In Vitro Mechanism of Action Test

Brief Description

Assessment of the in vitro effect of an investigational compound on voltage-gated sodium channels and receptor-gated ion channels.

Introduction

The mechanisms of action of currently marketed ASDs are not fully understood. Although numerous molecular targets exist wherein ASDs may exert an effect, most established ASDs appear to have a mechanism of action through modulation of voltage-gated and/or neurotransmitter-gated ion channels by various mechanisms [1-7]. One, they can reduce sustained, high-frequency repetitive firing of action potentials by modulating voltage-dependent sodium (Na+) channels. Second, they can enhance GABA-mediated inhibitory neurotransmission via a receptor-gated chloride channel [5]. Finally, they can modulate neurotransmitter release and neuronal bursting through an effect on voltage-gated and receptor-gated calcium (Ca2+) channels [1, 3]. In light of these common pathways, the ETSP has used electrophysiological techniques to assess the effect of promising investigational compounds on voltage-gated sodium channels and receptor-gated ion channels (NMDA, kainate, and GABA).

Methods

In an effort to gain some insight into the potential mechanism of action of the candidate substance, its potential for interaction with voltage- and receptor-gated ion channels is examined using whole-cell patch-clamp electrophysiology techniques with cell cultures. Briefly, the whole-cell patch clamp recording technique is used to examine the action of the test compound on neurotransmitter- and voltage-gated ion channels. Cortical cells are cultured from 15-gestational-day-old Swiss Webster mouse fetuses and are used 2-3 weeks after plating [8, 9]. Recordings are carried out at room temperature (23° C) according to previously described techniques [10]. Agonist-evoked current responses are measured in the absence and presence of the test compound. Whole-cell recordings from a murine neuroblastoma (N1E 115) cell line are used to characterize the effects of the test compound on voltage-gated sodium channels. For these studies, the test substance is dissolved in DMSO (50 mM stock) and diluted to make a final concentration of 100 µM. Final DMSO concentration is <1%. Cells are voltage clamped at -70 mV unless otherwise noted. A gravity-fed, three-barreled glass flow pipet is positioned 200-400 µm from the cell through which agonist, agonist plus test compound, and wash solution will flow continuously. GABAA receptor currents are evoked using 5 µM GABA, whereas ionotropic glutamate currents are evoked using 100 µM kainate and 10 µM NMDA plus 1 µM glycine. Agonists are applied for 1-5 sec and each application is separated by a 20-30 sec wash period. The agonist-evoked current values are measured using pClamp 10 software (Axon Instruments). The effect of an investigational compound on agonist-evoked currents is determined by comparing the agonist currents in the presence of drug with control agonist responses in the absence of drug. The effect of 100 µM investigational compound on voltage-gated sodium channels is assessed using N1E-115 murine neuroblastoma cells and whole-cell voltage-clamp recording techniques at -90 mV and -60 mV holding potentials, as previously described [11].

Results

For the electrophysiology studies, recording results from 5-7 individual cells in each group are averaged, S.E.M. calculated and statistical significance determined by the pairwise Student's t-test [12]. Differences are considered significant at p values <0.05. The effect of the investigational compound on GABA-mediated currents as well as glutamate receptor-mediated currents, including kainic acid- and NMDA-mediated currents are determined. The effect of the investigational compound on voltage-gated sodium currents at depolarized potentials is also examined.

Discussion

Of the many molecular mechanisms through which ASDs exert their effect, the majority of currently available drugs appear to target either voltage-gated or receptor-gated ion channels. The results from the limited electrophysiology studies conducted with investigational compounds are examined to determine whether there are notable effects on these voltage-gated and receptor-gated ion channels. Thus, the results obtained from limited in vitro studies may provide useful information to direct further investigation into the cellular mechanisms of the compound. Furthermore, such information may be useful in differentiating an investigational compound from ASDs currently available.