Biological and Pharmaceutical Bulletin

Challenges in Retinal Circuit Regeneration
Linking Neuronal Connectivity to Circuit Function

Fumitaka Osakada, Masayo Takahashi

Released: March 01, 2015

[Advance Publication] Released: January 19, 2015

p.341-357

Tremendous progress has been made in retinal regeneration, as exemplified by successful transplantation of retinal pigment epithelia and photoreceptor cells in the adult retina, as well as by generation of retinal tissue from embryonic stem cells and induced pluripotent cells. However, it remains unknown how new photoreceptors integrate within retinal circuits and contribute to vision restoration. There is a large gap in our understanding, at both the cellular and behavioral levels, of the functional roles of new neurons in the adult retina. This gap largely arises from the lack of appropriate methods for analyzing the organization and function of new neurons at the circuit level. To bridge this gap and understand the functional roles of new neurons in living animals, it will be necessary to identify newly formed connections, correlate them with function, manipulate their activity, and assess the behavioral outcome of these manipulations. Recombinant viral vectors are powerful tools not only for controlling gene expression and reprogramming cells, but also for tracing cell fates and neuronal connectivity, monitoring biological functions, and manipulating the physiological state of a specific cell population. These virus-based approaches, combined with electrophysiology and optical imaging, will provide circuit-level insight into neural regeneration and facilitate new strategies for achieving vision restoration in the adult retina. Herein, we discuss challenges and future directions in retinal regeneration research.

Flexibility of the Coordination Geometry at the N-Site of Cu(II)₂ Human Serum-Transferrin Induced by the Different Orientations of Arg124

Toshiyuki Hata, Yu Shibata, Miku Okano, Asako Kodera, Misato Ueda, Hiroshi Iwamoto, Hisao Tomida, Hiroyuki Iwamoto, Junzo Hirose

Released: March 01, 2015

[Advance Publication] Released: January 14, 2015

p.358-364

The ESR spectra of dicupric human serum-transferrin (serum-Tf) were measured from −20 to 37°C in the liquid state (56% glycerol at pH 7.6). Two coordination geometries (types B-1 and B-2) with different ESR parameters were present at the N-site. The contents of the coordination geometry of type B-1 at the N-site increased as the temperature increased. The equilibrium constant between the coordination geometries of types B-1 and B-2 was determined by ESR spectra. The enthalpy value from type B-2 to B-1 was +5.3 kcal/mol, as obtained from a van’t Hoff plot. The two conformational energies of the cluster models of the copper-binding site at the N-site of dicupric human serum-Tf, where the Arg124 residue was oriented in two different directions (conformations I and II), were calculated by Density Functional Theory, and the enthalpy value from conformation II to I was +2.1 kcal/mol. The enthalpy value was similar to that (+5.3 kcal/mol) obtained by the coordination geometrical change from type B-2 to B-1 in Cu(II)₂ serum-Tf. In conformations I and II, the residue of Arg124 at the N-site is located either far from or near the copper-binding site, respectively, and in both cases the coordination geometry of the cupric ions at the N-site has changed from a flattened tetrahedron to a trigonal bipyramid. This result implies that the ESR spectral change from type B-2 to B-1 is caused by the presence of two different orientations of Arg124 in the change from conformation II to I.

Improvement of Transdermal Delivery of Sumatriptan Succinate Using a Novel Self-dissolving Microneedle Array Fabricated from Sodium Hyaluronate in Rats

Dan Wu, Ying-shu Quan, Fumio Kamiyama, Kosuke Kusamori, Hidemasa Katsumi, Toshiyasu Sakane, Akira Yamamoto

Released: March 01, 2015

p.365-373

The purpose of the present study was to develop an alternative transdermal formulation containing sumatriptan succinate (SS) for the treatment of migraine. Novel self-dissolving SS-loaded microneedle arrays (MNs) were fabricated from sodium hyaluronate and their efficacy for transdermal delivery of SS was characterized. The resulting MNs maintained their skin piercing abilities for at least 30 min after being placed at a high relative humidity of 75%. Rapid release of SS from the MNs was also observed in vitro. Optical coherence tomography images demonstrated that MNs were able to successfully pierce into rat skin without any bending or cracking, and needles were completely dissolved within 1 h. MNs significantly increased transepidermal water loss; however, skin barrier function gradually recovered to control levels within 24 h, in contrast to the skin damage observed after tape stripping treatment. These findings indicated that the micropores created by MNs quickly resealed, and that the skin damage was reversible. Furthermore, a dose-dependent plasma concentration of SS was obtained after transdermal delivery using SS-loaded MNs in rats. Absorption of SS delivered by MNs was similar to that observed after subcutaneous injection and was associated with high bioavailability (ca. 90%), which was much higher than that produced by oral administration. These findings suggested that application of SS-loaded MNs to the skin provided an effective alternative approach to enhance the transdermal delivery of SS without serious skin damage, and would be likely to improve patient compliance.

