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Toward quasi-in vivo from in vitro assay (I): Development of spatial conductance fluctuation measurement assay using a human cardiomyocyte line-network cell chip with multielectrode array system for in vitro predictive proarrhythmic cardiotoxicity
Abstracts
doi:10.1016/j.vascn.2014.03.039
0035
Correlation of in vitro screening technologies for secondary pharmacodynamic assessment Duncan Armstronga, Jonathan Brighta, Thierry Jolasb, Jacques Migeonb, Mike Rolfa, Lyn Rosenbrier-Riberoa, Jean-Pierre Valentina, Joanne Bowesa a
AstraZeneca, Macclesfield, UK Cerep, SA, Celle l'Evescault, France
b
In vitro pharmacological profiling is a key component of drug discovery and involves screening compounds against a broad range of molecular targets including receptors, ion channels, enzymes and transporters in order to reduce off-target activity thus reducing the likelihood of adverse drug reactions (ADRs). Radioligand binding and functional assay technologies may be applied to the G protein-coupled receptor (GPCR) component of in vitro selectivity screening panels (Bowes et al. NRDD, 11, 909–922). The objective of this study was to compare the sensitivity of the two different methods for an exemplar group of 12 GPCRs, selected for their association with cardiovascular ADRs, using a large set of pharmacologically relevant compounds (between 282 and 616 compounds/ GPCR). Radioligand binding data were taken from the BioPrint (Cerep, SA) database and we generated concentration–response data in functional assays appropriate to each GPCR target. For all 12 GPCRs assessed, radioligand binding assays detected more active compounds than did the corresponding functional assessment. For all 12 GPCRs more compounds were antagonists than agonists. No consistent trend could be found in sensitivity where pA50 values were defined in both radioligand binding and functional assessments: for some GPCRs binding pA50 was more potent than agonist or antagonist pA50 while for other GPCRs the opposite was true. Differences in pA50 ranged from equipotent for α1A adrenoceptor radioligand binding/antagonism to 33-fold for β2 adrenoceptor radioligand binding/agonism. This data suggests that careful consideration should be given to the choice of technology for in vitro pharmacological profiling for early detection of off-target interactions that drive ADRs.
DP
Toward quasi-in vivo from in vitro assay (I): Development of spatial conductance fluctuation measurement assay using a human cardiomyocyte line-network cell chip with multielectrode array system for in vitro predictive proarrhythmic cardiotoxicity Tomoyo Hamadaa, Fumimasa Nomuraa, Hideyuki Terazonoa, Akihiro Hattoria, Peter Sartipyb, Mitsuhiro Edamurac, Thomas Meyerd, Kenji Yasudaa
0036
OF
large closed loop were formed in those shaped agarose microchambers fabricated on a multielectrode array chip for simultaneous measurement and comparison of their responses against Quinidine. The interspike intervals of the two-dimensional sheet was most durable and stable against administration of Quinidine even at 100 μM high dose, whereas the large closed loop network was most sensitive and showed lethal arrhythmia from 1 μM low dose. The results indicate the apparent spatial arrangement dependence of cell response and the necessity of the consideration of the proper community size and spatial arrangement pattern of cell networks for representing the proper response of cardiomyocytes against the proper amount of compounds.
