Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 44 , Issue 3
Showing 1-25 articles out of 25 articles from the selected issue
Communications to the Editor
  • Yoshitaka Saito, Kazuki Uchiyama, Tatsuhiko Sakamoto, Kosei Kubota, Hi ...
    2021 Volume 44 Issue 3 Pages 293-297
    Published: March 01, 2021
    Released: March 01, 2021
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    Chemotherapy regimen management is one of the most important oncology pharmacy practices, because chemotherapy is conducted according to the registered regimens. In this study, we evaluated the pharmaceutical practice that assumes the initial confirmation of chemotherapy regimens and the quality of practice sharing between oncology-specialized and non-specialized pharmacists in regimen management committee. Pharmacists initially confirmed the applied regimen prescribed by physicians regarding chemotherapeutic agents and prophylactic supportive care medicines. Following confirmation, the regimens were reviewed by the Hokkaido University Hospital Regimen Management Committee. A total of 233 regimens were reviewed by the committee over three years. In total, 110 pharmaceutical inquiries were conducted, 45% of inquiries were concerning chemotherapeutic agents, of which approximately half were regarding supportive care medicines. Most inquiries were regarding premedication, followed by those on administration time, solvent of infusion medicines, and dosage. Correction was performed for 84.5% of inquiries. There was no significant difference in inquiry rates between practice and trial regimens. We have entrusted the first basic regimen review according to the checklist, creation of the chemotherapy plan document, and registry of the adopted regimens in the ordering system from oncology-certified pharmacists to non-certified pharmacists. Basic regimen review was well conducted by a non-certified pharmacist, and a more advanced review was additionally performed by certified pharmacists. In conclusion, we demonstrated the utility of pharmaceutical confirmation in a chemotherapeutic regimen review, suitable review coverage, and quality practice sharing between oncology-certified and non-certified pharmacists, which is one of the recommended methods in chemotherapy regimen review.

  • Duc Dat Le, Duc Hung Nguyen, Eun Sook Ma, Jeong Hyung Lee, Byung Sun M ...
    2021 Volume 44 Issue 3 Pages 298-304
    Published: March 01, 2021
    Released: March 01, 2021
    [Advance publication] Released: December 23, 2020
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    Supplementary material

    The white-flowered leaves of Eclipta prostrata L. together with leaves of Scoparia dulcis and Cynodon dactylon are mixedly boiled in water and given to diabetic patients resulting in the significant improvement in the management of diabetes. However, the active constituents from this plant for antidiabetic and anti-obesity properties are remaining unclear. Thus, this study was to discover anti-diabetes and anti-obesity activities through protein tyrosine phosphatases (PTP)1B inhibitory effects. We found that the fatty acids (23, 24) showed potent PTP1B inhibition with IC50 values of 2.14 and 3.21 µM, respectively. Triterpenoid-glycosides (1215) also exhibited strong to moderate PTP1B inhibitory effects, with IC50 values ranging from 10.88 to 53.35 µM. Additionally, active compounds were investigated for their PTP1B inhibitory mechanism and docking analysis. On the other hand, the anti-inflammatory activity from our study revealed that compounds (14, 7, 8, 10) displayed the significant inhibition nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Especially, compound 9 showed the potent inhibitory effects in LPS-induced NO production on RAW264.7 cell. Therefore, further Western blot analysis was performed to identify the inhibitory expression including heme oxygenase-1 (HO-1) and inhibitor of kappaB (IκB) phosphorylation.

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Regular Articles
  • Xin Yang, Zejun Pei, Renjing Hu, Zhehao Zhang, Zaixiang Lou, Xin Sun
    2021 Volume 44 Issue 3 Pages 305-315
    Published: March 01, 2021
    Released: March 01, 2021
    [Advance publication] Released: January 13, 2021
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    Supplementary material

    In order to prevent and control the infection of Candida albicans, the antifungal activity, possible mechanism of myriocin against C. albicans and its biofilm were studied. The antifungal activity of myriocin was investigated by microdilution method. The effect of myriocin on fungal cell wall or membrane was evaluated by adding sorbitol, ergosterol or phytosphingosine (PHS). The damage to the cell membrane was investigated with propidium iodide (PI) staining and visualized by scanning electron microscope (SEM). The effects on biofilms and extracellular polysaccharides (EPS) were observed by crystal violet staining method and phenol-sulfuric acid method respectively. The adhesion of C. albicans cells to hydrocarbons was tested to evaluate cell surface hydrophobic (CSH). The combined effects of myriocin and antifungal drugs commonly used in clinical practice were investigated by using the checkerboard microdilution method. Minimal inhibitory concentrations (MICs) were found to be 0.125–4 µg/mL. Myriocin was found to affect both cell wall and cell membrane. After exposure to myriocin, biofilm and EPS were found to be inhibited and removed, and the CSH was decreased. The combined fungistasis of myriocin and voriconazole (VCZ) or amphotericin B (AMB) were additive. Myriocin had significant antifungal activity against C. albicans, and the antifungal mechanisms might be cell wall and membrane damage. Myriocin effectively inhibited and eliminated biofilms, and its mechanism may be related to the inhibition of EPS and CSH.