Evaluation of Antitumor Effects of Folate-Conjugated Methyl-β-cyclodextrin in Melanoma

Keiichi Motoyama, Risako Onodera, Nao Tanaka, Kazuhisa Kameyama, Taishi Higashi, Ryusho Kariya, Seiji Okada, Hidetoshi Arima

Released: March 01, 2015

p.374-379

Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-β-cyclodextrin (FA-M-β-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-β-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-β-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-β-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-β-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-β-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-β-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.

Overcoming Stability Challenges in the Quantification of Tissue Nucleotides: Determination of 2′-C-Methylguanosine Triphosphate Concentration in Mouse Liver

Hassan Rashidzadeh, Sanjeev Bhadresa, Steven Spencer Good, Marita Larsson Cohen, Kusum Sachdev Gupta, William Roger Rush

Released: March 01, 2015

p.380-388

A conventional, rapid and high throughput method for tissue extraction and accurate and selective LC-MS/MS quantification of 2′-C-methylguanosine triphosphate (2′-MeGTP) in mouse liver was developed and qualified. Trichloroacetic acid (TCA) was used as the tissue homogenization reagent that overcomes instability challenges of liver tissue nucleotide triphosphates due to instant ischemic degradation to mono- and diphosphate nucleotides. Degradation of 2′-MeGTP was also minimized by harvesting livers using in situ clamp-freezing or snap-freezing techniques. The assay also included a sample clean-up procedure using weak anion exchange solid phase extraction followed by ion exchange chromatography and tandem mass spectrometry detection. The linear assay range was from 50 to 10000 pmol/mL concentration in liver homogenate (250–50000 pmol/g in liver tissue). The method was qualified over three intraday batches for accuracy, precision, selectivity and specificity. The assay was successfully applied to pharmacokinetic studies of 2′-MeGTP in liver tissue samples after single oral doses of IDX184, a nucleotide prodrug inhibitor of the viral polymerase for the treatment of hepatitis C, to mice. The study results suggested that the clamp-freezing liver collection method was marginally more effective in preventing 2′-MeGTP degradation during liver tissue collection compared to the snap-freezing method.

Nano-mechanical Reinforcement in Drug-Resistant Ovarian Cancer Cells

Young Ho Seo, Yoo-na Jo, Yong Jin Oh, Soyeun Park

Released: March 01, 2015

p.389-395

The mechanical properties of cells are considered promising biomarkers for the early detection of cancer and the testing of drug efficacy against it. Nevertheless, generalized correlations between drug resistance and the nano-mechanical properties of cancer cells are yet to be defined due to the lack of necessary studies. In this study, we conducted atomic force microscopy (AFM)-based nano-mechanical measurements of cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian cancer cells. The difference in the efficacy of cisplatin between A2780 and A2780cis was confirmed in the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. We observed that the cisplatin-resistant ovarian cancer cells were more motile than cisplatin-sensitive cells based on the results of the wound closure experiment, and the AFM experiments showed that drug resistance induced nano-mechanical stiffening of the ovarian cancer cells. Increased mechanical stiffness caused by cisplatin resistance was consistent with the confocal microscopy images showing more distinct actin stress fibers in A2780cis than in A2780 cells. The down regulation of vinculin implicated the actin-driven elongation as a major motile mode for A2780cis cells. Our results consistently indicated that the acquisition of drug resistance in ovarian cancer cells induces an extensive reorganization of the actin cytoskeleton, which governs the cellular mechanical properties, motility, and possibly intracellular drug transportation.

Effects of miR-9 and Tetramethylpyrazine on Activation of Hepatic Stellate Cells

Shan-Gao Li, Jun Zhou, Ji-Hong Zhong, Hai-Jun Cao, Ling Zhu, Jun Liu, Hua-Jun Hu, Bin Lv

Released: March 01, 2015

[Advance Publication] Released: December 18, 2014

p.396-401

Micro-RNAs (miRNAs) are involved in regulation of the incidence and development of several hepatic diseases. Thus manipulating miRNAs may be a promising therapeutic strategy against these entities. In this study hepatic stellate cells (HSCs) were transfected with hsa-miR-9 or anti-hsa-miR-9, treated with tetramethylpyrazine (TMP), or subjected to treatment with TMP and hsa-miR-9 transfection (combined treatment group). Then, real-time polymerase chain reaction (PCR) was performed to measure mRNA levels of hsa-miR-9. Expression of hsa-miR-9 was highest in the combination treatment group compared with other groups, and significantly higher than TMP-treated and hsa-miR-9-transfected groups (both p<0.05). The anti-hsa-miR-9-transfected group expressed the lowest mRNA level of hsa-miR-9 with marked decrease versus control (p<0.05). Downstream factors that may be affected by miR-9 such as leptin, α-smooth muscle actin (SMA), and collagen I, as well as phosphorylation levels of Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) were investigated at the protein level. All these factors were regulated contrariwise to expression trends of hsa-miR-9, showing the lowest level in the combination treatment group and highest level in anti-hsa-miR-9-transfected group. These results suggest that both transfection of hsa-miR-9 and TMP can lead to upregulated endogenous expression of hsa-miR-9, inhibit activation of JAK1/STAT3 signal pathway induced by leptin, and lead to reduction of α-SMA and collagen I—thus impeding activation of HSC.

