Query String: CISAPRIDE
4-amino-5-chloro-N-(1-(3-(4-fluorophenoxy)propyl)-3-methoxypiperidin-4-yl)-2-methoxybenzamide PROPULSID cid_5311047 US10167299, Cisapride CISAPRIDE US10800776, Example Cisapride CHEMBL560739 US9221790, Cisapride 4-Amino-5-chloro-N-{1-[3-(4-fluoro-phenoxy)-propyl]-3-methoxy-piperidin-4-yl}-2-methoxy-benzamide BDBM50005836
- ChEMBL_978839 Inhibition of human ERG expressed in cisapride treated CHO cells by Ion Works assay
- Cell-Based Electrophysiology Assays All hERG percentage inhibitions at 10 uM compound concentration were determined by Millipore in a high-throughput cell-based electrophysiology assay for inhibition of hERG tail current41, and values are reported as a mean of multiple determinations. 0.3% DMSO aqueous vehicle negative control gave 7-16% inhibition. Cisapride (1 uM) positive control gave 96-104% inhibition. All hERG IC50 values were determined by Millipore,41 and the hERG IC50 for Example 1 was also determined by Cyprotex plc measuring hERG tail-currents by whole-cell voltage-clamping.
- hERG Assay Voltage clamp protocol: Computer software was used to set voltage clamp protocols. In whole-cell model, cells were held at −80 mV, first depolarized to −50 mV for 80 ms, and then depolarized to +20 mV for 4800 ms to activate the hERG channels. After that the cells were repolarized to −50 mV for 5000 ms to elicit the characteristic tail currents. Finally the cells were held at −80 mV again. The peak values of tail currents were sampled for analysis.Automated QPatch procedure: After achieving break-in (whole-cell) configuration, the cells were recorded for 120 sec to assess current stability. The voltage protocol described above was then applied to the cells every 15 sec throughout the whole procedure. Only stable cells with recording parameters above threshold were allowed to enter the drug application procedure. All experiments were conducted at room temperature (about 25° C.). External solution containing 0.1% DMSO (vehicle) was applied to the cells to establish the baseline. After allowing the current to stabilize for 3 minutes, compound was applied. Compound solution was added and the cells were kept in the test solution until the compound's effect reached a steady state or for a maximum of 4 min. For dose response assay, compound was applied to the cells accumulatively from low to high concentrations. Washout with external solution was performed after compound testing. Positive control cisapride is used in the experiments to test the same batch of cells used for test compounds to ensure the normal response and the good quality of the cells.Data were analyzed using Assay Software provided by Sophion, Microsoft Excel and Graphpad Prism.
- Inhibition Assay hERG study was conducted with an automated patch clamp machine, Qpatch-48HT (Sophion Biosciences, Denmark). Cultured CHO cells stably expressing hERG channels (provided by Sophion Biosciences, Denmark) were harvested from culture flasks of 70-90% cell confluence rate and prepared as cell suspension with a cell density of 3-8&timse;10^6 cells/mL in serum-free media (CHO-S-SFM II, cat#12052 Invitrogen; 25 mM HEPES). Cells in such condition were placed into a Qpatch cell stir chamber and used within 4 hours. For each run, cells were first spun down by the built-in Qpatch centrifuge and re-suspended in an extracellular solution (in mM, 2 CaCl2, 1 MgCl2, 4 KCl, 145 NaCl, 10 Glucose, 10 HEPES, pH 7.4, osmolarity ~305 mOsm). Qplate-48 that holds one cell in each of its 48 channels for the later voltage-clamped assay were primed with the extracellular solution and intracellular solution (in mM, 5.4 CaCl2, 1.75 MgCl2, 120 KCl, 10 HEPES, 5 EGTA, 4 NaATP, pH 7.25, Osmolarity ~280-295 mOsm). Cells were dispatched by Qpatch robotic dispensing guns into each Qplate channel and went through the process of giga-ohm sealing and whole cell configuration. Whole-cell recordings were performed in voltage-clamp mode at a holding potential of −80 mV. The hERG current was activated by depolarizing at +20 mV for 5 sec, after which the current was taken back to −50 mV for 5 sec to remove the inactivation and observe the deactivating tail current. The maximum amount of tail current size was used to determine the hERG current amplitude. The above voltage protocol was applied to the cells every 15 sec throughout the whole procedure. External solution containing 0.1% DMSO (vehicle) was applied to the cells to establish the baseline. Compound solution was added, and the cells were kept in the test solution until the compound's effect reached a steady state or for a maximum of 4 min. For dose response assays (0.1, 0.3, 1, 3, 10 and 30 μM), the compound was applied to the cells accumulatively from low to high concentrations. Washout with extracellular solution was performed after compound testing. Positive control cisapride 0.1 μM was used on each cell after compound testing to ensure the normal response and the good quality of the cell.