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  • Xiaohong Kang, Huijing Liang, Yating Luo, Zhouyong Li, Fang He, Xue Ha ...
    2021 Volume 44 Issue 3 Pages 316-324
    Published: March 01, 2021
    Released: March 01, 2021
    [Advance publication] Released: December 25, 2020
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    It is well documented that obesity and metabolic syndrome have a deep association with the intestinal immune system of the host animal. Recent studies indicate that some selected probiotics can modulate the immune responses of the host animal, thereby altering its lipid metabolism. However, the underlying mechanisms are still not fully understood. This study was conducted to investigate the possibility of probiotics to activate macrophages in the hosts, thus alter the differentiation of pre-adipocytes. In this study, Streptococcus thermophilus MN-ZLW-002 (MN-ZLW-002) was co-cultured with RAW264.7 macrophages, with Lactobacillus rhamnosus GG (LGG) as a control. The conditioned medium (CM) of the co-culture was collected and then added to 3T3-L1 pre-adipocytes. Viable and heat-killed (80 °C, 30 min) MN-ZLW-002 stimulated RAW264.7 cells to produce significant amounts of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and induced intense phosphorylation of P38, p44/42 mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase (ERK)) and nuclear factor κB (NF-κB). Cytokine production reduced dramatically when heat-killed MN-ZLW-002 was treated with Ribonuclease. Viable and heat-killed LGG induced less cytokine production and little signaling protein activation. Viable and heat-killed MN-ZLW-002-stimulated RAW264.7-CM notably suppressed pre-adipocytes differentiation. However, viable LGG-stimulated RAW264.7-CM had a weaker effect and heat-killed LGG-stimulated RAW264.7-CM had no effect. These findings suggest that viable and heat-killed (80 °C, 30 min) MN-ZLW-002 may alter its lipid metabolism by regulating its immune response, possibly via the release of cytokine, particularly TNF-α. The RNA of heat-killed MN-ZLW-002 may be a key component in its immune activation effect.

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  • Yueyi Li, Yan Dong, Li Meng, Panpan Yu, Penghui Zhao, Miao Gong, Qiang ...
    2021 Volume 44 Issue 3 Pages 325-331
    Published: March 01, 2021
    Released: March 01, 2021
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    The neuroprotective effects of heme oxygenase (HO) have been well investigated. The potential effects of exogenous supplementation of biliverdin (BVD), one of the main products catalyzed by HO, on neurobehaviors are still largely unknown. The present study aimed to investigate the effects of BVD treatment on depression, anxiety, and memory in adult mice. Mice were injected with BVD through tail vein daily for a total 5 d, and depression- and anxiety-like behaviors were conducted by using open field test (OFT), novelty suppressed feeding (NSF), forced swimming test (FST) and tail suspension test (TST) since the third day of BVD administration. Novel object recognition (NOR) paradigm was used for memory formation test. After the final test, serum and hippocampal levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) of mice were analyzed by enzyme-linked immunosorbent assay (ELISA). The results showed that BVD treatment at low dose (2 mg/kg) induced depression-like behaviors, and high dose (8 mg/kg) BVD injection increased anxiety-like behaviors and impaired memory formation in mice. ELISA data showed that BVD treatment significantly increased hippocampal IL-6 and TNF-α level while only decreasing serum IL-6 level of mice. The present data suggest that exogenous BVD treatment induced depression- and anxiety-like phenotypes, which may be related to inflammatory factors, providing BVD may be a potential target for the prevention of mental disorders.

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  • Takato Nikaido, Shota Muroga, Chikashi Maruyama, Yukiko Fujimaru, Tosh ...
    2021 Volume 44 Issue 3 Pages 332-337
    Published: March 01, 2021
    Released: March 01, 2021
    [Advance publication] Released: December 19, 2020
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    Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to cause gastric mucosal damage, in which gastric hypermotility has been reported to play a primary role. The antipyretic analgesic drug ethenzamide (ETZ) is widely used in combination with other NSAIDs and, in a recent study, was found to possess 5-hydroxytriptamine (5HT)2B receptor antagonistic activity. Therefore, the inhibition of gastric contraction via 5HT2B receptor blockade by ETZ might contribute to ETZ’s protective effect against NSAIDs-induced gastric mucosal damage. In the present study, we examined the effects of ETZ on gastric contraction and ibuprofen (IBP)-induced gastric mucosal damage in rats. We found that ETZ suppressed both 5HT- and α-methyl-5HT (5HT2 receptor agonist)-induced contractions of rat-isolated gastric fundus in a concentration-dependent manner. This suppressive effect of ETZ was not seen for either high-KCl- or acetylcholine-induced contractions. Furthermore, ETZ was confirmed to decrease ibuprofen-induced gastric mucosal damage in a dose-dependent manner in rats. Similarly, clonidine is known to reduce gastric motility, and methysergide (a 5HT2 receptor antagonist) is known to inhibit 5HT-induced contractions of the gastric fundus, which also decreases IBP-induced gastric mucosal damage, respectively. Although further research on other possible sites or mechanisms of action would be needed, these results suggest that ETZ exerts a protective effect against IBP-induced gastric mucosal damage and that suppressing the gastric contraction may play an important role in the gastroprotective effect of ETZ.