Pharmacokinetic–Pharmacodynamic Analysis of Sunitinib-Induced Thrombocytopenia in Japanese Patients with Renal Cell Carcinoma

Masashi Nagata, Yasuyoshi Ishiwata, Yutaka Takahashi, Hiromitsu Takahashi, Kazutaka Saito, Yasuhisa Fujii, Kazunori Kihara, Masato Yasuhara

Released: March 01, 2015

p.402-410

The aim of the present study was to clarify the therapeutic range and adequate dose of sunitinib in Japanese renal cell carcinoma patients by means of a pharmacokinetic–pharmacodynamic analysis of sunitinib-induced thrombocytopenia. Six patients with renal cell carcinoma were enrolled in this study. After starting the sunitinib treatment, between three and seven blood samples were obtained from each patient just before the administration of sunitinib. Serum concentrations of sunitinib and its active metabolite N-desethyl-sunitinib were fit to the 1-compartment model with first-order absorption. Changes in platelet counts were fit to the pharmacokinetic–pharmacodynamic model, in which the proliferation of platelet progenitor cells was assumed to be linearly inhibited by sunitinib and its metabolite. All patients using 50 mg as an initial dose of sunitinib developed grade 2 or 3 thrombocytopenia. The pharmacokinetic–pharmacodynamic model created successfully described the time course of sunitinib-induced thrombocytopenia and could predict changes in platelet counts after alterations to the dosage of sunitinib administered. The simulation results indicated that the total trough level of sunitinib to avoid severe thrombocytopenia should be <100 ng/mL, and also that the initial daily dose of sunitinib could be reduced to 37.5 mg or 25 mg in most Japanese patients. In addition to the pharmacokinetic-guided dosage adjustment, the careful monitoring of platelet counts is required for the safe use of sunitinib.

Comparative Study of the Anti-leukemic Effects of Imatinib Mesylate, Glivec™ Tablet and Its Generic Formulation, OHK9511

Masako Yokoo, Yasushi Kubota, Yoko Tabe, Shinya Kimura

Released: March 01, 2015

[Advance Publication] Released: January 08, 2015

p.411-416

Long-term treatment with imatinib mesylate (IM) allows patients with chronic myeloid leukemia (CML) to live a near-normal lifespan. However, the fact that tyrosine kinase inhibitors, including IM, are extremely expensive is a major cause of poor adherence, resulting in disease relapse or drug resistance. Therefore, physicians are encouraged to prescribe generic drugs to reduce the financial burden of medical expenses. In Japan, only generic drugs that have a basic chemical structure and pharmacokinetic data that are the same as those of the original drug are approved. However, it is not mandatory to demonstrate that generic drugs have adequate biological effects. This is one of the reasons why Japanese hematologists do not often use generic IM. The aim of the present study was to compare the anti-leukemic effects of Glivec™ (a commercial IM) and its generic formulation, OHK9511. The IC₅₀ values of OHK9511 and Glivec™ were comparable, and both induced similar levels of apoptosis in several CML cell lines. Furthermore, the overall survival of OHK9511-treated mice transplanted with BCR-ABL-positive cells was similar to that of mice treated with Glivec™. Although the experiments performed herein were basic, the results suggest that physicians should consider using generic IM.

Relationship between the Concentration of Anti-polyethylene Glycol (PEG) Immunoglobulin M (IgM) and the Intensity of the Accelerated Blood Clearance (ABC) Phenomenon against PEGylated Liposomes in Mice

Yosuke Hashimoto, Taro Shimizu, Amr Selim Abu Lila, Tatsuhiro Ishida, Hiroshi Kiwada

Released: March 01, 2015

p.417-424

PEGylation, which is the surface modification of nanocarriers with polyethylene glycol (PEG), has increased the circulation time and reduced the immunogenic responses to nanocarriers. However, many reports have demonstrated that the intravenous injection of sterically stabilized PEGylated liposome (SL) causes an accelerated blood clearance (ABC) of subsequent doses via anti-PEG immunoglobulin M (IgM)-mediated complement activation. In the present study, the relationships between serum anti-PEG IgM concentration, the intensity of complement activation and the hepatic clearance of SL were quantitatively investigated for their role in the ABC phenomenon. Interestingly, with increasing serum anti-PEG IgM concentrations, the intensity of complement activation increased linearly, while the intensity of the hepatic clearance of SL was increased and then saturated. In addition, only 15–17% of anti-PEG IgM in blood circulation induced by SL at different doses was associated with a second dose SL. The present results indicate that it is the hepatic uptake of SL that is the limiting step in the ABC phenomenon, rather than the association of anti-PEG IgM to the SL and a subsequent complement activation.

