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- HEK293 (1)
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- Kv 7.2/3 channel activators (1)
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- flupirtine (1)
- graphite furnace atomic absorption spectrometry (1)
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- retigabine (1)
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Flupirtine and retigabine were essential drugs to combat pain and epilepsy. However, the Kv7 potassium channel openers are fraught with hepatotoxicity and tissue discoloration, respectively, limiting their therapeutic value. Both adverse events are likely due to reactive metabolites arising from oxidative metabolism. Designing safer analogues lacking the structural elements leading to described side effects is an active area of current research. One of the main metabolites of flupirtine is the biologically inactive 4-fluorohippuric acid. Hitherto unexplained, the proposed metabolic pathway leading to the formation of 4-fluorohippuric acid from flupirtine is verified here. Through the use of eighteen flupirtine analogues, mechanistic details of this pathway could be elucidated. A possible connection with the in vitro hepatotoxicity of the flupirtine analogues and the levels of 4-fluorobenzoic acid formed in enzyme incubations was examined by correlation analysis. These findings provide important information for the design of new flupirtine analogues as potential drug candidates.
For the characterization of Kv7.2/3 channel activators, several analytical methods are available that vary in effort and cost. In addition to the technically elaborate patch-clamp method, which serves as a reference method, there exist several medium to high-throughput screening methods including a rubidium efflux flame-atomic absorption spectrometry (F-AAS) assay and a commercial thallium uptake fluorescence-based assay. In this study, the general suitability of a graphite furnace atomic absorption spectrometry (GF-AAS)-based rubidium efflux assay as a screening method for Kv7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known Kv7.2/3 activator retigabine were first classified as either active or inactive by using the GF-AAS-based rubidium (Rb) efflux assay. Then, the results were compared with a thallium (Tl) uptake fluorescence-based fluorometric imaging plate reader (FLIPR) potassium assay. Overall, 16 of 17 compounds were classified by the GF-AAS-based assay in agreement with their channel-activating properties determined by the more expensive Tl uptake, fluorescence-based assay. Thus, the performance of the GF-AAS-based Rb assay for primary drug screening of Kv7.2/3-activating compounds was clearly demonstrated, as documented by the calculated Z’-factor of the GF-AAS-based method. Moreover, method development included optimization of the coating of the microtiter plates and the washing procedure, which extended the range of this assay to poorly adherent cells such as the HEK293 cells used in this study.