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The unbound concentration of multimodal drugs in the assay medium used in the multi-electrode array system for cardiac risk assessment with iPS cell-derived cardiomyocytes
388
Abstracts
best biomarkers for predicting the TdP risk. However, recent increasing evidence suggests that the above currently-used biomarkers are not fully predictive for TdP, and do not address potential arrhythmia induced by other mechanisms including the disruption of hERG trafficking, the interaction with other ion channels including IKs, Nav1.5, Cav1.2 or IK1, or exchangers. In addition to QT and/or non-QTrelated arrhythmia, other cardiac safety liabilities such as dysfunction of exciting-contraction coupling (contractility) and structural damage (morphological damage to cardiomyocytes) are also major causes of drug attrition, but current in vitro assays do not cover all these liability. A recent advance in technology for large-scale production of human stem cell derived cardiomyocytes (hiPS/hES-CMs) has encouraged us to develop a new platform over traditional cell and ex vivo animal models for cardiac safety assessment. Indeed, the combination with hiPS/hESCMs and the new platforms such as multi-electrode arrays, patch clamps, impedance, motion vectors, or optical imaging (Ca transient or high content analysis) offer new comprehensive pre-clinical human models capable of predicting a wide range of cardiac liabilities. We introduce the latest progress on the new platforms and roadmap of CSAHi HEART activity. doi:10.1016/j.vascn.2016.02.171
0175 Electrophysiological characterization of iCell2 hiPSC derived cardiomyocytes: The new generation of CDI hiPSC-CMs Amy E. Taylora, Victor Zamoraa,b, Maria P. Hortigon-Vinagrea,b, Andrew Allana, Francis L. Burtona,b, Blake D. Ansonc, Margaret A. Craiga, Godfrey L. Smitha,b a
Clyde Biosciences Ltd., Glasgow, Scotland, UK University of Glasgow, Glasgow, Scotland, UK c Cellular Dynamics Inc., Madison, WI, USA b
Most of the efforts in cardiac safety pharmacology are orientated towards the improvement of early stage drug screening. In this regard, the creation of in vitro human ventricular electrophysiology has been accelerated by the commercial availability hiPSC-CMs. Many studies have validated iCell cardiomyocytes (Cellular Dynamics Inc.) as a robust model to carry out predictive cardiotoxicity, but the requirement of at least 10 days in vitro (DIV), increases the handling and associated risks. The electrophysiological characterization of 2nd generation of this cell type (iCell2 cardiomyocytes) with accelerated maturation processes that allows their use after 4–7 DIV is presented. Cellular electrophysiology was examined using CellOPTIQ (Clyde Biosciences Ltd.). The cells are cultured in 96-well plates. The electrical activity is recorded on cell monolayers loaded with di-4-ANEPPS (transient incubation in serum-free media). Baseline electrical recordings are registered after 4 and 7 DIV using CellOPTIQ (10 kHz, 15 s). A significant decrease on APD is reported on iCell2 at 7 DIV compared with iCell at 14 DIV (APD90 (mean ± SEM) = 384 ± 4.8 ms vs. 472 ± 4.8 ms, respectively; p b 0.001). Similar dose-dependent APD shortening is described on both when treated with the L-type Ca2 + channel blocker nifedipine. A dosedependent prolongation of APD is shown upon E4031 hERG blockade, but in contrast to the EADs reported on exposure to 30 nM E4301 in the previous iCell, no EADs were evident with iCell2. In summary, the electrophysiological characteristics of iCall2 cell line are easily recorded using the CellOPTIQ and show distinct differences from the earlier generation, compatible with their more efficient use in cardiotoxicology screening. doi:10.1016/j.vascn.2016.02.172
0176 Organ-on-Chip: A synthetic mimic for human myocardium Berend van Meer, Marcelo Ribeiro, Leon Tertoolen, Matthew Birket, Robert Passier, Christine Mummery Leiden University Medical Center, Leiden, The Netherlands Current drug testing methodology still fails to identify high-risk side effects, one third of which are related to cardiotoxicity, despite strict regulatory protocols. Apart from drugs withdrawn after FDA approval, 9 out of 10 experimental drugs already fail in Phase I clinical trials. These are strong indications that the animal models used in drug development do not accurately predict the effects of drugs on humans. Therefore, it is of utmost importance to develop