Anti-inflammatory Actions of Herbal Formula Gyejibokryeong-Hwan Regulated by Inhibiting Chemokine Production and STAT1 Activation in HaCaT Cells

Soo-Jin Jeong, Hye-Sun Lim, Chang-Seob Seo, Seong-Eun Jin, Sae-Rom Yoo, Nari Lee, Hyeun-Kyoo Shin

Released: March 01, 2015

p.425-434

Gyejibokryeong-hwan (GJBRH; Keishi-bukuryo-gan in Japan and Guizhi Fuling Wan in China) is a traditional herbal formula comprising five medicinal herbs and is used to treat climacteric syndrome. GJBRH has been shown to exhibit biological activity against diabetes, diabetic nephropathy, atherosclerosis, ischemia, and cancer. However, there is no scientific evidence of its activities against skin inflammation, including atopic dermatitis. We used the HaCaT human keratinocyte cell line to investigate the effects of GJBRH on skin inflammation. No significant cytotoxicity was observed in cells treated with GJBRH up to a concentration of 1000 µg/mL. Exposure to the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) significantly increased HaCaT cell production of the following chemokines: macrophage-derived chemokine (MDC)/CCL22; regulated on activation, normal T-cell expressed and secreted (RANTES)/CCL5; and interleukin-8 (IL-8). In contrast, GJBRH significantly reduced the production of MDC, RANTES, and IL-8 compared with control cells simulated with TNF-α and IFN-γ. Consistently, GJBRH suppressed the mRNA expression of MDC, RANTES, and IL-8 in TNF-α and IFN-γ-treated cells. Treatment with GJBRH markedly inhibited phosphorylation of signal transducer and activator of transcription 1 (STAT1) in HaCaT cells stimulated with TNF-α and IFN-γ. Our findings indicate that GJBRH impairs TNF-α and IFN-γ-mediated inflammatory chemokine production and STAT1 phosphorylation in keratinocytes. We suggest that GJBRH may be a potent therapeutic agent for inflammatory skin disorders.

Induction of Epithelial-Mesenchymal Transition and Down-Regulation of miR-200c and miR-141 in Oxaliplatin-Resistant Colorectal Cancer Cells

Shota Tanaka, Mika Hosokawa, Takeshi Yonezawa, Wataru Hayashi, Kumiko Ueda, Seigo Iwakawa

Released: March 01, 2015

[Advance Publication] Released: December 27, 2014

p.435-440

Epithelial-mesenchymal transition (EMT) and changes in the expression of the microRNA-200 (miR-200) family were examined in the human colorectal cancer (CRC) cell line SW620 with acquired oxaliplatin (L-OHP) resistance. Two CRC cell lines, SW480, derived from primary CRC, and SW620, derived from lymph node metastasis, which were obtained from the same patient, were used in the present study. L-OHP-resistant SW620 cells were obtained by exposure to L-OHP for 155 d. The concentration of L-OHP was increased to 80 µM in a stepwise manner. The IC₅₀ value of L-OHP was increased 16-fold in L-OHP-resistant SW620 cells, which also displayed mesenchymal cell-like characteristics, such as the down-regulation of E-cadherin and up-regulation of vimentin. However, L-OHP-resistant SW480 cells were not obtained when the concentration of L-OHP was increased in a similar stepwise manner. The expression levels of members of the miR-200 family (miR-200a, miR-200b, miR-429, miR-200c, and miR-141) were significantly higher in SW480 cells than in SW620 cells. The expression levels of miR-200c and miR-141 were significantly lower in L-OHP-resistant SW620 cells than in control SW620 cells. L-OHP-resistant SW620 cells did not exhibit cross-resistance to other anti-cancer drugs used to treat CRC, such as 5-fluorouracil, irinotecan, and the active metabolite of irinotecan (SN-38). These results suggest that the down-regulated expression of miR-200c and miR-141 plays a role in selective resistance to L-OHP and EMT in CRC cells during repeated treatments with L-OHP.

