced melanin spots and melanin content in the skin. Taken together, we successfully demonstrated that iontophoresis-mediated accumulation of RNPs in the epidermis prevented melanin production.

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