new, humanized heart models that are accurate. Human pluripotent stem cell (hPSC) derived cardiovascular cells could provide new tools for more reliable drug screening and as such, they could serve as an accurate alternative for animal models. Moreover, human Induced Pluripotent Cells (hiPSC) allow modelling of human genetic diseases since these can be obtained by reprogramming somatic cells of adult patients. In this research, a non-invasive measurement setup is being developed that optically assesses the membrane potential, calcium flux and force of contraction simultaneously in a single cell. The biophysical parameters are each sampled with a frequency of 333 Hz, allowing accurate evaluation of most important parameter values and kinetics. This provides a starting point to integrate the multiplexed measurement assays with a heterogeneous tissue population and engineered scaffold technology to create a synthetic mimic for myocardium, resulting in a so called “Organ-on-Chip”-model. Furthermore, with the use of hiPSC, genetic cardiac disease models can be made to use in drug development, reducing the use of animal models and increasing the efficiency of pharmaceutical drug discovery trajectories. doi:10.1016/j.vascn.2016.02.173
0177 The unbound concentration of multimodal drugs in the assay medium used in the multi-electrode array system for cardiac risk assessment with iPS cell-derived cardiomyocytes Takashi Yoshinagaa,d, Raku Shinkyob, Kiyomi Kikuchib, Tomohiko Taniguchia,d, Yuko Sekinoc,d, Kohei Sawadaa,d a
Global CV Assessment, Biopharmaceutical Assessments CFU, Eisai Co., Ltd., Tsukuba, Japan b Global DMPK, Biopharmaceutical Assessments CFU, Eisai Co., Ltd., Tsukuba, Japan c Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan d Japan iPS Cardiac Safety Assessment, Tokyo, Japan To characterize and predict the QT interval prolongation and proarrhythmia potential of drug candidates, investigations into the challenges of using multi-electrode array system and human stem cell-derived cardiomyocytes are on-going. Sometimes, discussions arise on whether assay results should be interpreted using total (applied) or unbound concentrations to understand the compounds cardiovascular liabilities. To provide useful suggestions, we measured the unbound concentrations of 59 drugs which include CiPA selected 29 compounds in the iCell Cardiomyocytes Maintenance Medium (CMM, Cellular Dynamics International) including fetal bovine serum
Abstracts
by equilibrium dialysis method using Rapid Equilibrium Dialysis device (ThermoFisher Scientific). Then, unbound fraction (fu) values at low and high concentrations (100-fold differences) in CMM were calculated. When saturation of binding at the high concentration was observed, an additional experiment was done at a middle concentration. Most of compounds showed similar fu values at low and high concentrations. On the other hand, several compounds such as Mibefradil, Thioridazine, Bepridil, Prenylamine, Tolterodine, Haloperidol, and Dronedarone showed more than 2-fold higher fu values at high concentrations than those at low ones. Tamoxifene and Amiodarone showed very low fu values (less than 0.01) which indicated very strong protein binding. Further analysis of the relationships between the fu values in CMM and human plasma was conducted. Good correlation in rank order with non-linier correlation was obtained. These results indicated that the actual unbound concentrations in CMM will be very useful to consider and interpret the relationship to unbound plasma concentrations which caused CV events in human. doi:10.1016/j.vascn.2016.02.174
0178 A comparison of drug-induced changes in electrical excitability using optogenetic sensors and conventional field stimulation in hiPS-derived cardiomyocytes (hiPSC-CMs) Victor Zamoraa,b, Maria Hortigon-Vinagrea,b, Andrew Allana, Hua Rong Luc, Francis Burtona,b, David Gallacherc, Godfrey Smitha,b a
Clyde Biosciences Ltd, Glasgow, Scotland, UK Institute of Cardiovascular and Medical Sciences, Glasgow, Scotland, UK c Global Safety Pharmacology, Discovery Sciences, Janssen Research & Development, Janssen Pharmaceutica NV, Beerse, Belgium b
