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Antangin Fit is Indonesian herbal medicine syrup which containing: Zingiber officinale, Phyllanthus niruri, Curcuma domestica, Blumea balsamifera, honey, and Menthae piperitae oil. This study was conducted to evaluate the acute and sub chronic oral toxicity of Antangin Fit in rats and the immunostimulatory activity in mice. The acute toxicity study was conducted on 50 Wistar rats, divided into 4 treatment groups and 1 control. The Antangin Fit syrup with dose of 4.7, 7.52, 12.03, and 19.25 mL kg^-1 was administered as a single dose orally. Each animal was observed for the first 24 h and continued for 14 days. There were no significant toxic effects and no death observed until the end of the study, showed that the lethal dose 50% (LD50) of Antangin Fit was > 19.25 mL kg^-1. The subchronic toxicity study was conducted on 80 Wistar rats. The Antangin Fit syrup with doses of 4.7, 9.5, and 19.25 mL kg^-1 day^-1 for each treatment group were administered for 90 days orally. There were no significant toxic effects observed at all dose. The immunostimulatory activity was observed on the ability of macrophage to stimulate phagocytic activity and secretion of Reactive Oxygen Intermediates (ROI), and lymphocyte proliferation on 80 male Swiss mice. The Antangin Fit syrup at dose of 18.36 kg^-1 day^-1 stimulate nonspecific phagocytic activity of normal mouse peritoneal macrophages. Phagocytic and production of ROI by peritoneal macrophages and lymphoproliferative response also increase during Listeria monocytogenes infection. These findings indicated the immunostimulatory activity of Antangin Fit.

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Background: Non-steroid anti-inflammatory drug (NSAID) is one of the most common medicines given before, during and after operation, including colon anastomosis operation. Some research shows that NSAID can delay wound healing process. However, research about NSAIDs contribution in the colon anastomosis wound healing is not known yet. Objective: We aim to investigate the effect of various NSAID on anastomosis wound healing process in rats. Methods: Study was conducted on rat model of colon anastomosis. The rats were divided into 4 different groups. The groups treated either with diclofenac, metamizole, paracetamol or placebo. Three days after colonic anastomosis procedures, rats were sacrificed. Histologic study was done on hematoxylin eosin stained specimen. Result: Diclofenac and metamizole groups showed less sign of anastomosis wound healing signs compare with those on placebo and paracetamol groups. Discussion: NSAIDs administration halted inflammation whereas it is needed in wound healing process. Inflammation contributes to the production of cytokines and growth factors needed for anastomosis wound healing. Therefore, NSAIDs with strong anti-inflammatory effect such as diclofenac and metamizole, show less colon anastomosis wound healing due to strong inflammation suppression. Conclusion: NSAIDs administration after colonic anastomosis affects anastomosis wound healing. Therefore choosing NSAIDs with sufficient analgesic effect but less anti-inflammatory are suggested.

Keywords: NSAIDs, wound healing, inflammation, colon anastomosis

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A large percentage of new drugs fail in clinical studies due to cardiac toxicity. Therefore development of highly predictive in vitro assays which use clinically relevant cell-based models and are suitable for high throughput screening (HTS) is extremely important for early lead optimization process. Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) are especially attractive because they express ion channels which are similar to those of adult hearts and demonstrate spontaneous mechanical and electrical activity. Actually, many publications showed that QTc prolonging effect in clinical correlates with prolongation of field potential duration in electrophysiological assessment with a multi-electrode array system. At the last JSOT annual meeting, we presented a method for the assessment of cardiac toxicity of anti-cancer agents by multi-spheroid imaging analysis using hiPS-CM. In the present study, we would like to introduce a new high throughput screening method to assess proarrhythmic potential of a drug candidate as application of multi-spheroid Ca-imaging analysis.