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  • Haruka Tsutsui, Shino Kuramoto, Kazuhisa Ozeki
    2021 Volume 44 Issue 3 Pages 338-349
    Published: March 01, 2021
    Released: March 01, 2021
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    Supplementary material

    Established guidelines have recommended a number of methods based on in vitro data to assess the CYP3A induction risk of new chemical entities in clinical practice. In this study, we evaluated the predictability of various assessment methods. We collected in vitro parameters from a variety of literature that includes data on 19 batches of hepatocytes. Clinical CYP3A induction was predicted using 3 direct approaches—the fold-change, basic model, and mechanistic static models—as well as 5 correlation approaches, including the relative induction score (RIS) and the relative factor (RF) method. These predictions were then compared with data from 30 clinical inductions. Collected in vitro parameters varied greatly between hepatocyte batches. Direct assessment methods using fixed cut-off values provided a lot of false predictions due to hepatocyte variability, which can overlook induction risk or lead to needless clinical drug–drug interaction (DDI) studies. On the other hand, correlation methods with the cut-off values set for each batch of hepatocytes accurately predicted the induction risk. Among these, the AUCu/inducer concentrations for half the maximum induction (EC50) and the RF methods which use the area under the curve (AUC) of the unbound inducers for calculating induction potential showed an especially good correlation with clinical induction. Correlation methods were better at predicting clinical induction risk than the other methods, regardless of hepatocyte variability. The AUCu/EC50 and the RF methods in particular had a small number of false predictions, and can therefore be used to assess induction risk along with the other correlation methods recommended in guidelines.

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    Editor’s picks

    The authors evaluated the predictability of various methods used to assess clinical CYP3A induction risk based on various in vitro parameters, and demonstrated that correlation methods were better at predicting clinical induction risk than direct methods recommended in guidance/guidelines. Among correlation approaches, the Relative Factor (RF) and AUC/F2 methods showed an especially good correlation with clinical induction, and can be used to assess induction risk along with other correlation methods recommended in guidance/guidelines. These findings may allow researchers to more confidently determine whether or not a clinical induction study should be performed before clinical trials.

  • Yanlin Chen, Jingxian Wu, Jin Zhu, Guoan Yang, Junying Tian, Yong Zhao ...
    2021 Volume 44 Issue 3 Pages 350-356
    Published: March 01, 2021
    Released: March 01, 2021
    [Advance publication] Released: December 25, 2020
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    Inflammation has an important role in ischemia–reperfusion (I/R) injury. Artesunate (ART) has anti-microbial and anti-inflammatory pharmacological activities, and it is used for various types of serious malaria, including cerebral malaria. ART maintains a high concentration in the brain but little is known about the neuroprotective effect of ART against brain I/R injury. We studied the neuroprotection of ART against brain I/R injury and its underlying mechanism. In this study, rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h. After 24 h of reperfusion, neurological deficits, cerebrum water content, infarct volume, hematoxylin–eosin (H&E)-staining, myeloperoxidase (MPO) activity, and proinflammatory cytokine levels were measured. Administration of 20, 40, 80, and 160 mg/kg ART intraperitoneally (i.p.) 10 min after MCAO significantly decreased brain water content and improved neurological deficits in a dose-dependent manner. An 80 mg/kg dosage was optimal. ART significantly reduced infarct volume, suppressed MPO activity and diminished the expressions of toll-like receptor (TLR)-4, MyD88, nuclear factor-κB (NF-κB), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in the area of the ischemic cortex. The neuroprotective action of ART against focal cerebral I/R injury might be due to the attenuation of inflammation through the TLR-4/NF-κB pathway.

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  • Chikako Matsumura, Masami Yamada, Yumi Jimaru, Rie Ueno, Kazushige Tak ...
    2021 Volume 44 Issue 3 Pages 357-362
    Published: March 01, 2021
    Released: March 01, 2021
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    Cancer pain is one of the most frequent and distressing symptoms associated with cancer and has a serious impact on the QOL of patients. However, inadequate pain treatment has also been reported in outpatients with cancer pain. The aims of this study were (1) to evaluate the relationship between pain intensity using the Numerical Rating Scale (NRS) and QOL scores using the Japanese version of the European Organization for Research and Treatment of Cancer (QOL Questionnaire Core 15 for Palliative Care (QLQ-C15-PAL)), and (2) to investigate their association with various pain patterns, especially with baseline and breakthrough pain. Forty outpatients who were receiving opioid therapy and obtained informed consent participated. We collected a total of 222 pharmacist consultations during the study period. Global QOL scores and pain scores (PA) in the QLQ-C15-PAL (PA score, 0–100) at the first visit were significantly correlated with worst pain intensity. In addition, the scores for the worst pain were significantly correlated with not only physical functioning scores but also with emotional functioning scores. The correlations between the worst pain NRS and PA scores were positive. Specifically, patients tended to report large variability of NRS scores when the PA score was less than 40 and also when they exhibited pain patterns with “baseline and breakthrough cancer pain in the same day” or “baseline pain throughout the day.” Reducing the worst pain NRS and relieving breakthrough pain appear to be important measures to improve the QOL of outpatients receiving opioid therapy for cancer pain.

