Physiological platelet aggregation assay to mitigate drug-induced thrombocytopenia using a microphysiological system.

Developing a reliable method to predict thrombocytopenia is imperative in drug discovery. Here, we establish an assay using a microphysiological system (MPS) to recapitulate the in-vivo mechanisms of platelet aggregation and adhesion. This assay highlights the role of shear stress on platelet aggregation and their interactions with vascular endothelial cells. Platelet aggregation induced by soluble collagen was detected under agitated, but not static, conditions using a plate shaker and gravity-driven flow using MPS. Notably, aggregates adhered on vascular endothelial cells under gravity-driven flow in the MPS, and this incident increased in a concentration-dependent manner. Upon comparing the soluble collagen-induced aggregation activity in platelet-rich plasma (PRP) and whole blood, remarkable platelet aggregate formation was observed at concentrations of 30 µg/mL and 3 µg/mL in PRP and whole blood, respectively. Moreover, ODN2395, an oligonucleotide, induced platelet aggregation and adhesion to vascular endothelial cells. SYK inhibition, which mediated thrombogenic activity via glycoprotein VI on platelets, ameliorated platelet aggregation in the system, demonstrating that the mechanism of platelet aggregation was induced by soluble collagen and oligonucleotide. Our evaluation system partially recapitulated the aggregation mechanisms in blood vessels and can contribute to the discovery of safe drugs to mitigate the risk of thrombocytopenia.

The mechanism of thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides.

In this study, we investigated thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides (Chol-ASO). First, we evaluated platelet activation induced by Chol-ASO in mice by flow cytometry after administration of platelet-rich plasma (PRP). An increase in the number of large particle-size events with platelet activation was detected in the Chol-ASO-treated group. In a smear study, numerous platelets were observed to attach to nucleic acid-containing aggregates. A competition binding assay showed that the conjugation of cholesterol to ASOs increased their affinity for glycoprotein VI. Platelet-free plasma was then mixed with Chol-ASO to form aggregates. The assembly of Chol-ASO was confirmed by dynamic light scattering measurements in the concentration range in which the formation of aggregates with plasma components was observed. In conclusion, the mechanism by which Chol-ASOs causes thrombocytopenia is proposed to be as follows: (1) Chol-ASOs form polymers, (2) the nucleic acid portion of the polymers interacts with plasma proteins and platelets, which cross-links them to form aggregates, and (3) platelets bound to aggregates become activated, resulting in platelet aggregation, leading to a decrease in platelet count in vivo. The details of the mechanism revealed in this study could contribute to creating safer oligonucleotide therapies without the risk of thrombocytopenia.

Cell-based high-throughput screening for the evaluation of reactive metabolite formation potential.

Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 µM D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 µM. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery.

Video-based assessment of drug-induced effects on contractile motion properties using human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Drug-induced inotropic change is a risk factor in drug development; thus, de-risking is desired in the early stages of drug development. Unlike proarrhythmic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), few in vitro models were validated to predict cardiac contractility. Motion field imaging (MFI), a high-resolution block matching-based optical flow technique, was expected to possess high quantitative predictivity in the detection of contraction speed. We aimed to establish an in vitro model to assess drug-induced contractile changes using hiPSC-CMs and MFI. METHODS: MFI was designed to noninvasively characterize cardiomyocyte contractile behavior by analyzing light microscope video images, and maximum contraction speed (MCS) was used as the index of contractility. Using MFI, 9 inactive compounds, 10 negative inotropes, and 10 positive inotropes were tested. Two negative chronotropes, ZD7288 and ivabradine, were also tested. To determine the sensitivity and specificity of the assay, the minimum effective concentration of the MCS was compared with the human effective total therapeutic concentration for 28 compounds in clinical use. RESULTS: For 8 negative and 8 positive inotropes, the effects observed in in vivo and clinical studies were detected in MFI assay. MFI assay showed negative chronotropic changes without inotropic changes. MFI assay presented sufficient specificity (83%) and sensitivity (88%), and RNA-sequence analysis provided an insight into the relationship between occurrence of the false compounds and target gene expression. DISCUSSION: We demonstrated the utility of MFI assay using hiPSC-CMs to assess drug-induced contractile function. These results will facilitate the de-risking of compounds during early drug development.

High-Throughput Screening to Evaluate Inhibition of Bile Acid Transporters Using Human Hepatocytes Isolated From Chimeric Mice.