Changes in myocardial electrical excitability are difficult to assess in medium–high throughput, due to the practical difficulty of using solid-state electrodes to provide stimulus to a large number of independent assays (100–200/day) while preventing contamination. Optogenetic probes, e.g. Channel Rhodopsin-2 (ChR2), offer a solution to this problem. ChR2 in excitable cell membranes allows the use of light (470 nm) to initiate an action potential and subsequent contraction. We used CellOPTIQ platform (Clyde Biosciences Ltd) to stimulate hiPSC-CMs with solid-state or optogenetic and compared changes in excitability caused by reference drugs. hiPSC-CMs (Axiogenesis AG) were transfected with AAV-ChR2 (Addgene# 20938M) at MOI 40,000 (48 h). 24 h post-transfection cells were stimulated to contract (2 ms pulse, 2 Hz and 3 Hz, 470 nm LED). The intensity of light was gradually increased until the myocytes responded to stimulation (assessed using contraction monitored). Cumulative concentrations of drug were applied and the threshold re-assessed after 30 min. Controls assays were done in parallel. Data were compared with threshold measurements using graphite electrodes (no ChR2). Moxifloxacin and Mexiletine (up to 10 μM) both caused frequency dependent decreases in excitability as assessed by both while quinidine and flecainide (up to 10 μM) showed comparable changes in excitability at both 2 Hz and 3 Hz using both techniques. Drugs not expected to alter excitability (atenolol and ebastine up to 3 μM) were without affect. This work demonstrates that optogenetic techniques applied to hiPS-CMs offers a sensitive method of detecting changes in cardiac excitability that can be applied at medium-high throughput. doi:10.1016/j.vascn.2016.02.175
389
0179 Use of inulin and PAH for renal drug safety evaluations in conscious dogs Matthew Abernathy, Jill Dalton, Marci Harter, David Gauvin, Theodore Baird MPI Research, Mattawan, MI, USA The glomerulus is composed of afferent and efferent arterioles allowing for blood filtration within the renal corpuscle. To measure a pharmacological impact on kidney function, glomerular filtration rate (GFR) can be quantified by examining the elimination rate of polysaccharides, like inulin. Similarly, renal blood flow (RBF) can be assessed by administration of para-aminohippurate (PAH), an acid also filtered from the blood and secreted into urine. Various methodologies for quantifying GFR and RBF rely on different analytic substrates, concentrations, loading durations, and/or the frequency of blood/urine sampling, making data comparisons difficult across laboratories. The objective of this study was to evaluate several dose regimens of inulin and PAH to determine optimal conditions for measurement of GFR and RBF. Four young adult male beagle dogs were intravenously administered three inulin/PAH conditions: 200 mg/kg inulin and 40 mg/kg PAH, 200 mg/kg/h inulin and 40 mg/kg/h PAH, and 70 mg/kg/h inulin and 13.5 mg/kg/h PAH. Serial paired- sampling of urine and blood were collected at 45, 75, 105, and 135 min after the initiation of each inulin/PAH administration. After the optimal inulin/PAH dose combination was identified, the GFR/RBF method was re-evaluated using oral administration of known renalactive compounds: 2 mg/kg hydrochlorothiazide, 2 mg/kg furosemide, 20 mg/kg acetazolamide, and sterile water. The results showed that sustained high concentrations of inulin and low concentrations of PAH produced more stable and reliable estimates of both GFR and RBF. Furthermore, the optimal concentrations of inulin and PAH generated data with the expected directionality following all three test compounds.
doi:10.1016/j.vascn.2016.02.176
0180 Gastroinstestinal motility: Motility and motor migrating complex (MMC) evaluations in rats, dogs and non-human primates Simon Authiera,c, Hai-Ming Tangb, Samir Abtouta, Alexis Ascaha, Mylene Pouliota, Kim Bujolda, Eric Troncyb, Michael K. Pugsleya, Roy Forstera a
CiToxLAB North America, Laval, QC, Canada Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA c Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, QC, Canada b
Introduction: Drug-induced effects on gastrointestinal motility are observed with a number of approved drugs but available nonclinical assays for assessing such effects are limited in drug development. Methodology: Fluoroscopic video imaging was used to assess oesophageal and gastric motility in rats using a buccal iodixanol radio-opaque meal. In Beagle dogs and cynomolgus monkeys, this methodology was used to monitor gastric and intestinal motor migrating complexes and gastric emptying times using 10 × 3 mm radio-opaque beads and/or barium solution. In dogs, gastric pH was sequentially measured from fasted animals or after a wet food meal.
