Abstract
Biological, genetic, and clinical evidence provide validation for N-type calcium channels (CaV2.2) as therapeutic targets for chronic pain. A state-dependent CaV2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl CaV2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent CaV2 inhibitor (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1) has an improved therapeutic window compared with ziconotide. Here we characterize TROX-1 inhibition of Cav2.2 channels in more detail. When channels are biased toward open/inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits CaV2.2 channels with an IC50 of 0.11 μM. The voltage dependence of CaV2.2 inhibition was examined using automated electrophysiology. TROX-1 IC50 values were 4.2, 0.90, and 0.36 μM at −110, −90, and −70 mV, respectively. TROX-1 displayed use-dependent inhibition of CaV2.2 with a 10-fold IC50 separation between first (27 μM) and last (2.7 μM) pulses in a train. In a fluorescence-based calcium influx assay, TROX-1 inhibited CaV2.2 channels with an IC50 of 9.5 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the CaV2 subfamily. Depolarized IC50 values were 0.29, 0.19, and 0.28 μM by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 μM by calcium influx for CaV2.1, CaV2.2, and CaV2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non–subtype-selective CaV2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent CaV2.2-selective inhibitor ziconotide in preclinical models of chronic pain.
Footnotes
A.M.S., J.H., R.M.B., G.D., R.J.H., K.S.R., M.M.S., V.A.W., S.B.H., C.L., J.L.D., G.J.K., and O.B.M. are present or former employees of Merck Research Laboratories and may hold stock or stock options in Merck and Co. S.P.A., C.E., D.P., and T.P.S. are present or former employees in Zalicus Pharmaceuticals and may hold stock or stock options in Zalicus Pharmaceuticals.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
-
ABBREVIATIONS:
- Cav
- voltage-gated calcium channel
- DMSO
- dimethyl sulfoxide
- HEK
- human embryonic kidney
- FLIPR
- fluorometric imaging plate reader
- TROX-1
- (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one.
- Received August 10, 2011.
- Accepted December 21, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|