At first, hiPS-CM (iCell® Cardiomyocytes) was pre-cultured 7-10 days in gelatin-coated 6 well plate and then the number of 15,000 cells was seeded into 96-half well plate which has fibronectin-coated spots in each well. The spheroids formation was confirmed in a plate on all fibronectin spots after 7 days culture. Ca sensing fluorescence dye and test compounds solutions, such as E-4031, terfenadine, flecainide, chromanol293B, moxifloxacin, verapamil, cisaplide, isoproterenol, propranolol and aspirin, were added and the dynamic changes in fluorescent intensity (Ca peak) of all spheroids were measured using Cellvoyager CV7000 system. The beat rate, Ca peak duration and sign of arrhythmia were analyzed from 30 seconds captured live-image for each well at 20 minutes after compound addtiton. Detail results will be introduced in JSOT annual meeting.

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The inotropic state (IS) of the heart is a fundamental property of the cardiovascular system due to its role in determining cardiac output. Normal hearts can compensate for small decrements in IS and maintain cardiac output, but in disease states, such as heart failure or diabetic cardiomyopathy, the capacity to compensate for IS depression is limited and may adversely affect these patient groups. For this reason it is advisable to evaluate drugs in pre-clinical stages for inotropic actions. CellOPTIQ^® (Clyde Biosciences Ltd), a multiparametric medium-high throughput assay platform was used in conjunction with hiPSC derived cardiomyocytes, to develop a suitable biological assay for screening inotropic actions on the heart. Both electrical activity and contractility were assessed using iCell² hiPSC cardiomyocytes (Cellular Dynamics Inc.) using a Voltage Sensitive Dyes (VSD) and a cell motion-based contractility assay. The cells were paced at constant rate (1Hz) rate throughout. After recording the baseline activity, the cells were treated with a set of drugs with known inotropic effect. For example, the L-type calcium channel blocker Nifedipine (negative inotropic) decreased the amplitude of contraction to 51.9±5.3% of baseline at 0.1µM (n=8); this was accompanied by shortening of APD (APD90= 47.1±6.9% of baseline). Blebbistatin decreased contractility (to 59.7±6.9% of baseline) at 3µM, with minimal changes in electrical activity (APD90=86.5±2% of baseline). In conclusion, the implementation of an IS assay to the CellOPTIQ^® was tested using CDI iCell² hiPSC-CMs. The utility of this assay for cardiac safety assessment was established using several drugs with established inotropic actions.

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The ICH S7B guidelines recommend that all new chemical entities should be subjected to hERG repolarization assay due to its association with life-threatening Torsades de Pointes (TdP) arrhythmia. However, it has become evident that not all hERG channel inhibitors result in TdP and not all compounds that induce QT prolongation and TdP necessarily inhibit hERG. In order to address the limitations of the S7B/E14 guidelines, the FDA through a public/private partnership initiated the Comprehensive in vitro Proarrhytmia assay (CiPA) initiative to examine the possible modification of the ICH E14/S7B guidelines. The three components of CiPA include voltage clamp assessment of human ion channels in addition to hERG channel, in silico modeling of electrophysiological activity and confirmation using in vitro assays on human stem cell-derived cardiomyocytes (hSC-CMs). During CiPA phase I study, 3 independent sites used iPSC-derived cardiomyocytes (iCell Cardiomyocytes) in conjunction with xCELLigence RTCA CardioECR system that allows for simultaneous measurement of cardiomyocyte field potential (FP) signal using extracellular recording (ECR) electrodes and contraction using impedance electrodes to assess the effect of 8 well-known reference compounds with different potency of proarrhythmia liability. Our data clearly shows hERG channel blockers, such as E4031 and Moxifloxacin, prolonged field potential duration (FPD) at low concentration and induced arrhythmic beating activity in both FP and impedance recordings at higher concentrations. On the contrary, Nifedipine, an inhibitor of Calcium channel, didn’t disrupt the periodicity of cell beating. However, it weakened cell contractile activity and shortened FPD. The multichannel inhibitors, such as Flecainide, Qunidine and Mexiletine, not only increased FPD, induced arrhythmia but also significantly reduced amplitude of FP spike. JNJ303, a I_Ks inhibitor, only affected cell electrophysiological activity through an increase in FPD. In addition, the FPD results of 8 compounds across 3 different evaluation sites appears very consistent. Taken together, this multi-parameter assay using hSC-CMs in conjunction with simultaneous measurement of ion channel activity, contractility can be a reliable tool for prediction of drug-induced proarrhythmia.