RO
320
a
Tokyo Medical and Dental University, Tokyo, Japan Cellectis Stem Cells, Göteborg, Sweden c Bio Research Center, Nagoya, Japan d Multi Channel Systems MCS GmbH, Reutlingen, Germany
TE
b
UN
CO
RR
EC
Recent studies show the limitations of proarrhythmic potential prediction in drug induced fatal ventricular tachycardia (VT) or fibrillation (VF) in conventional in vitro assays. Hence, we have developed on-chip in vitro cardiomyocyte network spatiotemporal signal-fluctuation screening assays using human embryonic stem cell-derived cardiomyocytes (hES-CMs) for more precise prediction of fatal arrhythmia. The results showed that the temporal fluctuation (short term variability) of the field potential duration was more effective and precise for the arrhythmogenic prediction against conventional in vitro assays, and that the spatial fluctuation of propagation (conduction) in a lined-up hES-CM network in a rectangular agarose-microchamber on a multielectrode array (MEA) chip was even better than that of the temporal fluctuation assay, e.g. terfenadine, which was difficult to detect proarrhythmic potential in the temporal fluctuation assay. Moreover, simultaneous monitoring of tension generation in the lined-up cardiomyocytes was combined with the electrophysiological measurement set-up in the temperature-controlled long-term observation unit. We also have evaluated the portability of the sealed cardiomyocyte lined-up MEA chip for international transportation between Sweden and Japan and confirmed no distinguishable damage for measurement. We have examined the compatibility of our spatial fluctuation measurement procedures to the existing MEA assay using the cardiomyocyte lined-up MEA chip and the propagation fluctuation analysis software and found that similar predictive tendencies could be acquired from this set-up. The results indicate that the spatial propagation fluctuation assay is the simpler and more precise and could improve the ability of in vitro pro-arrhythmia measurement including TdP/VT/VF as quasi-in vivo assays.
doi:10.1016/j.vascn.2014.03.040
doi:10.1016/j.vascn.2014.03.041
0037 Review: Rat models of pulmonary hypertension Kristy Bruse Lovelace Respiratory Research Institute, Albuquerque, NM, USA Currently, there is no definitive animal disease model of pulmonary arterial hypertension (PAH) that mimics clinical idiopathic PAH. Adult nonrodent animal models such as nonhuman primate (Bruse, Poster at SPS 2012 annual meeting) have essentially not been successful. Transgenic mice models have had partial success. There are currently three rat models of PAH that we have utilized: (1) monocrotaline (MCT; 50 or 60 mg/kg, IP, assessed 28–33 days later), (2) 3 weeks chronic hypoxia (CH; PIO2 = 12%) + 2 weeks normoxia, and (3) Sugen (SU-5416; 20 mg/kg, IP) + 3 weeks chronic hypoxia + 2 weeks normoxia (S-CH). Male rats (Sprague–Dawley or Wistar) aged 9–12 weeks of age were utilized at our facility located at 5355 ft (1632 m) above sea level. Pathophysiological endpoints included: decreased body weight increase (beginning Day 7), elevated right ventricular systolic pressure (RVSP) without systemic blood
doi:10.1016/j.vascn.2014.03.039
0035
Correlation of in vitro screening technologies for secondary pharmacodynamic assessment Duncan Armstronga, Jonathan Brighta, Thierry Jolasb, Jacques Migeonb, Mike Rolfa, Lyn Rosenbrier-Riberoa, Jean-Pierre Valentina, Joanne Bowesa a
AstraZeneca, Macclesfield, UK Cerep, SA, Celle l'Evescault, France
b
In vitro pharmacological profiling is a key component of drug discovery and involves screening compounds against a broad range of molecular targets including receptors, ion channels, enzymes and transporters in order to reduce off-target activity thus reducing the likelihood of adverse drug reactions (ADRs). Radioligand binding and functional assay technologies may be applied to the G protein-coupled receptor (GPCR) component of in vitro selectivity screening panels (Bowes et al. NRDD, 11, 909–922). The objective of this study was to compare the sensitivity of the two different methods for an exemplar group of 12 GPCRs, selected for their association with cardiovascular ADRs, using a large set of pharmacologically relevant compounds (between 282 and 616 compounds/ GPCR). Radioligand binding data were taken from the BioPrint (Cerep, SA) database and we generated concentration–response data in functional assays appropriate to each GPCR target. For all 12 GPCRs assessed, radioligand binding assays detected more active compounds than did the corresponding functional assessment. For all 12 GPCRs more compounds were antagonists than agonists. No consistent trend could be found in sensitivity where pA50 values were defined in both radioligand binding and functional assessments: for some GPCRs binding pA50 was more potent than agonist or antagonist pA50 while for other GPCRs the opposite was true. Differences in pA50 ranged from equipotent for α1A adrenoceptor radioligand binding/antagonism to 33-fold for β2 adrenoceptor radioligand binding/agonism. This data suggests that careful consideration should be given to the choice of technology for in vitro pharmacological profiling for early detection of off-target interactions that drive ADRs.