- hERG Analysis-Method 1 Cell CultureThe hERG-expressing Chinese hamster ovary K1 (CHO) cells described by (Persson, Carlsson, Duker, & Jacobson, 2005) were grown to semi-confluence at 37° C. in a humidified environment (5% CO2) in F-12 Ham medium containing L-glutamine, 10% foetal calf serum (FCS) and 0.6 mg/mL hygromycin (all available from Sigma-Aldrich). Prior to use, the monolayer was washed using a pre-warmed (37° C.) 3 mL aliquot of Versene 1:5,000 (Invitrogen) After aspiration of this solution the flask was incubated at 37° C. in an incubator with a further 2 mL of Versene 1:5,000 for a period of 6 minutes. Cells were then detached from the bottom of the flask by gentle tapping and 10 mL of Dulbecco's Phosphate-Buffered Saline containing calcium (0.9 mM) and magnesium (0.5 mM) (PBS; Invitrogen) was then added to the flask and aspirated into a 15 mL centrifuge tube prior to centrifugation (50 g, for 4 mins). The resulting supernatant was discarded and the pellet gently re-suspended in 3 mL of PBS. A 0.5 mL aliquot of cell suspension was removed and the number of viable cells (based on trypan blue exclusion) was determined in an automated reader (Cedex; Innovatis) so that the cell re-suspension volume can be adjusted with PBS to give the desired final cell concentration. It is the cell concentration at this point in the assay that is quoted when referring to this parameter. CHO-Kv1.5 cells, which were used to adjust the voltage offset on IONWORKS HT, were maintained and prepared for use in the same way.ElectrophysiologyThe principles and operation of this device have been described by (Schroeder, Neagle, Trezise, & Worley, 2003). Briefly, the technology is based on a 384-well plate (PATCHPLATE) in which a recording was attempted in each well by using suction to position and hold a cell on a small hole separating two isolated fluid chambers. Once sealing had taken place, the solution on the underside of the PATCHPLATE was changed to one containing amphotericin B. This permeablises the patch of cell membrane covering the hole in each well and, in effect, allowed a perforated, whole-cell patch clamp recording to be made.A β-test IONWORKS HT from Essen Instrument was used. There is no capability to warm solutions in this device hence it is operated at r.t. (21° C.), as follows. The reservoir in the Buffer position was loaded with 4 mL of PBS and that in the Cells position with the CHO-hERG cell suspension described above. A 96-well plate (V-bottom, Greiner Bio-one) containing the compounds to be tested (at 3-fold above their final test concentration) was placed in the Plate 1 position and a PATCHPLATE was clamped into the PATCHPLATE station. Each compound plate was laid-out in 12 columns to enable ten, 8-point concentration-effect curves to be constructed; the remaining two columns on the plate were taken up with vehicle (final concentration 0.33% DMSO), to define the assay baseline, and a supra-maximal blocking concentration of cisapride (final concentration 10 μM) to define the 100% inhibition level. The fluidics-head (F-Head) of IONWORKS HT then added 3.5 μL of PBS to each well of the PATCHPLATE and its underside was perfused with internal solution that had the following composition (in mM): K-Gluconate (100 parts), KCl (40 parts), MgCl2 (3.2 parts), EGTA (3 parts) and HEPES (5 parts, pH 7.25-7.30 using 10M KOH) After priming and de-bubbling, the electronics-head (E-head) then moved around the PATCHPLATE performing a hole test (i.e. applying a voltage pulse to determine whether the hole in each well is open). The F-head then dispensed 3.5 μL of the cell suspension described above into each well of the PATCHPLATE and the cells were given 200 seconds to reach and seal to the hole in each well. Following this, the E-head moved around the PATCHPLATE to determine the seal resistance obtained in each well.