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  • Daisuke Yasuda, Yoshinori Hiraoka, Mikiko Ohno, Kiyoto Nishi, Hirotaka ...
    2021 Volume 44 Issue 3 Pages 363-371
    Published: March 01, 2021
    Released: March 01, 2021
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    Nardilysin (NRDC) has been shown to be involved in post-translational histone modifications, in addition to enhancement in ectodomain shedding of membrane-anchored protein, which play significant roles in various pathophysiology, including glucose homeostasis, inflammatory diseases and cancer. The present study sought to determine roles of NRDC in the liver on lipid and lipoprotein metabolism. We established liver-specific NRDC deficient mice by use of NRD1 floxed mice and albumin promoter-Cre recombinase (Cre) transgenic mice, and found that their serum low-density lipoprotein (LDL) cholesterol levels were significantly lower than those in control littermate mice. In the liver, LDL receptor (LDLR) mRNA expression was significantly upregulated, while inducible degrader of LDLR (IDOL) and microsomal triglyceride transfer protein (MTP) mRNA expression was significantly downregulated, in liver-specific NRDC deficient mice. Hepatic cell-surface LDLR expression levels were significantly elevated and serum pro-protein convertase subtilisin–kexin type 9 (PCSK9) levels were significantly reduced in mice with hepatic NRDC deficiency. In cultured hepatocytes, NRDC deficiency significantly reduced secreted PCSK9 and increased cell-surface LDLR expression. On the other hand, NRDC overexpression in cultured hepatocytes significantly increased secreted PCSK9 and lowered cell-surface LDLR expression. Thus, NRDC in murine hepatocytes appears to play key roles in cholesterol homeostasis, although the precise molecular mechanisms remain to be determined.

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  • Maaged Akiel, Jawaher Alsughayyir, Ahmed M. Basudan, Hassan S. Alamri, ...
    2021 Volume 44 Issue 3 Pages 372-378
    Published: March 01, 2021
    Released: March 01, 2021
    [Advance publication] Released: January 09, 2021
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    Supplementary material

    The prevalence of cancer-associated anemia (CIA) is high, and the mechanisms governing its development remain poorly understood. Eryptosis, the suicidal cell death of red blood cells (RBCs), may account for CIA as it is triggered by clinically approved chemotherapeutics including cisplatin and paclitaxel. Physcion (PSN), an anthraquinone extracted from rhubarb and other plants, has shown great promise as an anticancer agent. However, the potential toxicity of PSN to RBCs remains elusive. RBCs were isolated from heparinized blood, and incubated with 10–100 µM of PSN for 24 h at 37 °C. Hemolysis was photometrically calculated from hemoglobin concentration in the medium at 405 nm, while flow cytometry was employed to investigate cardinal markers of eryptosis. Phosphatidylserine (PS) exposure was detected by Annexin-V-fluorescein isothiocyanate (FITC), intracellular calcium by Fluo4/AM, cellular volume from forward scatter (FSC), and oxidative stress by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). PSN induced overt hemolysis at 50 and 100 µM which was not mediated through calcium influx, protein kinase C, casein kinase 1α, or receptor-interacting protein 1. Moreover, PSN caused significant increase in Annexin-V-FITC and Fluo4 fluorescence with no appreciable influence on FSC or DCF values. Accordingly, PSN stimulates premature eryptosis characterized by PS externalization and intracellular calcium overload without cell shrinkage or oxidative damage. In conclusion, this report shows, for the first time, that PSN is cytotoxic to RBCs by inducing hemolysis and programmed cell death which may limit its success as a chemotherapeutic agent.

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  • Huan Ding, Jing-jing Wang, Xiao-Ya Zhang, Lei Yin, Tao Feng
    2021 Volume 44 Issue 3 Pages 379-388
    Published: March 01, 2021
    Released: March 01, 2021
    [Advance publication] Released: December 26, 2020
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    Lipopolysaccharide (LPS)-induced inflammation is the leading cause of multiple organ failure in sepsis. Pyruvate kinase 2 (PKM2) is a protein kinase and transcriptional coactivator that plays an important role in glycolysis. Recent studies have confirmed that glycolysis maintains the M1 differentiation and induces immune activation in macrophages. Lycium barbarum polysaccharide (LBP), the main bioactive component of Chinese wolfberry, suppresses glycolysis and inflammation. Here, RAW264.7 macrophages were treated with LBP for evaluating its effects against LPS-induced inflammation. The differentiation of M1/M2 macrophages was assessed by flow cytometry for assessing the cell surface markers, CD86 and CD206. The enrichment of hypoxia inducible factor (HIF)-1α and ubiquitin in the PKM2 protein complex was determined by co-immunoprecipitation. LBP suppressed LPS-induced glycolysis, differentiation of M1 macrophages, and the production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and high mobility group (HMG) 1 proteins. The suppressive effects of LBP were similar to those of PKM2 knockdown, but were abolished by the overexpression of PKM2. LPS elevated the mRNA and protein levels of PKM2. LBP reduced the LPS-induced expression of PKM2 protein, but had no effects on the expression of PKM2 mRNA. LPS inhibited the ubiquitination of PKM2, probably by downregulating the expression of ubiquitin ligases, including Nedd4L, Nedd4, and Gnb2. LBP interfered with the inhibition of PKM2 ubiquitination by upregulating the expression of Nedd4L, Nedd4, and Gnb2. In conclusion, LBP suppressed the LPS-induced inflammation by altering glycolysis and the M1 differentiation of macrophages. The effects of LBP were mediated by the downregulation of PKM2 via enhanced ubiquitination.