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl-l-lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/Cmax) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/Cmax>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI.

Effect of hydrothermal treatment on light transmission of translucent zirconias.

STATEMENT OF PROBLEM: Studies of the light transmission of translucent zirconias after hydrothermal treatment are limited. PURPOSE: The purpose of this in vitro study was to evaluate the effect of hydrothermal treatment on the light transmission of translucent zirconias for monolithic restorations. MATERIAL AND METHODS: Four commercially available zirconia products, BruxZir Anterior Solid Zirconia (BruxAnt, BA), Lava Plus High Translucency (LPHT), Katana Zirconia Super Translucent (KST), and Katana Zirconia Ultra Translucent (KUT) were assessed and 1 type of lithium disilicate, e.max Press LT (LDLT) was used as a control. Plate specimens, 20×20×1 mm (n=80) for the translucency assessment were sectioned from postsintered zirconia bulk materials and ground with a #400-grit diamond wheel and coolant. The specimens were placed under hydrothermal conditions of 134°C at 0.2 MPa (n=5 per group at 0, 5, 50, and 100 hours). Percentage of total transmittance of light (Tt%) of each specimen was measured using a spectrophotometer with an integrating sphere. X-ray diffraction analyses were used to measure tetragonal-monoclinic phase transformation. Surfaces were examined by scanning electron microscopy and energy dispersive spectrometry. Data were analyzed using 2-way ANOVA followed by the Tukey test (α=.05). RESULTS: The Tt% ranged from 6.5% to 28.3%. Group LDLT obtained significantly higher transmittance than other tested groups, whereas groups KST and KUT had significantly higher Tt% than groups BA and LPHT (P<.05). A statistically significant increase in the amount of monoclinic phase was revealed within all translucent zirconia groups (P<.05), and the increase in group LPHT was significantly higher than those of the other 3 translucent zirconias (P<.05). Minimal changes in the percentages of light transmittance were revealed after 100-hour hydrothermal treatment for all tested translucent zirconias and a lithium disilicate glass-ceramic control. CONCLUSIONS: Hydrothermal treatment had minimal effects on the translucency of translucent zirconias. The tetragonal-monoclinic phase transformation rate of translucent zirconias was found to be low, except in group LPHT.

A comparative evaluation of the translucency of zirconias and lithium disilicate for monolithic restorations.

STATEMENT OF PROBLEM: Studies comparing the translucency of zirconias and lithium disilicates are limited. PURPOSE: The purpose of this in vitro study was to measure the translucency of recently developed translucent zirconias and compare them with lithium disilicate. MATERIAL AND METHODS: Five types of zirconia, Prettau Anterior (Zirkonzahn GmbH), BruxZir (Glidewell Laboratories), Katana HT, Katana ST, and Katana UT (Kurary Noritake Dental Inc), and 1 type of lithium disilicate, e.max CAD LT (Ivoclar Vivadent AG), were assessed. Non-colored zirconia test specimens (n=5) were prepared as rectangles with dimensions of 15×10×0.5 and 15×10×1.0 mm. The shade of lithium disilicate was B1. A spectrophotometer (Evolution 300 UV-Vis) with an integrating sphere was used to evaluate the total transmittance of light as a percentage (Tt%) at a wavelength of 555 nm for comparison among groups. The Welch robust test for equality of means was used to compare group means (α=.025) and post hoc pairwise comparisons among groups were performed with the Dunnett T3 method. RESULTS: For the 0.5 mm thickness groups, the Tt% was 31.90 ±0.49 for Prettau Anterior, 28.82 ±0.22 for BruxZir, 28.49 ±0.14 for Katana HT, 31.67 ±0.24 for Katana ST, 33.73 ±0.13 for Katana UT, and 40.32 ±0.25 for e-max CAD LT. Post hoc tests indicated that all groups were significantly different from each other, except for between BruxZir and Katana HT, and between Prettau Anterior and Katana ST. Katana UT was significantly more translucent than all other zirconias, and e-max CAD LT was significantly more translucent than all zirconias. For the 1.0 mm thickness groups, the Tt% was 22.58 ±0.41 for Prettau Anterior, 20.13 ±0.22 for BruxZir, 20.18 ±0.39 for Katana HT, 21.86 ±0.39 for Katana ST, 23.37 ±0.27 for Katana UT, and 27.05 ±0.56 for e-max CAD LT. Post hoc tests indicated that all materials were significantly different from each other, except for between BruxZir and Katana HT, and among Prettau Anterior, Katana ST and Katana UT which were significantly more translucent than all other zirconias and less translucent than e-max CAD LT. CONCLUSION: At a thickness of 0.5 mm, Katana UT was significantly more translucent than all other zirconias, and e-max CAD LT was significantly more translucent than all zirconias. At a thickness of 1.0 mm, Prettau Anterior, Katana ST, and Katana UT were significantly more translucent than all other zirconias and less than e-max CAD LT.