Abstracts
best biomarkers for predicting the TdP risk. However, recent increasing evidence suggests that the above currently-used biomarkers are not fully predictive for TdP, and do not address potential arrhythmia induced by other mechanisms including the disruption of hERG trafficking, the interaction with other ion channels including IKs, Nav1.5, Cav1.2 or IK1, or exchangers. In addition to QT and/or non-QTrelated arrhythmia, other cardiac safety liabilities such as dysfunction of exciting-contraction coupling (contractility) and structural damage (morphological damage to cardiomyocytes) are also major causes of drug attrition, but current in vitro assays do not cover all these liability. A recent advance in technology for large-scale production of human stem cell derived cardiomyocytes (hiPS/hES-CMs) has encouraged us to develop a new platform over traditional cell and ex vivo animal models for cardiac safety assessment. Indeed, the combination with hiPS/hESCMs and the new platforms such as multi-electrode arrays, patch clamps, impedance, motion vectors, or optical imaging (Ca transient or high content analysis) offer new comprehensive pre-clinical human models capable of predicting a wide range of cardiac liabilities. We introduce the latest progress on the new platforms and roadmap of CSAHi HEART activity. doi:10.1016/j.vascn.2016.02.171
0175 Electrophysiological characterization of iCell2 hiPSC derived cardiomyocytes: The new generation of CDI hiPSC-CMs Amy E. Taylora, Victor Zamoraa,b, Maria P. Hortigon-Vinagrea,b, Andrew Allana, Francis L. Burtona,b, Blake D. Ansonc, Margaret A. Craiga, Godfrey L. Smitha,b a
Clyde Biosciences Ltd., Glasgow, Scotland, UK University of Glasgow, Glasgow, Scotland, UK c Cellular Dynamics Inc., Madison, WI, USA b
Most of the efforts in cardiac safety pharmacology are orientated towards the improvement of early stage drug screening. In this regard, the creation of in vitro human ventricular electrophysiology has been accelerated by the commercial availability hiPSC-CMs. Many studies have validated iCell cardiomyocytes (Cellular Dynamics Inc.) as a robust model to carry out predictive cardiotoxicity, but the requirement of at least 10 days in vitro (DIV), increases the handling and associated risks. The electrophysiological characterization of 2nd generation of this cell type (iCell2 cardiomyocytes) with accelerated maturation processes that allows their use after 4–7 DIV is presented. Cellular electrophysiology was examined using CellOPTIQ (Clyde Biosciences Ltd.). The cells are cultured in 96-well plates. The electrical activity is recorded on cell monolayers loaded with di-4-ANEPPS (transient incubation in serum-free media). Baseline electrical recordings are registered after 4 and 7 DIV using CellOPTIQ (10 kHz, 15 s). A significant decrease on APD is reported on iCell2 at 7 DIV compared with iCell at 14 DIV (APD90 (mean ± SEM) = 384 ± 4.8 ms vs. 472 ± 4.8 ms, respectively; p b 0.001). Similar dose-dependent APD shortening is described on both when treated with the L-type Ca2 + channel blocker nifedipine. A dosedependent prolongation of APD is shown upon E4031 hERG blockade, but in contrast to the EADs reported on exposure to 30 nM E4301 in the previous iCell, no EADs were evident with iCell2. In summary, the electrophysiological characteristics of iCall2 cell line are easily recorded using the CellOPTIQ and show distinct differences from the earlier generation, compatible with their more efficient use in cardiotoxicology screening. doi:10.1016/j.vascn.2016.02.172
0176 Organ-on-Chip: A synthetic mimic for human myocardium Berend van Meer, Marcelo Ribeiro, Leon Tertoolen, Matthew Birket, Robert Passier, Christine Mummery Leiden University Medical Center, Leiden, The Netherlands Current drug testing methodology still fails to identify high-risk side effects, one third of which are related to cardiotoxicity, despite strict regulatory protocols. Apart from drugs withdrawn after FDA approval, 9 out of 10 experimental drugs already fail in Phase I clinical trials. These are strong indications that the animal models used in drug development do not accurately predict the effects of drugs on humans. Therefore, it is of utmost importance to develop new, humanized heart models that are accurate. Human pluripotent stem cell (hPSC) derived cardiovascular cells could provide new tools for more reliable