In Vitro Inhibition of CYP2C9-Mediated Warfarin 7-Hydroxylation by Iguratimod
Possible Mechanism of Iguratimod–Warfarin Interaction

Satoshi Yamaori, Ken Takami, Ayaka Shiozawa, Kanako Sakuyama, Naoki Matsuzawa, Shigeru Ohmori

Released: March 01, 2015

[Advance Publication] Released: January 19, 2015

p.441-447

Iguratimod is a novel disease-modifying antirheumatic drug. A blue letter (safety advisory) for drug interaction between iguratimod and warfarin was issued by the Ministry of Health, Labour and Welfare of Japan in May 2013. Iguratimod may affect warfarin metabolism catalyzed by CYP. However, it is not clear whether iguratimod inhibits warfarin oxidation. This study was performed to investigate the effects of iguratimod on warfarin 7-hydroxylation with human liver microsomes (HLMs) and recombinant CYP enzymes. Iguratimod concentration-dependently inhibited R,S-warfarin 7-hydroxylase activity of HLMs with an IC₅₀ value of 15.2 µM. The inhibitory effect was examined with S-warfarin and R-warfarin to determine which enantiomer was more potently inhibited by iguratimod. Iguratimod potently inhibited the S-warfarin 7-hydroxylase activity of HLMs with an IC₅₀ value of 14.1 µM, but showed only slight inhibition of R-warfarin 7-hydroxylation. Furthermore, iguratimod inhibited the S-warfarin 7-hydroxylase activity of recombinant CYP2C9.1 (rCYP2C9.1) and rCYP2C9.3 in a concentration-dependent manner with IC₅₀ values of 10.8 and 20.1 µM, respectively. Kinetic analysis of the inhibition of S-warfarin 7-hydroxylation by iguratimod indicated competitive-type inhibition for HLMs and rCYP2C9.1 but mixed-type inhibition for rCYP2C9.3. The K_i values for HLMs, rCYP2C9.1, and rCYP2C9.3 were 6.74, 4.23, and 14.2 µM, respectively. Iguratimod did not exert metabolism-dependent inhibition of S-warfarin 7-hydroxylation. These results indicated that iguratimod is a potent direct inhibitor of CYP2C9-mediated warfarin 7-hydroxylation and that its inhibitory effect on CYP2C9.1 was more sensitive than that on CYP2C9.3.

Serotonin Regulates β-Casein Expression via 5-HT₇ Receptors in Human Mammary Epithelial MCF-12A Cells

Takeshi Chiba, Soichiro Kimura, Katsuo Takahashi, Yasunori Morimoto, Tomoji Maeda, Atsushi Sanbe, Hideo Ueda, Kenzo Kudo

Released: March 01, 2015

p.448-453

We previously reported that serotonin (5-hydroxytryptamine; 5-HT) suppresses β-casein expression, a differentiation marker in mammary epithelial cells, via inhibition of the signal transducer and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial cell line, MCF-12A. In this study, we investigated the expression pattern of the different 5-HT receptor subtypes in MCF-12A cells, and identified the receptors involved in 5-HT-mediated suppression of β-casein protein expression. β-Casein mRNA expression was inhibited by 30 µM 5-HT in a time-dependent manner. Treatment with 30 µM 5-HT for 72 h decreased β-casein protein levels and STAT5 phosphorylation (pSTAT5). The cells expressed four 5-HT receptors subtypes (5-HTR_{1D, 2B, 3A, and 7}) at the mRNA and protein level, and their expression was elevated by prolactin (PRL) treatment. Additionally, the mRNA levels of 5-HTR_1D and 5-HTR₇ were significantly higher than the other 5-HT receptors in the cells. Tryptophan hydroxylase 1 mRNA was detectable in the cells in the absence of PRL, and PRL treatment significantly increased its expression. β-Casein and pSTAT5/STAT5 levels in the cells co-treated with 5-HT and a selective 5-HTR_1D inhibitor, BRL15572, were equal to those observed in cells treated with 5-HT alone. However, in the cells co-treated with 5-HT and a selective 5-HTR₇ inhibitor, SB269970, β-casein and pSTAT5/STAT5 levels increased in a SB269970 concentration-dependent manner. In conclusion, we showed that 5-HT regulates β-casein expression via 5-HTR₇ in MCF-12A human mammary epithelial cells.

Effects of Menthol on the Pharmacokinetics of Triazolam and Phenytoin

Motohiro Hoshino, Nobutomo Ikarashi, Ryuta Hirobe, Mami Hayashi, Hironori Hiraoka, Kohsuke Yokobori, Takumi Ochiai, Yoshiki Kusunoki, Risako Kon, Masataka Tajima, Wataru Ochiai, Kiyoshi Sugiyama