Effect of accelerated aging on the fracture toughness of zirconias.

STATEMENT OF PROBLEM: Low temperature degradation (LTD) of yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) is of concern. PURPOSE: The purpose of this in vitro study was to assess the effect of accelerated aging on the Vickers hardness and fracture toughness of a newly developed Y-TZP and 2 primary Y-TZPs. MATERIAL AND METHODS: Two primary 3 mol% Y-TZP, Lava (LA), Everest Zirconium Soft (EV), and a new 3 mol% Y-TZP, ZirTough (NZ) were assessed. Specimens (n=30 each brand) of 10 × 10 × 3 mm were hydrothermally treated for accelerated aging to examine LTD. Five conditions were used (n = 5 per condition) as follows: control group (no aging); 5 hours at 134°C/0.2 MPa (5h-134°C); 100 hours at 134°C/0.2 MPa (100 h-134°C); 5 hours at 180°C/1.0 MPa (5 h-180°C); and 20 hours at 180°C/1.0 MPa (20 h-180°C). Fracture toughness was measured by using the indentation fracture (IF) method under a loading of 294 N and calculated from the obtained measurements. To observe differences in particle composition and fracture patterns, mirror-polished test specimens (n=5 each brand) were re-sintered at 1200°C for 1 hour as a thermal etching process, and a Vickers indenter was pressed into the test specimens according to the IF method. Test piece surfaces and cracks were observed with scanning electron microscopy (SEM). One-way ANOVA and the post- hoc (Scheffé test were used to examine) interlevel significant differences (α=.05). RESULTS: The Vickers hardness and fracture toughness were as follows: 1319 HV and 7.36 MPa · m(1/2) for LA, and 1371 HV and 6.76 MPa · m(1/2) for EV in no aging; 1334 HV and 7.02 MPa · m(1/2) for LA, and 1346 HV and 6.07 MPa · m(1/2) for EV in 5h-134°C. No significant differences were found between no aging and 5h-134°C for LA and EV for Vickers hardness and fracture toughness. Measurements could not be made for LA and EV for 100 h-134°C, 5h-180°C, or 20 h-180°C because of fractures in the surface layer. For NZ, Vickers hardness and fracture toughness were as follows: 1261 HV and 15.60 MPa · m(1/2) in no aging; 1217 HV and 14.98 MPa · m(1/2) in 5h-134°C; 1231 HV and 15.13 MPa · m(1/2) in 100 h-134°C; 1252 HV and 15.51 MPa · m(1/2) in 5h-180°C; 1224 HV and 15.01 MPa · m(1/2) in 20 h-180°C. No significant differences were shown in the Vickers hardness and fracture toughness. SEM observations after the thermal etching processing of NZ showed zirconia particles and scattered alumina particles. CONCLUSION: Measurements with LA and EV could only be made for no aging and 5h-134°C, and no significant differences were found in Vickers hardness and fracture toughness. Measurements were made with NZ under all conditions and no significant differences were found in Vickers hardness and fracture toughness.

Effect of loading conditions on the fracture toughness of zirconia.

PURPOSE: A Vickers hardness indenter was pressed into yttria-stabilized zirconia (Y-TZP) by the indentation fracture method (IF method). METHODS: The effect on the calculated Vickers hardness, fracture toughness values, and indentation fracture load (9.8, 49, 98, 196, and 294 N) was examined to deduce the optimum conditions of the IF method. Calculated Vickers hardness and fracture toughness values were analyzed with one-way analysis of variance and then multiple comparisons (Scheffe). The appearance of on indentation and cracks was also evaluated using a scanning electron microscopy (SEM). RESULTS: Indentation of Y-TZP was generated by 9.8 and 49 N of indentation fracture load, however cracks could not be confirmed with the microscope attached to the Vickers hardness tester. Both indentation and cracks were observed at 98, 196 and 294 N of indentation fracture load obtained values of 7.1 and 6.8 MPam(1/2). Cracks noted at the 98 N were not clear, whereas the 196 and 294 N showed especially clear cracks. Due to the hardness of zirconia and the light loads, fracture toughness values for 9.8, 49, and 98 N could not be calculated. There was no significant difference between 196 and 294 N, when calculated fracture toughness values were analyzed with multiple comparisons. SEM revealed clear indentation and cracks, that extended linearly, but no chips or fractures were observed. Surface changes were observed at 196 and 294 N that are presumed to be accompanied by phase transition around the cracks. CONCLUSIONS: Optimum experimental conditions of the indentation fracture load in the IF method were determined as 196 and 294 N.

