- Email: [email protected]
Review: Rat models of pulmonary hypertension
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
Abstracts
0038 An in silico transmural model for arrhythmias originated from the Purkinje network/endocardium interface Pascal Champeroux, Sebastien Jude, Christine Laigot, Anne Maurin, Arnaud Laveissière, Serge Richard
OF
doi:10.1016/j.vascn.2014.03.042
Sildenafil (5.625 mg/kg) significantly decreased the serum lipid parameters including total lipid, triacylglycerols, cholesterol, and HDL-C, LDL-C, and VLDL-C concentrations of rats fed on fat-enriched diet. However, it increased their values in the serum of negative control rats. In addition, the administration of sildenafil to normal rats caused insignificant changes in serum liver enzymes' ALT and AST concentrations all over the period of the experiment; as well as serum urea and creatinine; yet, it significantly decreased their serum concentrations in animals fed on fat-enriched diet compared to the +ve untreated ones, upon its administration starting from the 30th day of the experiment. However, concurrent administration of sildenafil with high-fat diet (group-iv) failed to guard against the rise in such liver and kidney function biomarkers. These data suggest that sildenafil may act as a mixed blessing drug; therefore it must be used carefully and under physician supervision to get its therapeutic benefits and guard against its adverse effects. Keywords: sildenafil, liver function, kidney function, lipid profile, erectile dysfunction, side effect. doi:10.1016/j.vascn.2014.03.044
CERB, Baugy, France 0040
N-(2-hydroxy phenyl) acetamide induces apoptosis of brain cancer cells by increasing Bax/Bcl-2 ratio Farina Hanifa, Kahkashan Perveena, Shabana Usman Simjeea,b
DP
The proarrhythmic profile of a drug is determined by its electrophysiological properties on cardiac cells in most cases. We built an in silico canine model to reproduce a transmural heterogeneity in terms of action potential pattern from the Purkinje network, endocardial cells, M cells and up to epicardial cells. This model also includes possible differences in terms of electrophysiological sensitivity depending on the cellular type and involved ionic channels. The initial objective was to rebuild the T wave pattern in order to anticipate the impact of ex vivo finding collected in isolated Purkinje fibres and endocardial cells on the QT interval. Our first results show that QT prolongation or shortening can be modelled from these ex vivo data with good correlations with in vivo QT studies. Then, we integrate in this model a possible influence of the autonomic nervous system (ANS) on electrophysiological properties of known proarrhythmic drugs. Indeed, changes in the autonomic balance are known as triggering arrhythmias. We used data that were derived from in vivo power spectral analysis of heart rate variability and from ex vivo experiments in isolated cardiac tissues combining agents known for their interaction with ANS. Our results suggest that our model is able to trigger in silico arrhythmias driven by the ANS in the presence of proarrhythmic drugs (dofetilide). Moreover, our data suggest that the Purkinje network/endocardial cells interface could play a major role through reentry mechanisms.
RO
pressure changes, increased right heart weight (Fulton Index), heavier lung weight and lung vascular wall thickening. The S-CH and MCT models of severe PAH are similar in RVSP (65 and 58 mm Hg) and Fulton Index (0.561 and 0.585) compared to control and CH (RVSP 28.1 and 34.3 mm Hg; Fulton Index 0.377 and 0.398). In summary, these rat models are effective at the altitude of our facility with the MCT model utilized for initial efficacy screening and the S-CH model utilized to confirm efficacy of novel therapies to treat clinical idiopathic PAH.
321
a
TE
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Sind, Pakistan b HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Sind, Pakistan
0039
CO
doi:10.1016/j.vascn.2014.03.043
RR
EC
Despite the modern therapies available for treating glioblastoma (GBM), it is still a deadly disease. The development of new therapeutic strategies for the management of gliomas is therefore crucial. The present study is designed to analyze the therapeutic potentials of synthetic compound N-(2-hydroxyphenyl) acetamide in the treatment of GBM alone or in combination with temozolomide on glioblastoma cells. MTT and TUNEL assays were used to detect the growth inhibitory effect and apoptotic activity of NA-2 alone and in combination with temozolomide. Synergy was assessed using combination index method. The expression of apoptosis related markers BAX and BCL-2 were assessed by RT-PCR. Chromatin condensation was observed using DAPI staining. Both NA-2 and temozolomide dose dependently inhibited the growth of U87 cells. The combined administration of NA-2 (50 μg/ml) and temozolomide (100 μM) significantly enhanced the cell growth inhibition and apoptosis. Furthermore RT-PCR and immunocytochemistry data revealed that cooperative apoptosis induction was associated with increased BAX/ BCL-2 ratio. Our findings support that NA-2 possesses strong apoptotic activity and the combined administration of NA-2 and TMZ may be therapeutically exploited for the management of GBM.
a
UN
Safety profile after long-term administration of sildenafil to hyperlipidemic albino rats Abubakr El-Mahmoudya, Amer Elgerwib Department of Pharmacology, Benha University Faculty of Veterinary Medicine, Moshtohor, Qalioubeya, Egypt b Department of Pharmacology, Toxicology & Forensic Medicine, Tripoli University Faculty of Veterinary Medicine, Tripoli, Libya The present study was adopted to investigate the possible biochemical alterations in lipid metabolic profile and organ function profiles that may result from continuous treatment with the drug, sildenafil in normal and hyperlipidemic rats. Blood samples were taken for biochemical analysis on days 30, 45 and 60 of the experiment.