Released: March 01, 2015

[Advance Publication] Released: January 14, 2015

p.454-460

We have previously shown that menthol attenuates the anticoagulant effect of warfarin by increasing the expression levels of CYP3A and CYP2C in the liver. This study evaluated the effects of menthol on the pharmacokinetics of the CYP3A substrate triazolam and the CYP2C substrate phenytoin. Menthol was orally administered to mice for 7 d. Twenty-four hours after the administration of menthol, triazolam was orally administered, and the plasma concentration was measured. In addition, the CYP3A metabolic activity for triazolam and the CYP3A expression level in the liver were determined. The effects of menthol on the pharmacokinetics of phenytoin were assessed in the same manner. In the menthol-treated group, the area under the blood concentration–time curve (AUC) of triazolam was lower and its clearance was higher compared with the control group. The CYP3A metabolic activity and CYP3A expression level in the liver were significantly increased in the menthol-treated group compared with the control group. Similarly, the AUC of phenytoin was lower and the hepatic CYP2C expression level was higher in the menthol-treated group. Thus, menthol lowered the plasma concentrations of triazolam and phenytoin when concurrently administered. These effects may be attributed to an increased metabolic activity for these drugs due to the increased expression of CYP3A and CYP2C in the liver.

Liposomal Pemetrexed: Formulation, Characterization and in Vitro Cytotoxicity Studies for Effective Management of Malignant Pleural Mesothelioma

Noha Essam Eldin, Hanan Mohamed Elnahas, Mahmoud Abd-Elghany Mahdy, Tatsuhiro Ishida

Released: March 01, 2015

p.461-469

Pemetrexed (PMX) is a newly developed multi-targeted anti-folate with promising clinical activity in many solid tumors including malignant pleural mesothelioma (MPM). However, PMX does not show sufficient anti-tumor activity in vivo when used alone either due to inefficient delivery of adequate concentrations to tumor tissue or dose-limiting side effects. In order to overcome these problems and to achieve potent anti-tumor activity, PMX was encapsulated into a liposomal delivery system. In the present study, various formulations of liposomal PMX were prepared. The effect of formulation parameters on the encapsulation efficiency of PMX within liposomes was evaluated. In addition, the influence of drug release rate on the in vitro cytotoxicity was investigated. Encapsulation of PMX within liposomes was remarkably increased by the incorporation of cholesterol within liposomal membranes and by increasing the total lipid concentration. Encapsulation efficiency was found to be unaffected by the type of phospholipid used or the inclusion of a cation lipid, DC-6-14. Interestingly, encapsulation of PMX within “fluid” liposomes was found to allow efficient release of PMX from liposomes resulting in a potent in vitro cytotoxicity against MPM MSTO-211H cell line. On the other hand, entrapment of PMX within “solid” liposomes substantially hindered PMX release from liposomes, and thus PMX failed to exert any in vitro cytotoxicity. These results suggest that encapsulation of PMX within “fluid” liposomes might represent a novel strategy to enhance the therapeutic efficacy of PMX while minimizing the side effect encountered by the non selective delivery of free PMX to various body tissues.

Metabolic Changes in Paraquat Poisoned Patients and Support Vector Machine Model of Discrimination

Xianqin Wang, Meiling Zhang, Jianshe Ma, Yuan Zhang, Guangliang Hong, Fa Sun, Guanyang Lin, Lufeng Hu

Released: March 01, 2015

p.470-475

Numerous people die of paraquat (PQ) poisoning every year in the world. Although several studies regarding paraquat (PQ) poisoning have been conducted, the metabolic changes in plasma remain unknown. In this study, the metabolomics of 15 PQ poisoned patients with plasma PQ concentrations in excess of 0.1 µg/mL and 16 healthy volunteers were investigated. The plasma samples were evaluated through the use of gas chromatography-mass spectrometry (GC/MS) and analyzed by partial least-squares discriminant analysis (PLS-DA). Based on the metabolomics data, a support vector machine (SVM) discrimination model was developed. The results showed the plasma levels of urea, glucose oxime and L-phenylalanine decreased and cholesterol increased in PQ poisoned patients in comparison to healthy volunteers. The SVM discrimination model was developed, and performed with a high degree of accuracy, to distinguish PQ poisoned patients from healthy volunteers. In conclusion, metabolic pathways including the urea cycle, and amino acid, glucose, and cholesterol metabolism were impaired after PQ poisoning. An SVM discrimination model, based on metabolomics data, was established and may become a new powerful tool for the diagnosis of PQ poisoning.