Combination of reactive oxygen species and tissue-type plasminogen activator enhances the induction of gelatinase B in brain endothelial cells.

Tissue-type plasminogen activator (t-PA) increases the risk of intracranial hemorrhage by gelatinase B (matrix metalloprotease-9; MMP-9) production in brain endothelial cells. It was recently reported that the free-radical scavenger edaravone significantly decreases t-PA-mediated MMP-9 production. Therefore, by using cultured brain endothelial cells (b.End3), we investigated whether t-PA-mediated MMP-9 production was enhanced by reactive oxygen species (ROS) and its signaling pathways. Moreover, we also investigated that whether this combined enhancement is reduced by edaravone. The b.End3 cells were exposed to t-PA (10 µg/mL), followed by H2O2 (30 µM); further, the MMP-9 protein level was measured. ROS enhanced MMP-9 production, and ROS plus t-PA significantly increased MMP-9 production more than t-PA or ROS alone. The results showed that H2O2 or t-PA alone caused a significant increase in NF-κB translocation to the nucleus, whereas the combination of t-PA and H2O2 increased the translocation of NF-κB to an even greater extent. Moreover, the combination of t-PA and ROS significantly increased I-κB degradation as well as NF-κB expression. Edaravone completely decreased the ROS plus t-PA-mediated MMP-9 enhancement. In conclusion, ROS enhanced t-PA-mediated MMP-9 production in brain endothelial cells; this MMP-9 production was decreased by the addition of edaravone, which inhibited the NF-κB pathway, specifically by enhancing I-κB degradation.

Ring cleavage and successive aldol reaction of 3-[(trialkylsilyl)methyl]cyclobutanones.

3-[(Trialkylsilyl)methyl]cyclobutanones reacted with aldehydes by activation with titanium(IV) chloride to give acyclic ß,γ-unsaturated ß'-hydroxyketones.

Adenosine diphosphate receptor P2Y12-mediated migration of host smooth muscle-like cells and leukocytes in the development of transplant arteriosclerosis.

BACKGROUND: We have recently reported that platelet P2Y12 receptors may play a role in the development of transplant arteriosclerosis (TA). In the present study, we investigated the role of P2Y12 receptors on host-derived smooth muscle-like cells (SMLCs, including bone-marrow-derived SMLCs) and CD45+ leukocytes, both of which are believed to be associated with the development of TA, using P2Y12-deficient (KO) mice. METHODS: Orthotopic carotid artery transplantation was performed from C3H/He (H-2k) donors into KO or wild-type (WT) recipient mice (129S:C57BL/6, H-2b). Grafts were harvested at 8 weeks after transplantation for histology. Plasma monocyte chemoattractant protein-1 (MCP-1) levels were analyzed with a kit. Cell migration was examined using a Boyden chamber system. The expression of MCP-1 messenger RNA was assessed by real-time polymerase chain reaction. RESULTS: Eight weeks after allotransplantation, KO recipient mice showed a significant reduction of luminal occlusion, host-derived SMLCs, CD45+ leukocytes, MCP-1+ cells in the grafts, and of plasma MCP-1 levels. In addition, the migration of host-derived SMLCs (including bone-marrow-derived SMLCs) and CD45+ leukocytes stimulated with adenosine diphosphate (ADP) or 2-methylthio-ADP (2MeSADP, a stable ADP analog) was significantly decreased in KO mice. There were no significant changes in MCP-1-induced cell migration between WT and KO mice. The low concentration of 2MeSADP plus MCP-1 significantly increased cell migration in WT but not KO mice. Furthermore, 2MeSADP-induced MCP-1 messenger RNA expression was significantly reduced in the cells of KO mice. CONCLUSIONS: Thus, the P2Y12-mediated migration of host-derived SMLCs and CD45+ leukocytes may play an important role in the development of TA, partly by MCP-1 pathways.