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  • Genki Nakamura, Kazuhisa Ozeki, Hiroaki Takesue, Mitsuyasu Tabo, Ken-i ...
    2021 Volume 44 Issue 3 Pages 389-395
    Published: March 01, 2021
    Released: March 01, 2021
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    Supplementary material

    Human pharmacokinetics (PK) profiles of monoclonal antibodies (mAbs) are usually predicted using non-human primates (NHP), but this comes with drawbacks in terms of cost and throughput. Therefore, we established a human PK profile prediction method using human neonatal Fc receptor (hFcRn) transgenic mice (TgM). We administered launched 13 mAbs to hFcRn TgM and measured the concentration in plasma using electro-chemiluminescence immunoassay. This was then used to calculate PK parameters and predict human PK profiles. The mAbs showed a bi-phased elimination pattern, and clearance (CL) (mL/d/kg) and distribution volume at steady state (Vdss) (mL/kg) ranges were 11.0 to 131 and 110 to 285, respectively. There was a correlation in half-life at elimination phase (t1/2β) between hFcRn TgM and humans for 10 mAbs showing CL of more than 80% in the elimination phase (R2 = 0.714). Human t1/2β was predicted using hFcRn TgM t1/2β; 9 out of 10 mAbs were within 2-fold the actual values, and all mAbs were within 3-fold. Regarding the predicted CL values, 7 out of 10 mAbs were within 2-fold the human values and all mAbs were within 3-fold. Furthermore, even on day 7 the predicted CL values of 8 out of 10 mAbs were within 2-fold the observed value, with all mAbs within 3-fold. These results suggest human PK profiles can be predicted using hFcRn TgM data. These methods can accelerate the development of antibody drugs while also reducing cost and improving throughput.

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  • Xi-juan Zhang, Zhong-hua Cui, Yan-xin Zhao, Ting-ting He, Ling Wang, X ...
    2021 Volume 44 Issue 3 Pages 396-403
    Published: March 01, 2021
    Released: March 01, 2021
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    Ferulic acid (FA) has potential therapeutic effects in multiple diseases including cardiovascular diseases. However, the effect and molecular basis of FA in heart failure (HF) has not been thoroughly elucidated. Herein, we investigated the roles and mechanisms of FA in HF in isoproterenol (ISO)-induced HF rat model. Results found that FA ameliorated cardiac dysfunction, alleviated oxidative stress, reduced cell/myocardium injury-related enzyme plasma level, inhibited cardiocyte apoptosis in ISO-induced HF rat models. Moreover, FA reduced the co-localization of Keap1 and nuclear factor-E2-related factor 2 (Nrf2) in heart tissues of ISO-induced HF rats, and FA alleviated the inhibitory effects of ISO on expressions of p-Nrf2, heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate quinone dehydrogenase 1 (NQO1). Additionally, Nrf2 signaling pathway inhibitor ML385 showed adverse effects. FA weakened the effects of ML385 in ISO-induced HF rat models. Collectively, FA ameliorated HF by decreasing oxidative stress and inhibiting cardiocyte apoptosis via activating Nrf2 pathway in ISO-induced HF rats. Our data elucidated the underling molecular mechanism and provided a novel insight into the cardioprotective function of FA, thus suggested the therapeutic potential of FA in HF treatment.

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  • Jumpei Ishibashi, Kana Saito, Takako Ishizaki, Ichiro Horie, Yoichiro ...
    2021 Volume 44 Issue 3 Pages 404-409
    Published: March 01, 2021
    Released: March 01, 2021
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    Supplementary material

    Mucus hypersecretion is a hallmark of respiratory diseases, and excess airway mucus can worsen these conditions. Therefore, it is important to control the production of airway mucus in the treatment of respiratory diseases. The phosphodiesterase inhibitor ibudilast has been reported to be effective in treating sputum and postnasal drip in patients with chronic airway inflammation. On the basis of the hypothesis that ibudilast could inhibit mucus production in the airway, in the present study, we examined the effects of ibudilast on the production of MUC5AC, a major protein component of mucus. In in vitro studies using NCI-H292 cells, ibudilast suppressed MUC5AC production induced by various stimuli. In addition, ibudilast inhibited extracellular signal-regulated kinase (ERK)1/2 phosphorylation and MUC5AC gene transcription. Furthermore, it attenuated MUC5AC production and Muc5ac mRNA expression in lipopolysaccharide-treated mice in vivo. Collectively, these findings demonstrate that ibudilast has an inhibitory effect on mucus production, which could at least partly be attributed to the inhibition of ERK1/2 phosphorylation and the repression of MUC5AC gene transcription.

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  • Toshie Tanaka, Kohei Sano, Masayuki Munekane, Toshihide Yamasaki, Hito ...
    2021 Volume 44 Issue 3 Pages 410-415
    Published: March 01, 2021
    Released: March 01, 2021
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    Supplementary material

    Melanoma is a highly malignant skin cancer that frequently metastasizes to the lung, bone, and brain at an early phase. Therefore, noninvasive detection of metastasized melanoma could be beneficial to determine suitable therapeutic strategies. We previously reported a biocompatible ternary anionic complex composed of plasmid DNA (pDNA), polyethyleneimine (PEI), and γ-polyglutamic acid (γ-PGA) based on an electrostatic interaction, which was highly taken up by melanoma cells (B16-F10), even if it was negatively charged. Here, we developed a radiolabeled γ-PGA complex by using indium-111 (111In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 (125I)-labeled PEI instead of native PEI, and evaluated its effectiveness as a melanoma-targeted imaging probe. This ternary complex was synthesized at a theoretical charge ratio; carboxyl groups of 111In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125I-PEI : carboxyl groups of γ-PGA was 1 : 8 : 16, and the size and zeta potential were approximately 29 nm and −33 mV, respectively. This complex was taken up by B16-F10 cells with time. Furthermore, a biodistribution study, using normal mice, demonstrated its accumulation in the liver, spleen, and lung, where macrophage cells are abundant. Almost the same level of radioactivity derived from both 111In and 125I was observed in these organs at an early phase after probe injection. Compared with the normal mice, significantly higher lung-to-blood ratios of radioactivity were observed in the B16-F10-lung metastatic cancer model. In conclusion, the radiolabeled γ-PGA complex would hold potentialities for nuclear medical imaging of lung metastatic melanoma.