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The modifying potential of di(n-butyl)phthalate (DBP) on the second stage, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-initiated urinary bladder carcinogenesis were investigated in male Sprague-Dawley rats. Six-week-old rats received 0.05 % BBN in their drinking water for 4 weeks and then DBP (0.00001, 0.0001 and 0.001% DBP group) or vehicle (tap water: Vehicle group) were given during experimental weeks 10-26. All rats were killed at the end of weeks 26 of the experiment, after then the densities of putative preneoplastic, papillary or nodular (PN) hyperplasia were revealed in the vehicle control group. The incidences of PN hyperplasia / 10 cm (basement membrane) of the low DBP dose group were similar to those of the Vehicle group, but those of the high DBP dose group was significantly lower compared to those of the Vehicle group. The present study indicated that 0.001% DBP group inhibiting effects on BBN-initiated urinary bladder carcinogenesis, and these events showed dose relationship.

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System L is a major nutrient transport system responsible for the Na+ -independent transport of large neutral amino acid including several essential amino acids, consisted of the L-type amino acid transporter1 (LAT1) and the heavy chain of 4F2 cell surface antigen (4F2hc), and up-regulate to support several malignant tumor cell growth in vitro. As tumor angiogenesis is critical for tumor cell growth in vivo, the present study employed an ultrastructural immunohistochemicaly analysis for clarify the LAT1/4F2hc expression at microvessels in N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced rat bladder carcinomas composed of many LAT1/F2hc expressed tumor cells. Normal, hyperplastic, and papilloma microvessels composed non-fenestrated typed endothelial cell, while bladder carcinoma microvessels composed fenestrated or non-fenestrated typed endothelial cells. LAT1/4F2hc exclusively distributed the luminal and abluminal cell membranes including some membranous vesicles and some lysosomes of the fenestrated typed endothelial cells, but the non-fenestrated typed endothelial cells and pericytes did not. LAT1/4F2hc expression at tumor angiogenic microvessels seemed to be responsible to supply nutrition to BBN bladder carcinoma.

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Epidemiologic studies have reported PM_2.5 extracts show detrimental effects on respiratory health. The components and/or factors of PM_2.5 that contribute to respiratory health have not been identified. It is necessary to determine whether different types of components of seasonally variable ambient PM_2.5 extracts affect respiratory and immune system. Our study elucidates the effects of aqueous and organic extracts of PM_2.5 collected from four different seasons during November 2014-December 2015 in Kawasaki and Fukuoka cities of Japan. Human airway epithelial cells, murine splenocytes and bone marrow derived cells (BMDC) were exposed to extracts of PM_2.5. The cell viability and release of IL-6, IL-8 and sICAM-1 in airway epithelial cells, proliferation, TCR and CD19 expression in splenocytes and DEC205 and CD86 expression on BMDC were measured. The aqueous extracts especially of fall from Kawasaki had more cytotoxic effect than organic extracts in airway epithelial cells, however, caused almost no pro-inflammatory response. Aqueous extracts of fall, summer and spring from Fukuoka significantly increased cell proliferation of splenocytes. Organic extracts of spring and summer from Kawasaki significantly elevated TCR expression, while CD19 expression slightly decreased. Furthermore, extracts from fall, especially aqueous extracts from Fukuoka, increased expression of DEC205 and CD86. These results suggest that PM_2.5 extracts can be responsible for cytotoxicity in airway epithelial cells and activation of immune cells via T-cells and BMDC. These effects can differ by components, collection areas and seasons.
This study was performed as a part of the study project on PM_2.5 by the Ministry of the Environment, Japan.

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