To assess the utility of flupirtine for neonatal seizures, we have used two animal models of induced seizures that have been extensively employed in previous neonatal seizures studies. Either flurothyl or kainic acid robustly induced seizures in rats at P10, an age thought comparable in developmental maturity to human neonates. When give prior to seizure induction by kainic acid, flupirtine was significantly more effective than either diazepam or phenobarbital in preventing progression to status epilepticus (). Flupirtine was also significantly more effective than either approved drug in preventing seizure induction by flurothyl (). Even when administerered after kainic acid seizures that were allowed to progress to a state of continuous status epilepticus, flupirtine was very effective, by behavioral measures, qualitative EEG, and several tests of EEG power (-). In this post-treatment paradigm, suppression of total EEG power by flupirtine and high dose phenobarbital (50 mg/kg, yielding a serum level of approximately 40 μg/ml) did not differ significantly, but the effect of phenobarbital treatment was significantly delayed in onset and was associated with persistent continuous high frequency EEG activity and brief behavioral seizures that were not seen after flupirtine.
The current study confirms results of an earlier study by Dzhala et al. that showed that phenobarbital pretreatment was relatively ineffective for prevention of kainic acid induced seizures in neonatal rodents,38
and extends the findings to a second agent, diazepam. Dzhala used analysis of EEG power to compare the ability of phenobarbital and the transporter blocking agent, bumetamide, to suppress kainate induced seizures in neonatal rats. Although our approach was similar in many respects, there are some salient differences. In the current study, phenobarbital was used at a higher dose (50 mg/kg versus 25 mg/kg), and trial drugs were given after seizures had progressed to SE (rather than prior to kainate injection). We used a test of significance that imposed several statistical power-weakening corrections (see Methods), where the previous study used a less stringent, ANOVA-based approach. Indeed, pair-wise comparison of flupirtine and phenobarbital treatment using ANOVA and data from yields strong numerical support for superior power reduction by flupirtine (p = 0.0033). Because of these methodological differences, and the limitations of power measurement as a marker of more important clinical efficacy endpoints, drawing conclusions about the relative merits of bumetamide and flupirtine must await additional studies. At present, it is clear that flupirtine is strongly anticonvulsant in P10 rats.
Although our kainate induction protocol was designed to limit mortality to ~10%, it is noteworthy that 8 of 55 control animals (14.5%), 11 of 35 diazepam-treated animals (31.4%), 2 of 48 phenobarbital treated animals (4.2%), and 0 of 56 flupirtine-treated animals (0%) died within the experimental observation period. Factors underlying these observed mortality rate differences may include, for example, respiratory and hemodynamic effects we have not yet characterized. These may be clinically significant and deserve further study.
Flupirtine has not been approved for use in the United States. However, retigabine is a close structural analogue () that is currently undergoing phase 3 clinical trial as adjunctive therapy for adult partial epilepsy, after a large stage 2 trial showed tolerability and dose-dependent suppression of seizure frequency.21
Our findings do not imply that KCNQ openers such as retigabine and flupirtine are more likely to be effective in neonates than in older patients. KCNQ channels continue to be expressed throughout childhood and adulthood 40-42
. Although neonatal brain is unique in its high degree of seizure susceptibility after blockade
of KCNQ channels, 13-16, 43
this finding does not imply progressive diminishment in the experimental or clinical utility of openers
with age. Evidence has been presented that flupirtine and retigabine are capable of antagonizing glutamate induced toxicity and enhancing inhibitory synaptic currents,27, 44
in addition to their well-established direct effects as KCNQ openers. Additional studies are required to better specify the mechanisms relevant to the anti-epileptic effects of these drugs. As noted above, in vivo experiments have raised concerns about potential pro-apoptotic effects of anticonvulsants currently utilized in neonates.6-8
Although in vitro results suggesting anti-apoptotic and anti-oxidative effects of flupirtine and retigabine are encouraging26, 27
, it will be important in future studies to directly examine the effects of flupirtine and other KCNQ openers on apoptosis and other aspects of developmental plasticity in the neonatal brain.
The current translational investigation was made possible by earlier genetic and basic research9
. The benign familial neonatal seizures syndrome was the first idiopathic epilepsy for which genetic loci were identified.45-47
Cloning of KCNQ2
, two potassium channel genes, at these loci has allowed these novel channels to be studied both using in vitro
and in vivo methods.48
Such studies have shown that the channels mutated in BNFS are widely expressed in the central nervous system, are critical regulators of excitability not only in infancy but throughout life, and can be pharmacologically activated by many drugs.41, 49-51
It is somewhat paradoxical that KCNQ2 and KCNQ3 mutations are rare and typically cause a phenotype restricted to the first weeks of life, but KCNQ channels exhibit widespread, lifelong expression and pivotal importance for brain function. This offers an important general lesson for translational neuroscientists: clinical disease can offer clues allowing the discovery of essential brain proteins and pathways, without fully revealing the range of functional activity of these components. This is the case with BFNS and the underlying KCNQ channels.
Neuronal KCNQ channels remain quite poorly understood, and many questions regarding their subunit composition, biological roles, and pharmacology are unanswered. Continued development of KCNQ channel openers with distinctive subunit specificity and potency profiles will be critical for achieving better understanding of channel functions. Flupirtine is a low potency KCNQ channel opener22
, and is representative of a class of drugs that exert their greatest effects on the KCNQ3 subunit.52
Currently disclosed KCNQ channel openers include compounds of greater overall potency (e.g., retigabine, various ethyl acrylamides, ICA-27243), and agents that differ from flupirtine in their kinetic mechanisms and selectivity among the neuronal KCNQ subunits (e.g., zinc pyrithione).50
Further in vitro and in vivo testing of novel openers will help clarify differences in biological functions between the KCNQ subunits. In turn, this may allow drug action upon these channels to be rationally tailored to maximize therapeutic benefits while reducing unwanted side effects, for various indications in pediatric and adult patients.
Our studies join recent efforts to translate understanding of the distinctive cellular and molecular features of developing brain into mechanistically novel neonatal seizure therapies. Thus, developmental differences in GABA(A) receptor subunit expression and the transmembrane Cl-
gradient have been found to make synaptic inhibition relatively less effective in the immature compared to the adult brain.38, 53
As noted above, the use of bumetamide, which promotes a more negative Cl-
equilibrium potential and thereby enhances the inhibitory effects of GABAergic neurotransmission, has been investigated to circumvent this.38, 54
In contrast to the functional diminishment of GABAergic inhibition at birth, some excitatory glutamate receptors are overexpressed during early development, making blockade of these receptors another rational treatment approach. The AMPA receptor antagonist NBQX and kainate receptor antagonist topiramate have shown efficacy in the treatment of experimental neonatal seizures1, 36, 39
, and do not cause enhanced apoptosis.55
Our results further validate the notion that neonatal seizure therapy may be improved through targeting molecules and mechanisms confirmed by laboratory or genetic studies to be of particular importance during early brain development.