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  • Mutsuko Sakai, Ryoichi Yoshimura
    2021 Volume 44 Issue 3 Pages 416-421
    Published: March 01, 2021
    Released: March 01, 2021
    [Advance publication] Released: December 16, 2020
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    Supplementary material

    In hepatitis, activated hepatic stellate cells (HSCs) produce collagens, causing liver fibrosis. Microenvironmental stiffness is a known trigger of HSC activation and is communicated through mechanotransduction. Cell proliferation, alpha smooth muscle actin (α-SMA) and collagen type Iα (Col1α) are indicative of activated HSCs. We hypothesized that certain compounds could interfere with the HSC’s recognition of microenvironmental stiffness by blocking cell adhesion signaling. To verify the potential of mechanotransduction, and in particular of focal adhesion proteins, as liver fibrosis drug targets, we evaluated existing drugs. We examined the effects of the integrin antagonist, BS-1417; the focal adhesion kinase (FAK) inhibitor, defactinib; the cyclin-dependent kinase (CDK) inhibitor, roscovitine; and two microtubule modulators, paclitaxel and colchicine, on stiffness-induced HSC activation. To determine the extent of transforming growth factor β (TGF-β) participation in mechanotransduction, we measured gene expression levels of α-SMA and Col1α. We also measured ATP levels to determine cell number. Results revealed that interestingly, although TGF-β did not show additional HSC activation after stiffness stimulation, the TGF-β receptor inhibitor, SB525334, markedly suppressed stiffness-induced α-SMA and Col1α mRNA expression. BS-1417, roscovitine, defactinib and colchicine suppressed α-SMA and Col1α mRNA expression as well as the number of HSCs. Paclitaxel also suppressed stiffness-induced α-SMA mRNA expression and the number of HSCs, but mildly reduced that of Col1α mRNA. Together, these results show that an integrin antagonist and mechanotransduction-targeting drugs reduced stiffness-induced HSC activation in a dose-dependent fashion. The targeting of focal adhesion proteins involved in mechanotransduction is promising in liver fibrosis drug development.

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  • Hiroki Yoshida, Rika Tsuhako, Chihiro Sugita, Masahiko Kurokawa
    2021 Volume 44 Issue 3 Pages 422-430
    Published: March 01, 2021
    Released: March 01, 2021
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    Glucosyl hesperidin (GH) is a water-soluble derivative of hesperidin, a citrus flavonoid. GH has various pharmacological effects, such as hypolipidemic and hypouricemic effects, and may therefore be a useful supplement or drug. In the present study, we evaluated the effects of long- and short-term intake of GH on hyperglycemia and macrophage infiltration into the adipose tissue of high-fat diet (HFD)-fed mice. Long-term (11-week) consumption of GH tended to reduce body weight and the fasting blood glucose concentration of the HFD-fed mice, and ameliorated glucose intolerance and insulin resistance, according to glucose and insulin tolerance tests. Additionally, although GH did not affect fat pad weight, it reduced HFD-induced macrophage infiltration into adipose tissue. Short-term (2-week) consumption of GH did not affect the HFD-induced increases in body weight or fasting blood glucose, and it did not ameliorate glucose intolerance or insulin resistance. However, short-term intake did reduce the HFD-induced macrophage infiltration and monocyte chemotactic protein 1 (MCP-1) expression in adipose tissue. Furthermore, hesperetin, which is an aglycone of GH, inhibited MCP-1 expression in 3T3-L1 adipocytes, 3T3-L1 adipocytes co-cultured with RAW264 macrophages, and tumor necrosis factor-α-treated 3T3-L1 adipocytes. The present findings suggest that daily consumption of GH may have preventive and/or therapeutic effects on obesity-related diseases, such as diabetes mellitus.

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    Editor’s picks

    Obese adipose tissue is characterized by increased immune cell infiltration. Adipocyte-immune cell interaction overproduces inflammatory adipokine, which contribute to the development of type 2 diabetes mellitus. The regulation of immune cells infiltration and inflammation in adipose tissue may exert preventive and therapeutic effects on obesity-related diseases. Flavonoids such as hesperidin have anti-inflammatory properties, but their low bioavailability limits their use as drugs and supplements. The authors report that glucosyl hesperidin (GH), a water-soluble derivative of hesperidin, ameliorated glucose intolerance and reduced macrophage infiltration into adipose tissue in high-fat diet-fed mice. These results suggest the usefulness of GH against obesity-related diseases.