Structure-Dependent Inhibitory Effects of Green Tea Catechins on Insulin Secretion from Pancreatic β-Cells

Yukiko K. Kaneko, Miki Takii, Yumiko Kojima, Hiroko Yokosawa, Tomohisa Ishikawa

Released: March 01, 2015

p.476-481

The effects of green tea catechins on glucose-stimulated insulin secretion (GSIS) were investigated in the β-cell line INS-1D. Epigallocatechin gallate (EGCG) at 10 µM or gallocatechin gallate (GCG) at 30 µM caused significant inhibitory effects on GSIS, and each of these at 100 µM almost abolished it. In contrast, epicatechin (EC) or catechin (CA) had no effect on GSIS at concentrations up to 100 µM. We thus investigated the structure–activity relationship by using epigallocatechin (EGC) and gallocatechin (GC) containing a trihydroxyl group in the B-ring, and epicatechin gallate (ECG) and catechin gallate (CG) containing the gallate moiety. EGC, GC, and ECG caused an inhibition of GSIS, although significant effects were obtained only at 100 µM. At this concentration, EGC almost abolished GSIS, whereas GC and ECG partially inhibited it. In contrast, CG did not affect GSIS at concentrations up to 100 µM. EGCG also abolished the insulin secretion induced by tolbutamide, an ATP-sensitive K⁺ channel blocker, and partially inhibited that induced by 30 mM K⁺. Moreover, EGCG, but not EC, inhibited the oscillation of intracellular Ca²⁺ concentration induced by 11.1 mM glucose. These results suggest that some catechins at supraphysiological concentrations have inhibitory effects on GSIS, the potency of which depends on their structure; the order of potency was EGCG>GCG>EGC>GC≈ECG. The inhibitory effects seem to be mediated by the inhibition of voltage-dependent Ca²⁺ channels, which is caused, at least in part, by membrane hyperpolarization resulting from the activation of K⁺ channels.

ISG15 Regulates RANKL-Induced Osteoclastogenic Differentiation of RAW264 Cells

Tomoharu Takeuchi, Genki Shimakawa, Mayumi Tamura, Hideyoshi Yokosawa, Yoichiro Arata

Released: March 01, 2015

p.482-486

Interferon-stimulated gene 15 kDa (ISG15) is a protein upregulated by interferon-β that negatively regulates osteoclastogenesis. We investigated the role of ISG15 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenic differentiation of murine RAW264 cells. RANKL stimulation induced ISG15 expression in RAW264 cells at both the mRNA and protein levels. Overexpression of ISG15 in RAW264 cells resulted in suppression of cell fusion in RANKL-stimulated cells as well as the reduced expression of ATP6v0d2, a gene essential for cell fusion in osteoclastogenic differentiation. These results suggest that ISG15 suppresses RANKL-induced osteoclastogenesis, at least in part, through inhibition of ATP6v0d2 expression.

Direct Radical Scavenging Activity of Benzbromarone Provides Beneficial Antioxidant Properties for Hyperuricemia Treatment

Daisuke Kadowaki, Shoichi Sakaguchi, Yohei Miyamoto, Kazuaki Taguchi, Nanako Muraya, Yuki Narita, Keizo Sato, Victor Tuan Giam Chuang, Toru Maruyama, Masaki Otagiri, Sumio Hirata

Released: March 01, 2015

[Advance Publication] Released: January 15, 2015

p.487-492

Uric acid exerts an important antioxidant effect against external oxidative stress under physiological conditions. However, uric acid itself can increase oxidative stress via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in adipocytes and vascular cells. Uric acid transporter 1 is involved in the generation of this oxidative stress. Furthermore, uric acid locally activates the renin–angiotensin system, thus producing angiotensin II and subsequently increasing intracellular oxidative stress. Benzbromarone has been reported to suppress uric acid reabsorption via uric acid transporter 1 inhibition in renal tubular cells. In this study we evaluated the in vitro antioxidant effect of benzbromarone from several perspectives. First, the direct radical-trapping capacity of benzbromarone was measured by chemiluminescence assay and electron paramagnetic resonance spectroscopy. Second, the intracellular antioxidant activity of benzbromarone in hyperuricemia was evaluated using endothelial cells. In light of these results, benzbromarone is hypothesized directly to scavenge the superoxide anion radical. In addition, benzbromarone inhibited reactive oxygen species production that was induced by angiotensin II or uric acid in endothelial cells. These findings suggest that benzbromarone possesses the ability directly to scavenge radicals and may act as an antioxidant against uric acid and angiotensin II-induced oxidative stresses in endothelial cells at therapeutically achievable levels in blood.

Antibacterial Drug Treatment Increases Intestinal Bile Acid Absorption via Elevated Levels of Ileal Apical Sodium-Dependent Bile Acid Transporter but Not Organic Solute Transporter α Protein

Masaaki Miyata, Kenjiro Hayashi, Hiroki Yamakawa, Yasushi Yamazoe, Kouichi Yoshinari

Released: March 01, 2015

[Advance Publication] Released: December 27, 2014

p.493-496

Antibacterial drug treatment increases the bile acid pool size and hepatic bile acid concentration through the elevation of hepatic bile acid synthesis. However, the involvement of intestinal bile acid absorption in the increased bile acid pool size remains unclear. To determine whether intestinal bile acid absorption contributes to the increased bile acid pool in mice treated with antibacterial drugs, we evaluated the levels of bile acid transporter proteins and the capacity of intestinal bile acid absorption. Ileal apical sodium-dependent bile acid transporter (ASBT) mRNA and protein levels were significantly increased in ampicillin (ABPC)-treated mice, whereas organic solute transporter α (OSTα) mRNA levels, but not protein levels, significantly decreased in mice. Similar alterations in the expression levels of bile acid transporters were observed in mice treated with bacitracin/neomycin/streptomycin. The capacity for intestinal bile acid absorption was evaluated by an in situ loop method. Increased ileal absorption of taurochenodeoxycholic acid was observed in mice treated with ABPC. These results suggest that intestinal bile acid absorption is elevated in an ASBT-dependent manner in mice treated with antibacterial drugs.