drug screening and as such, they could serve as an accurate alternative for animal models. Moreover, human Induced Pluripotent Cells (hiPSC) allow modelling of human genetic diseases since these can be obtained by reprogramming somatic cells of adult patients. In this research, a non-invasive measurement setup is being developed that optically assesses the membrane potential, calcium flux and force of contraction simultaneously in a single cell. The biophysical parameters are each sampled with a frequency of 333 Hz, allowing accurate evaluation of most important parameter values and kinetics. This provides a starting point to integrate the multiplexed measurement assays with a heterogeneous tissue population and engineered scaffold technology to create a synthetic mimic for myocardium, resulting in a so called “Organ-on-Chip”-model. Furthermore, with the use of hiPSC, genetic cardiac disease models can be made to use in drug development, reducing the use of animal models and increasing the efficiency of pharmaceutical drug discovery trajectories. doi:10.1016/j.vascn.2016.02.173
0177 The unbound concentration of multimodal drugs in the assay medium used in the multi-electrode array system for cardiac risk assessment with iPS cell-derived cardiomyocytes Takashi Yoshinagaa,d, Raku Shinkyob, Kiyomi Kikuchib, Tomohiko Taniguchia,d, Yuko Sekinoc,d, Kohei Sawadaa,d a
Global CV Assessment, Biopharmaceutical Assessments CFU, Eisai Co., Ltd., Tsukuba, Japan b Global DMPK, Biopharmaceutical Assessments CFU, Eisai Co., Ltd., Tsukuba, Japan c Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan d Japan iPS Cardiac Safety Assessment, Tokyo, Japan To characterize and predict the QT interval prolongation and proarrhythmia potential of drug candidates, investigations into the challenges of using multi-electrode array system and human stem cell-derived cardiomyocytes are on-going. Sometimes, discussions arise on whether assay results should be interpreted using total (applied) or unbound concentrations to understand the compounds cardiovascular liabilities. To provide useful suggestions, we measured the unbound concentrations of 59 drugs which include CiPA selected 29 compounds in the iCell Cardiomyocytes Maintenance Medium (CMM, Cellular Dynamics International) including fetal bovine serum
Abstracts
by equilibrium dialysis method using Rapid Equilibrium Dialysis device (ThermoFisher Scientific). Then, unbound fraction (fu) values at low and high concentrations (100-fold differences) in CMM were calculated. When saturation of binding at the high concentration was observed, an additional experiment was done at a middle concentration. Most of compounds showed similar fu values at low and high concentrations. On the other hand, several compounds such as Mibefradil, Thioridazine, Bepridil, Prenylamine, Tolterodine, Haloperidol, and Dronedarone showed more than 2-fold higher fu values at high concentrations than those at low ones. Tamoxifene and Amiodarone showed very low fu values (less than 0.01) which indicated very strong protein binding. Further analysis of the relationships between the fu values in CMM and human plasma was conducted. Good correlation in rank order with non-linier correlation was obtained. These results indicated that the actual unbound concentrations in CMM will be very useful to consider and interpret the relationship to unbound plasma concentrations which caused CV events in human. doi:10.1016/j.vascn.2016.02.174
0178 A comparison of drug-induced changes in electrical excitability using optogenetic sensors and conventional field stimulation in hiPS-derived cardiomyocytes (hiPSC-CMs) Victor Zamoraa,b, Maria Hortigon-Vinagrea,b, Andrew Allana, Hua Rong Luc, Francis Burtona,b, David Gallacherc, Godfrey Smitha,b a
Clyde Biosciences Ltd, Glasgow, Scotland, UK Institute of Cardiovascular and Medical Sciences, Glasgow, Scotland, UK c Global Safety Pharmacology, Discovery Sciences, Janssen Research & Development, Janssen Pharmaceutica NV, Beerse, Belgium b
