Characterization of the K⁺ channel opening effect of the anticonvulsant retigabine in PC12 cells

Abstract

Retigabine (D-23129) is a new anticonvulsant compound which acts as a K⁺ channel opener in neuronal cells. The aim of the present study was to further characterize the retigabine induced K⁺ current. In nerve growth factor treated PC12 cells and in rat cortical neurones the application of retigabine activated a K⁺ current. In contrast, however, no K⁺ current activation was observed in untreated PC12 and in glial cells which were cultivated together with the neuronal cells. To characterise the retigabine activated K⁺ current, K⁺ channel blockers were used. The retigabine induced current was not affected by 1 and 10 mM 4-aminopyridine (4AP). Ba²⁺ 1 mM resulted in a reduction of 88.6±3.0% (n=5); 10 mM abolished the current. Tetraetylamonium (TEA), 1 and 10 mM, reduced the current by 23.6±3.1 and 61.6±3.7%, respectively. To investigate the current/voltage (I/V) relation of the current initiated by retigabine (10 μM), cells were clamped to a holding potential of −80 mV and a ramp stimulation protocol (−120 to +60 mV in 5 s) was applied prior to and during application of retigabine. Subtraction of the two traces yielded the current induced by retigabine. A nearly linear relationship was determined between −120 and −40 mV. At potentials positive to −40 mV, the response was variable. This was due to the additionally observed weak blocking effect of retigabine on delayed rectifier (K_dr) currents. If the ramp was applied in the presence of 10 mM 4AP to block K_dr, a nearly linear I/V-relationship was present from −120 to +60 mV. The comparison of the I/V relation and pharmacology with published K⁺ channel subtypes gives evidence that an unknown neuronal K⁺ channel subtype may be involved.

Introduction

Epilepsy is a chronic disease which is characterized by a condition of recurrent paroxysmal seizures of cerebral origin. The prevalence of epilepsy is found to be 0.5–1% of the world’s population (over 50 million people) which makes it one of the most common neurological disorders (Porter, 1993).

A large percentage (20–30%) of patients diagnosed as having epilepsy are not adequately controlled by presently available medications (Dam, 1986, Loiseau, 1988, Schmidt et al., 1992). In many cases, seizure control is limited by occurrence of severe side effects directly related to the antiepileptic drug treatment. There is therefore a need to develop novel, more effective and less toxic pharmacological agents rather than variations based on currently available but inadequate therapeutic regimes to increase control of seizures in currently drug resistant patients and to reduce side effects in patients which are only seizure free at the expense of chronic side effects.

Retigabine (D-23129) is a new anticonvulsant currently undergoing phase 2 clinical trials. The drug is chemically unrelated to currently marketed anticonvulsants. It exhibited pronounced anticonvulsant activity in a broad range of in vivo and in vitro seizure models and in genetic models of epileptic syndromes (Dailey et al., 1995, Kapetanovic and Rundfeldt, 1996, Rostock et al., 1996). In a model discussed to be the most predictive for complex partial seizures, retigabine was particularly effective in increasing the threshold (Tober et al., 1996). While in previous studies direct and indirect facilitating effects on GABAergic neurotransmission and weak blocking effects on neuronal sodium channels were reported (Kapetanovic et al., 1995, Rundfeldt et al., 1995, Kapetanovic and Rundfeldt, 1996), we recently found that retigabine acts as a potent opener of K⁺ channels in neuronal cells (Rundfeldt, 1997). Several lines of evidence can be found that the opening of K⁺ channels may be promising strategy for new anticonvulsant drug therapy (Doupnik et al., 1995, Sah, 1996, Meldrum, 1997). Opening K⁺ channels tends to hyperpolarize cells and decreases the effect of excitatory input. In both animal models of epilepsy and epileptic patients, mutations of K⁺ channels were reported as possible cause (Patil et al., 1995, Beck et al., 1996, Janigro et al., 1997, Charlier et al., 1998); an in vivo virus mediated transfection of CNS neurones with a voltage gated K⁺ channel resulted in a reduction in seizure susceptibility (Kirkby et al., 1996) and K⁺ channel openers of ATP sensitive K⁺ channels (given intracerebroventricularly) provide anticonvulsant action in some models (Gandolfo et al., 1989, Popoli et al., 1991). However, currently known K⁺ channel openers are hampered by their pronounced effects on blood pressure and other peripheral organ systems (Weston and Edwards, 1992).

The present work was aimed at characterizing the K⁺ channel opening effect of retigabine. Part of the work was already published in abstract form (Rundfeldt and Dost, 1997).