Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Nav1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC50 ‫؍‬ 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Nav1.8 (IC50 ‫؍‬ 8 nM) and was >100-fold selective vs. human Nav1.2, Nav1.3, Nav1.5, and Nav1.

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are voltage-gated K ϩ channel subunits that underlie the neuronal M current. In humans, mutations in these genes lead to a rare form of neonatal epilepsy (Biervert et al., 1998;Singh et al., 1998), suggesting that KCNQ2/Q3 channels may be attractive targets for novel antiepileptic drugs. In the present study, we have identified the compound N- (6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243) as a selective activator of the neuronal M current and KCNQ2/Q3 channels. In SH-SY5Y human neuroblastoma cells, ICA-27243 produced membrane potential hyperpolarization that could be prevented by coadministration with the M-current inhibitors 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE-991) and linopirdine. ICA-27243 enhanced both 86 Rb ϩ efflux (EC 50 ϭ 0.2 M) and whole-cell currents in Chinese hamster ovary cells stably expressing heteromultimeric KCNQ2/Q3 channels (EC 50 ϭ 0.4 M). Activation of KCNQ2/Q3 channels was associated with a hyperpolarizing shift of the voltage dependence of channel activation (V 1/2 shift of Ϫ19 mV at 10 M). In contrast, ICA-27243 was less effective at activating KCNQ4 and KCNQ3/Q5 and was selective over a wide range of neurotransmitter receptors and ion channels such as voltagedependent sodium channels and GABA-gated chloride channels. ICA-27243 (1-10 M) was found to reversibly suppress seizure-like activity in an ex vivo hippocampal slice model of epilepsy and demonstrated in vivo anticonvulsant activity (ED 50 ϭ 8.4 mg/kg) in the mouse maximal electroshock epilepsy model. In conclusion, ICA-27243 represents the first member of a novel chemical class of selective KCNQ2/Q3 activators with anticonvulsant-like activity in experimental models of epilepsy.

BACKGROUND AND PURPOSENaV1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF-01247324, a new generation, selective, orally bioavailable Nav1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACHThe inhibition of Nav1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain. KEY RESULTSPF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50: 331 nM) and in recombinantly expressed h Nav1.8 channels (IC50: 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav1.5 channels (IC50: ∼10 μM) and 65-100-fold selectivity over TTX-sensitive (TTX-S) channels (IC50: ∼10-18 μM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC50 448 nM, and the block of both human recombinant Nav1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models. CONCLUSIONS AND IMPLICATIONSUsing PF-01247324, we have confirmed a role for Nav1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.

Openers or activators of neuronal KCNQ2/Q3 potassium channels decrease neuronal excitability and may provide benefit in the treatment of disorders of neuronal excitability such as epilepsy. In the present study, we evaluate the effects of ICA-27243 [N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide], an orally bioavailable, potent, and selective KCNQ2/Q3 opener, in a broad range of rodent seizure models. ICA-27243 was effective against maximal electroshock (MES) and pentylenetetrazole (PTZ)-induced seizures in both rats (MES, ED 50 ϭ 1.5 mg/kg p.o.; PTZ, ED 50 ϭ 2.2 mg/kg p.o.) and mice (MES, ED 50 ϭ 8.6 mg/kg p.o.; PTZ, ED 50 ϭ 3.9 mg/kg p.o.) in the rat amygdala kindling model of partial seizures (full protection from seizure at 9 mg/kg p.o.) and in the 6-Hz model of psychomotor seizures in mice (active at 10 mg/kg i.p.). Antiseizure efficacy in all models was observed at doses significantly less than those shown to effect open-field locomotor activity (rat ED 50 ϭ 40 mg/kg p.o.) or ability to remain on a Rotorod (no effect in rat at doses up to 100 mg/kg p.o.). There was no evidence of cognition impairment as measured in the Morris water maze in the rat (10 and 30 mg/kg p.o.), nor was there evidence of the development of tolerance after multiple doses of ICA-27243. Our findings suggest that selective KCNQ2/Q3 opening activity in the absence of effects on KCNQ3/Q5 or GABA-activated channels may be sufficient for broad-spectrum antiepileptic activity in rodents.KCNQ2-5 (Kv7.2-7.5) voltage-dependent potassium channels are potentially attractive targets for novel antiepileptic drugs. These channels are expressed at high levels in the brain, including regions linked to seizure disorders, such as cortex, hippocampus, and thalamus. They represent the molecular correlate of the neuronal M current

The design, optimization, and evaluation of a series of novel imidazo­pyridazine-based subtype-selective positive allosteric modulators (PAMs) for the GABAA ligand-gated ion channel are described. From a set of initial hits multiple subseries were designed and evaluated based on binding affinity and functional activity. As designing in the desired level of functional selectivity proved difficult, a probability-based assessment was performed to focus the project’s efforts on a single subseries that had the greatest odds of delivering the target profile. These efforts ultimately led to the identification of two precandidates from this subseries, which were advanced to preclinical safety studies and subsequently to the identification of the clinical candidate PF-06372865.