| Assay Method Information | |
| | Pharmacological Assay |
| Description: | 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), then returned to the holding voltage of −100 mV. This voltage protocol was repeated at a frequency of every 15 seconds, the current magnitude was quantified at the P2 step throughout the recording. Inhibition of the measured current amplitude with compounds of the invention was analyzed by fitting a 4-6 points dose-response curve allowing determination of the fifty percent inhibition concentration (IC50). P2 currents were normalized according to measurements made at baseline (Baseline, vehicle only), after compound (Input, at each test concentration), and after positive reference compound (FullResponse, to achieve complete block), fit to the following equation:n.ICPD=NormalizedCurrent=(Input-Baseline)(FullResponse-Baseline)[1570]To assess current run-down over the course of the experiment vehicle-only wells were utilized and the normalized current with vehicle-only (n.IVEH) was determined. To correct the compound response for run-down, the currents were corrected according to the following formula:n.IRD_Correct=(n.ICPD-n.IVEH)(1-n.IVEH) |
| Affinity data for this assay | |
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