  • Kazuhiro Nishiyama, Chiemi Toyama, Yuri Kato, Tomohiro Tanaka, Akiyuki ...
    2021 Volume 44 Issue 3 Pages 431-436
    Published: March 01, 2021
    Released: March 01, 2021
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    Supplementary material

    Non-alcoholic steatohepatitis (NASH) is a disease that has progressed from non-alcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. Two transient receptor potential canonical (TRPC) subfamily members, TRPC3 and TRPC6 (TRPC3/6), reportedly participate in the development of fibrosis in cardiovascular and renal systems. We hypothesized that TRPC3/6 may also participate in NASH fibrosis. We evaluated the effects of TRPC3 or TRPC6 functional deficiency in a NASH mouse model using choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Wild-type (WT) and TRPC3 or TRPC6 gene-deficient (KO) mice were fed with CDAHFD or standard diet for 6 weeks. The CDAHFD-induced body weight loss in TRPC6 KO mice was significantly lower compared with WT mice with CDAHFD. CDAHFD treatment significantly increased TRPC3 mRNA expression level and tissue weight in WT liver, which were suppressed in TRPC3 KO mice. However, either systemic deletion of TRPC3 or TRPC6 failed to attenuate liver steatosis, inflammation and fibrosis. These results imply that TRPC3 and TRPC6 are unlikely to be involved in liver dysfunction and fibrosis of NASH model mice.

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    Editor’s picks

    Two transient receptor potential canonical (TRPC) subfamily members, TRPC3 and TRPC6, reportedly participate in the development of fibrosis in cardiovascular and renal systems. This study is to investigate whether TRPC3 and TRPC6 channels also contribute to the formation of nonalcoholic steatohepatitis (NASH) which includes liver fibrosis, using TRPC3 or TRPC6 systemic knockout mice fed with the choline-deficient, L-amino acid-defined, high-fat diet. The authors found that systemic deletion of TRPC3 or TRPC6 gene alone failed to attenuate liver dysfunction and fibrosis in NASH model mice.

  • Koji Nishi, Keiki Sakurama, Hiroshi Watanabe, Toru Maruyama, Keishi Ya ...
    2021 Volume 44 Issue 3 Pages 437-441
    Published: March 01, 2021
    Released: March 01, 2021
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    We recently reported that aripiprazole (ARP), an antipsychotic drug, binds strongly to human serum albumin (HSA), the major drug binding protein in serum. It is known that uremic toxins that accumulate during renal disease affect the interaction between HSA and drug binding. In this study, the issue of how uremic toxins (indoxyl sulfate, indole acetic acid and p-cresyl sulfate) affect the binding of ARP to HSA was investigated. Equilibrium dialysis experiments revealed that all uremic toxins inhibited the binding of ARP to HSA although the inhibitory effects differed, depending on the specific uremic toxin. The potency of inhibition can be partially explained by the affinities of uremic toxins to HSA. Fluorescence displacement experiments suggested that ARP as well as all uremic toxins bind to site II of HSA. The inhibitory effects of the toxins on the binding of ARP for the drugs binding to the diazepam subsite are significantly larger, comparing with those for binding to arylpropionic acids subsite. Interestingly, induced circular dichroism (CD) spectra indicated that the spatial orientation of p-cresyl sulfate in the binding pocket is different from that for indoxyl sulfate and indole acetic acid. The limited findings obtained herein are important data in considering the effects of uremic toxins on the pharmacokinetics of ARP and the drugs that bind to site II on HSA, particularly drugs binding to diazepam binding site in site II.

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    Editor’s picks

    Aripiprazole (ARP), an antipsychotic drug, binds strongly to site II on human serum albumin (HSA). In this study, the issue of how uremic toxins (indoxyl sulfate, indole acetic acid and p-cresyl sulfate) affect the binding of ARP to HSA were investigated. Authors demonstrated that these uremic toxins inhibit the binding of ARP to diazepam subsite within site II, and these inhibitory effects were more significant, comparing with those on the drug binding to arylpropionic acids subsite. These findings provide important information for considering the pharmacokinetics of ARP and the drugs that bind to site II during renal diseases.

  • Misaki Niu, Atsushi Kasai, Kaoru Seiriki, Misuzu Hayashida, Masato Tan ...
    2021 Volume 44 Issue 3 Pages 442-447
    Published: March 01, 2021
    Released: March 01, 2021
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    The dopamine system plays an important role in regulating many brain functions, including the motor function. The blockade of dopamine receptors results in a serious motor dysfunction, such as catalepsy and Parkinsonism. However, the neuronal mechanism underlying the drug-induced motor dysfunction is not well understood. Here, we examine brain-wide activation patterns in Fos-enhanced green fluorescent protein reporter mice that exhibit cataleptic behavior induced by SCH39166, a dopamine D1-like receptor antagonist, and raclopride, a dopamine D2-like receptor antagonist. Support vector classifications showed that the orbital cortex (ORB) and striatum including the caudoputamen (CP) and nucleus accumbens (ACB), prominently contribute to the discrimination between brains of the vehicle-treated and both SCH39166- and raclopride-treated mice. Interregional correlations indicated that the increased functional connectivity of functional networks, including the ORB, CP, and ACB, is the common mechanism underlying SCH39166- and raclopride-induced cataleptic behavior. Moreover, the distinct mechanisms in the SCH39166- and raclopride-induced cataleptic behaviors are the decreased functional connectivity between three areas above and the cortical amygdala, and between three areas above and the anterior cingulate cortex, respectively. Thus, the alterations of functional connectivity in diverse brain regions, including the ORB, provide new insights on the mechanism underlying drug-induced movement disorders.