Changes in myocardial electrical excitability are difficult to assess in medium–high throughput, due to the practical difficulty of using solid-state electrodes to provide stimulus to a large number of independent assays (100–200/day) while preventing contamination. Optogenetic probes, e.g. Channel Rhodopsin-2 (ChR2), offer a solution to this problem. ChR2 in excitable cell membranes allows the use of light (470 nm) to initiate an action potential and subsequent contraction. We used CellOPTIQ platform (Clyde Biosciences Ltd) to stimulate hiPSC-CMs with solid-state or optogenetic and compared changes in excitability caused by reference drugs. hiPSC-CMs (Axiogenesis AG) were transfected with AAV-ChR2 (Addgene# 20938M) at MOI 40,000 (48 h). 24 h post-transfection cells were stimulated to contract (2 ms pulse, 2 Hz and 3 Hz, 470 nm LED). The intensity of light was gradually increased until the myocytes responded to stimulation (assessed using contraction monitored). Cumulative concentrations of drug were applied and the threshold re-assessed after 30 min. Controls assays were done in parallel. Data were compared with threshold measurements using graphite electrodes (no ChR2). Moxifloxacin and Mexiletine (up to 10 μM) both caused frequency dependent decreases in excitability as assessed by both while quinidine and flecainide (up to 10 μM) showed comparable changes in excitability at both 2 Hz and 3 Hz using both techniques. Drugs not expected to alter excitability (atenolol and ebastine up to 3 μM) were without affect. This work demonstrates that optogenetic techniques applied to hiPS-CMs offers a sensitive method of detecting changes in cardiac excitability that can be applied at medium-high throughput. doi:10.1016/j.vascn.2016.02.175
389
0179 Use of inulin and PAH for renal drug safety evaluations in conscious dogs Matthew Abernathy, Jill Dalton, Marci Harter, David Gauvin, Theodore Baird MPI Research, Mattawan, MI, USA The glomerulus is composed of afferent and efferent arterioles allowing for blood filtration within the renal corpuscle. To measure a pharmacological impact on kidney function, glomerular filtration rate (GFR) can be quantified by examining the elimination rate of polysaccharides, like inulin. Similarly, renal blood flow (RBF) can be assessed by administration of para-aminohippurate (PAH), an acid also filtered from the blood and secreted into urine. Various methodologies for quantifying GFR and RBF rely on different analytic substrates, concentrations, loading durations, and/or the frequency of blood/urine sampling, making data comparisons difficult across laboratories. The objective of this study was to evaluate several dose regimens of inulin and PAH to determine optimal conditions for measurement of GFR and RBF. Four young adult male beagle dogs were intravenously administered three inulin/PAH conditions: 200 mg/kg inulin and 40 mg/kg PAH, 200 mg/kg/h inulin and 40 mg/kg/h PAH, and 70 mg/kg/h inulin and 13.5 mg/kg/h PAH. Serial paired- sampling of urine and blood were collected at 45, 75, 105, and 135 min after the initiation of each inulin/PAH administration. After the optimal inulin/PAH dose combination was identified, the GFR/RBF method was re-evaluated using oral administration of known renalactive compounds: 2 mg/kg hydrochlorothiazide, 2 mg/kg furosemide, 20 mg/kg acetazolamide, and sterile water. The results showed that sustained high concentrations of inulin and low concentrations of PAH produced more stable and reliable estimates of both GFR and RBF. Furthermore, the optimal concentrations of inulin and PAH generated data with the expected directionality following all three test compounds.
doi:10.1016/j.vascn.2016.02.176
0180 Gastroinstestinal motility: Motility and motor migrating complex (MMC) evaluations in rats, dogs and non-human primates Simon Authiera,c, Hai-Ming Tangb, Samir Abtouta, Alexis Ascaha, Mylene Pouliota, Kim Bujolda, Eric Troncyb, Michael K. Pugsleya, Roy Forstera a
CiToxLAB North America, Laval, QC, Canada Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA c Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, QC, Canada b
Introduction: Drug-induced effects on gastrointestinal motility are observed with a number of approved drugs but available nonclinical assays for assessing such effects are limited in drug development. Methodology: Fluoroscopic video imaging was used to assess oesophageal and gastric motility in rats using a buccal iodixanol radio-opaque meal. In Beagle dogs and cynomolgus monkeys, this methodology was used to monitor gastric and intestinal motor migrating complexes and gastric emptying times using 10 × 3 mm radio-opaque beads and/or barium solution. In dogs, gastric pH was sequentially measured from fasted animals or after a wet food meal.