doi:10.1016/j.vascn.2014.03.045
0041 Predicting cardiac toxicity from data generated via high-throughput screening Hitesh Mistry, Frances Brightman, Eric Fernandez, David Orrell, Jonathan Swinton, Christophe Chassagnole Physiomics PLC, Oxford, UK
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
Abstracts
0038 An in silico transmural model for arrhythmias originated from the Purkinje network/endocardium interface Pascal Champeroux, Sebastien Jude, Christine Laigot, Anne Maurin, Arnaud Laveissière, Serge Richard
OF
doi:10.1016/j.vascn.2014.03.042
Sildenafil (5.625 mg/kg) significantly decreased the serum lipid parameters including total lipid, triacylglycerols, cholesterol, and HDL-C, LDL-C, and VLDL-C concentrations of rats fed on fat-enriched diet. However, it increased their values in the serum of negative control rats. In addition, the administration of sildenafil to normal rats caused insignificant changes in serum liver enzymes' ALT and AST concentrations all over the period of the experiment; as well as serum urea and creatinine; yet, it significantly decreased their serum concentrations in animals fed on fat-enriched diet compared to the +ve untreated ones, upon its administration starting from the 30th day of the experiment. However, concurrent administration of sildenafil with high-fat diet (group-iv) failed to guard against the rise in such liver and kidney function biomarkers. These data suggest that sildenafil may act as a mixed blessing drug; therefore it must be used carefully and under physician supervision to get its therapeutic benefits and guard against its adverse effects. Keywords: sildenafil, liver function, kidney function, lipid profile, erectile dysfunction, side effect. doi:10.1016/j.vascn.2014.03.044
CERB, Baugy, France 0040
N-(2-hydroxy phenyl) acetamide induces apoptosis of brain cancer cells by increasing Bax/Bcl-2 ratio Farina Hanifa, Kahkashan Perveena, Shabana Usman Simjeea,b
DP
The proarrhythmic profile of a drug is determined by its electrophysiological properties on cardiac cells in most cases. We built an in silico canine model to reproduce a transmural heterogeneity in terms of action potential pattern from the Purkinje network, endocardial cells, M cells and up to epicardial cells. This model also includes possible differences in terms of electrophysiological sensitivity depending on the cellular type and involved ionic channels. The initial objective was to rebuild the T wave pattern in order to anticipate the impact of ex vivo finding collected in isolated Purkinje fibres and endocardial cells on the QT interval. Our first results show that QT prolongation or shortening can be modelled from these ex vivo data with good correlations with in vivo QT studies. Then, we integrate in this model a possible influence of the autonomic nervous system (ANS) on electrophysiological properties of known proarrhythmic drugs. Indeed, changes in the autonomic balance are known as triggering arrhythmias. We used data that were derived from in vivo power spectral analysis of heart rate variability and from ex vivo experiments in isolated cardiac tissues combining agents known for their interaction with ANS. Our results suggest that our model is able to trigger in silico arrhythmias driven by the ANS in the presence of proarrhythmic drugs (dofetilide). Moreover, our data suggest that the Purkinje network/endocardial cells interface could play a major role through reentry mechanisms.
RO
pressure changes, increased right heart weight (Fulton Index), heavier lung weight and lung vascular wall thickening. The S-CH and MCT models of severe PAH are similar in RVSP (65 and 58 mm Hg) and Fulton Index (0.561 and 0.585) compared to control and CH (RVSP 28.1 and 34.3 mm Hg; Fulton Index 0.377 and 0.398). In summary, these rat models are effective at the altitude of our facility with the MCT model utilized for initial efficacy screening and the S-CH model utilized to confirm efficacy of novel therapies to treat clinical idiopathic PAH.
321
a
TE
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Sind, Pakistan b HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Sind, Pakistan
0039
CO
doi:10.1016/j.vascn.2014.03.043
RR
EC
Despite the modern therapies available for treating glioblastoma (GBM), it is still a deadly disease. The development of new therapeutic strategies for the management of gliomas is therefore crucial. The present study is designed to analyze the therapeutic potentials of synthetic compound N-(2-hydroxyphenyl) acetamide in the treatment of GBM alone or in combination with temozolomide on glioblastoma cells. MTT and TUNEL assays were used to detect the growth inhibitory effect and apoptotic activity of NA-2 alone and in combination with temozolomide. Synergy was assessed using combination index method. The expression of apoptosis related markers BAX and BCL-2 were assessed by RT-PCR. Chromatin condensation was observed using DAPI staining. Both NA-2 and temozolomide dose dependently inhibited the growth of U87 cells. The combined administration of NA-2 (50 μg/ml) and temozolomide (100 μM) significantly enhanced the cell growth inhibition and apoptosis. Furthermore RT-PCR and immunocytochemistry data revealed that cooperative apoptosis induction was associated with increased BAX/ BCL-2 ratio. Our findings support that NA-2 possesses strong apoptotic activity and the combined administration of NA-2 and TMZ may be therapeutically exploited for the management of GBM.
a
UN
Safety profile after long-term administration of sildenafil to hyperlipidemic albino rats Abubakr El-Mahmoudya, Amer Elgerwib Department of Pharmacology, Benha University Faculty of Veterinary Medicine, Moshtohor, Qalioubeya, Egypt b Department of Pharmacology, Toxicology & Forensic Medicine, Tripoli University Faculty of Veterinary Medicine, Tripoli, Libya The present study was adopted to investigate the possible biochemical alterations in lipid metabolic profile and organ function profiles that may result from continuous treatment with the drug, sildenafil in normal and hyperlipidemic rats. Blood samples were taken for biochemical analysis on days 30, 45 and 60 of the experiment.
doi:10.1016/j.vascn.2014.03.045
0041 Predicting cardiac toxicity from data generated via high-throughput screening Hitesh Mistry, Frances Brightman, Eric Fernandez, David Orrell, Jonathan Swinton, Christophe Chassagnole Physiomics PLC, Oxford, UK