DP
Toward quasi-in vivo from in vitro assay (I): Development of spatial conductance fluctuation measurement assay using a human cardiomyocyte line-network cell chip with multielectrode array system for in vitro predictive proarrhythmic cardiotoxicity Tomoyo Hamadaa, Fumimasa Nomuraa, Hideyuki Terazonoa, Akihiro Hattoria, Peter Sartipyb, Mitsuhiro Edamurac, Thomas Meyerd, Kenji Yasudaa
0036
OF
large closed loop were formed in those shaped agarose microchambers fabricated on a multielectrode array chip for simultaneous measurement and comparison of their responses against Quinidine. The interspike intervals of the two-dimensional sheet was most durable and stable against administration of Quinidine even at 100 μM high dose, whereas the large closed loop network was most sensitive and showed lethal arrhythmia from 1 μM low dose. The results indicate the apparent spatial arrangement dependence of cell response and the necessity of the consideration of the proper community size and spatial arrangement pattern of cell networks for representing the proper response of cardiomyocytes against the proper amount of compounds.
RO
320
a
Tokyo Medical and Dental University, Tokyo, Japan Cellectis Stem Cells, Göteborg, Sweden c Bio Research Center, Nagoya, Japan d Multi Channel Systems MCS GmbH, Reutlingen, Germany
TE
b
UN
CO
RR
EC
Recent studies show the limitations of proarrhythmic potential prediction in drug induced fatal ventricular tachycardia (VT) or fibrillation (VF) in conventional in vitro assays. Hence, we have developed on-chip in vitro cardiomyocyte network spatiotemporal signal-fluctuation screening assays using human embryonic stem cell-derived cardiomyocytes (hES-CMs) for more precise prediction of fatal arrhythmia. The results showed that the temporal fluctuation (short term variability) of the field potential duration was more effective and precise for the arrhythmogenic prediction against conventional in vitro assays, and that the spatial fluctuation of propagation (conduction) in a lined-up hES-CM network in a rectangular agarose-microchamber on a multielectrode array (MEA) chip was even better than that of the temporal fluctuation assay, e.g. terfenadine, which was difficult to detect proarrhythmic potential in the temporal fluctuation assay. Moreover, simultaneous monitoring of tension generation in the lined-up cardiomyocytes was combined with the electrophysiological measurement set-up in the temperature-controlled long-term observation unit. We also have evaluated the portability of the sealed cardiomyocyte lined-up MEA chip for international transportation between Sweden and Japan and confirmed no distinguishable damage for measurement. We have examined the compatibility of our spatial fluctuation measurement procedures to the existing MEA assay using the cardiomyocyte lined-up MEA chip and the propagation fluctuation analysis software and found that similar predictive tendencies could be acquired from this set-up. The results indicate that the spatial propagation fluctuation assay is the simpler and more precise and could improve the ability of in vitro pro-arrhythmia measurement including TdP/VT/VF as quasi-in vivo assays.
doi:10.1016/j.vascn.2014.03.040
doi:10.1016/j.vascn.2014.03.041
0037 Review: Rat models of pulmonary hypertension Kristy Bruse Lovelace Respiratory Research Institute, Albuquerque, NM, USA Currently, there is no definitive animal disease model of pulmonary arterial hypertension (PAH) that mimics clinical idiopathic PAH. Adult nonrodent animal models such as nonhuman primate (Bruse, Poster at SPS 2012 annual meeting) have essentially not been successful. Transgenic mice models have had partial success. There are currently three rat models of PAH that we have utilized: (1) monocrotaline (MCT; 50 or 60 mg/kg, IP, assessed 28–33 days later), (2) 3 weeks chronic hypoxia (CH; PIO2 = 12%) + 2 weeks normoxia, and (3) Sugen (SU-5416; 20 mg/kg, IP) + 3 weeks chronic hypoxia + 2 weeks normoxia (S-CH). Male rats (Sprague–Dawley or Wistar) aged 9–12 weeks of age were utilized at our facility located at 5355 ft (1632 m) above sea level. Pathophysiological endpoints included: decreased body weight increase (beginning Day 7), elevated right ventricular systolic pressure (RVSP) without systemic blood