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    Editor’s picks

    By combining whole-brain activation mapping of neurons activated in response to dopamine D1 and D2 receptor antagonists with non-bias analysis, Niu et al. provide the direct evidence that the orbital cortex in addition to the striatum are important brain areas associated with DA antagonists-induced movement abnormality.

Notes
  • Shungo Imai, Kenji Momo, Hitoshi Kashiwagi, Takayuki Miyai, Mitsuru Su ...
    2021 Volume 44 Issue 3 Pages 448-452
    Published: March 01, 2021
    Released: March 01, 2021
    JOURNALS FREE ACCESS FULL-TEXT HTML

    Antibiotic-associated diarrhea (AAD) is a typical side effect of antibiotic treatment, especially in children. Amoxicillin (AMPC) and amoxicillin/clavulanate (AMPC/CVA) are associated with high risk of AAD; however, these antibiotics are important in the pediatric field. Recent research suggests that probiotics prevent pediatric AAD, including that caused by AMPC and AMPC/CVA. Indeed, guidelines for acute otitis media in children recommend the concomitant use of probiotics. However, the prescription status of probiotics for pediatric patients with otitis media receiving oral AMPC and AMPC/CVA remains unknown. We therefore conducted a survey to clarify the current status of these prescriptions and, in particular, to identify specific populations with a low proportion of probiotic prescriptions. Pediatric patients (≤15 years of age) newly prescribed oral AMPC or AMPC/CVA for otitis media between April 2016 and March 2017 were identified from a Japanese health insurance claims database. Eligible patients were divided into the AMPC (1303 patients) and AMPC/CVA (424 patients) groups, in which 659 (50.6%) and 293 (69.1%) patients were prescribed probiotics, respectively. Of the patients receiving probiotic prescriptions in the AMPC and AMPC/CVA groups, 632 (95.9%) and 286 (97.6%) patients received antibiotic-resistant probiotic prescriptions, respectively. When classified by the prescribing clinical department and patient age, the proportions of probiotic prescriptions in Internal Medicine and Pediatrics departments were lower than those in the Otorhinolaryngology department regardless of age. These results indicate the probability of insufficient probiotic prescriptions for pediatric patients with otitis media. Solving this issue may lead to the provision of safer antimicrobial therapy.

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  • Erika Saito, Madoka Kage, Yoshihiro Tokudome
    2021 Volume 44 Issue 3 Pages 453-457
    Published: March 01, 2021
    Released: March 01, 2021
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    The aim of this study was to examine the effects of carba cyclic phosphatidic acid (ccPA) on cornified envelope (CE) formation and keratinocyte differentiation. ccPA-treated keratinocytes showed higher mRNA and protein levels of differentiation markers and CE components than untreated cells. These results suggest that ccPA could serve as therapeutic targets for treating skin barrier dysfunction because of their roles in upregulating genes and proteins associated with CE formation and keratinocyte differentiation.

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  • Shogo Hamaguchi, Marin Kariya, Aya F. Ozaki, Iyuki Namekata, Hikaru Ta ...
    2021 Volume 44 Issue 3 Pages 458-460
    Published: March 01, 2021
    Released: March 01, 2021
    JOURNALS FREE ACCESS FULL-TEXT HTML

    The functional role of ATP released from sympathetic nerve terminals was examined in isolated guinea pig ventricular papillary muscles. The contractile force of papillary muscles was increased by field electrical stimulation of sympathetic nerve endings. This increase was attenuated by pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) or suramin, blockers of the P2X receptor, and was abolished by propranolol and prazosin. PPADS, suramin, and ATP affected neither the basal contractile force nor the positive inotropic effect of noradrenaline. These results provide functional evidence that ATP released from sympathetic nerve terminals enhances noradrenaline release and contributes to sympathetic nerve-induced inotropy.

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  • Maho Tsubota, Kazuki Matsui, Saaya Fukushi, Kyoko Okazaki, Fumiko Seki ...
    2021 Volume 44 Issue 3 Pages 461-464
    Published: March 01, 2021
    Released: March 01, 2021
    JOURNALS FREE ACCESS FULL-TEXT HTML

    T-Type Ca2+ channels (T-channels), particularly Cav3.2, are now considered as therapeutic targets for treatment of intractable pain including visceral pain. Among existing medicines, bepridil, a multi-channel blocker, used for treatment of arrhythmia and angina, and pimozide, a dopamine D2 receptor antagonist, known as a typical antipsychotic, have potent T-channel blocking activity. We thus tested whether bepridil and pimozide could suppress visceral pain in mice. Colonic and bladder pain were induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) and systemic administration of cyclophosphamide (CPA), respectively. Referred hyperalgesia was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load with intracolonic water injection and spontaneous bladder pain were evaluated by observing nociceptive behaviors in conscious mice. The mice exhibited referred hyperalgesia and colonic hypersensitivity to distension on day 6 after TNBS treatment. Systemic administration of bepridil at 10–20 mg/kg or pimozide at 0.1–0.5 mg/kg strongly reduced the referred hyperalgesia on the TNBS-induced referred hyperalgesia and colonic hypersensitivity to distension. CPA treatment caused bladder pain-like nociceptive behavior and referred hyperalgesia, which were reversed by bepridil at 10–20 mg/kg or pimozide at 0.5–1 mg/kg. Our data thus suggest that bepridil and pimozide, existing medicines capable of blocking T-channels, are useful for treatment of colonic and bladder pain, and serve as seeds for the development of new medicines for visceral pain treatment.

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