| Assay Method Information | |
| | Biological Assay |
| Description: | Human embryonic kidney 293 cells (HEK293) expressing human Nav1.8, human Navβ1 and human TREK1 (HEK293-Nav1.8) were grown at 37° C., 5% CO2 in 150 cm2 flasks. HEK293-Nav1.8 were passaged every 2-3 days into T175 cell culture flasks when confluency reached 80-90%. Pharmacological assessment of the compounds of the invention was performed using HEK293-Nav1.8 in combination with an assay developed on the QPatch 48 HTX electrophysiological system. HEK293-Nav1.8 were prepared on the day of use by removing culture media, washing in DPBS, adding Accutase (2 ml to cover the surface, aspirate 1 ml then 1.5 min at 37° C.) followed by addition of CHO-SFM II to stop the enzyme digestion and in order to obtain a suspension of 3×106 cell/mL. Compound was prepared in an extracellular solution of the following composition: NaCl (145 mM), KCl (4 mM), CaCl2 (2 mM), MgCl2 (2 mM), HEPES (1 mM), Glucose (10 mM), pH 7.4 with NaOH Osmolality 300 mOsM/L. The intracellular solution was used of the following composition: CsF (115 mM), CsCl (20 mM), NaCl (5 mM), EGTA (10 mM), HEPES (10 mM), Sucrose (20 mM), pH 7.2 with CsOH Osmolality 310 mOsm/L. Utilizing the voltage-clamp mode in the QPatch 48 HTX system a half inactivation state voltage protocol (V1/2) was used to determine pharmacological activity of compounds of the invention at Nav1.8 ion channels. A V1/2 protocol was utilized with the following voltage steps: a holding voltage of −100 mV was established followed by a 20 ms voltage step to 0 mV (P1), followed by an inactivating voltage step at −46 mV for 8 seconds, followed by a step to −100 mV for 20 ms, before a 20 ms step to 0 mV (P2) before returning to the holding voltage of −100 mV. This voltage protocol was repeated at a frequency of 0.07 Hz., current magnitude was quantified at the P2 step throughout the recording. Inhibition of the measured current amplitude with the compounds of the invention was analyzed by fitting a 6-8 point dose-response curve allowing determination of the fifty percent inhibition concentration (IC50). |
| Affinity data for this assay | |
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