Volume And Issue List
Biological and Pharmaceutical Bulletin

Predecessor

Biological and Pharmaceutical Bulletin Vol. 38(2015) No. 3

Review

Challenges in Retinal Circuit Regeneration
Linking Neuronal Connectivity to Circuit Function

Regular Article

Flexibility of the Coordination Geometry at the N-Site of Cu(II)₂ Human Serum-Transferrin Induced by the Different Orientations of Arg124

Improvement of Transdermal Delivery of Sumatriptan Succinate Using a Novel Self-dissolving Microneedle Array Fabricated from Sodium Hyaluronate in Rats

Evaluation of Antitumor Effects of Folate-Conjugated Methyl-β-cyclodextrin in Melanoma

Overcoming Stability Challenges in the Quantification of Tissue Nucleotides: Determination of 2′-C-Methylguanosine Triphosphate Concentration in Mouse Liver

Nano-mechanical Reinforcement in Drug-Resistant Ovarian Cancer Cells

Effects of miR-9 and Tetramethylpyrazine on Activation of Hepatic Stellate Cells

Pharmacokinetic–Pharmacodynamic Analysis of Sunitinib-Induced Thrombocytopenia in Japanese Patients with Renal Cell Carcinoma

Comparative Study of the Anti-leukemic Effects of Imatinib Mesylate, Glivec™ Tablet and Its Generic Formulation, OHK9511

Relationship between the Concentration of Anti-polyethylene Glycol (PEG) Immunoglobulin M (IgM) and the Intensity of the Accelerated Blood Clearance (ABC) Phenomenon against PEGylated Liposomes in Mice

Anti-inflammatory Actions of Herbal Formula Gyejibokryeong-Hwan Regulated by Inhibiting Chemokine Production and STAT1 Activation in HaCaT Cells

Induction of Epithelial-Mesenchymal Transition and Down-Regulation of miR-200c and miR-141 in Oxaliplatin-Resistant Colorectal Cancer Cells

In Vitro Inhibition of CYP2C9-Mediated Warfarin 7-Hydroxylation by Iguratimod
Possible Mechanism of Iguratimod–Warfarin Interaction

Serotonin Regulates β-Casein Expression via 5-HT₇ Receptors in Human Mammary Epithelial MCF-12A Cells

Effects of Menthol on the Pharmacokinetics of Triazolam and Phenytoin

Liposomal Pemetrexed: Formulation, Characterization and in Vitro Cytotoxicity Studies for Effective Management of Malignant Pleural Mesothelioma

Metabolic Changes in Paraquat Poisoned Patients and Support Vector Machine Model of Discrimination

Structure-Dependent Inhibitory Effects of Green Tea Catechins on Insulin Secretion from Pancreatic β-Cells

Notes

ISG15 Regulates RANKL-Induced Osteoclastogenic Differentiation of RAW264 Cells

Direct Radical Scavenging Activity of Benzbromarone Provides Beneficial Antioxidant Properties for Hyperuricemia Treatment

Antibacterial Drug Treatment Increases Intestinal Bile Acid Absorption via Elevated Levels of Ileal Apical Sodium-Dependent Bile Acid Transporter but Not Organic Solute Transporter α Protein

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Volume And Issue List Biological and Pharmaceutical Bulletin

Predecessor

Biological and Pharmaceutical Bulletin Vol. 38(2015) No. 3

Review

Challenges in Retinal Circuit Regeneration Linking Neuronal Connectivity to Circuit Function

Regular Article

In Vitro Inhibition of CYP2C9-Mediated Warfarin 7-Hydroxylation by Iguratimod Possible Mechanism of Iguratimod–Warfarin Interaction

Notes

System Maintenance

Announcement From J-STAGE

Journal Tools

Share this Title

Volume And Issue List
Biological and Pharmaceutical Bulletin

Challenges in Retinal Circuit Regeneration
Linking Neuronal Connectivity to Circuit Function

In Vitro Inhibition of CYP2C9-Mediated Warfarin 7-Hydroxylation by Iguratimod
Possible Mechanism of Iguratimod–Warfarin Interaction