Discovery of the First Orally Available, Selective KNa1.1 Inhibitor: In Vitro and In Vivo Activity of an Oxadiazole Series.

The gene KCNT1 encodes the sodium-activated potassium channel KNa1.1 (Slack, Slo2.2). Variants in the KCNT1 gene induce a gain-of-function (GoF) phenotype in ionic currents and cause a spectrum of intractable neurological disorders in infants and children, including epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Effective treatment options for KCNT1-related disease are absent, and novel therapies are urgently required. We describe the development of a novel class of oxadiazole KNa1.1 inhibitors, leading to the discovery of compound 31 that reduced seizures and interictal spikes in a mouse model of KCNT1 GoF.

Discovery of Clinical Candidate 4-[2-(5-Amino-1H-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfonamide (PF-05089771): Design and Optimization of Diaryl Ether Aryl Sulfonamides as Selective Inhibitors of NaV1.7.

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective NaV1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective NaV1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.

The Selective Nav1.7 Inhibitor, PF-05089771, Interacts Equivalently with Fast and Slow Inactivated Nav1.7 Channels.

Voltage-gated sodium (Nav) channel inhibitors are used clinically as analgesics and local anesthetics. However, the absence of Nav channel isoform selectivity of current treatment options can result in adverse cardiac and central nervous system side effects, limiting their therapeutic utility. Human hereditary gain- or loss-of-pain disorders have demonstrated an essential role of Nav1.7 sodium channels in the sensation of pain, thus making this channel an attractive target for new pain therapies. We previously identified a novel, state-dependent human Nav1.7 selective inhibitor (PF-05089771, IC50 = 11 nM) that interacts with the voltage-sensor domain (VSD) of domain IV. We further characterized the state-dependent interaction of PF-05089771 by systematically varying the voltage, frequency, and duration of conditioning prepulses to provide access to closed, open, and fast- or slow-inactivated states. The current study demonstrates that PF-05089771 exhibits a slow onset of block that is depolarization and concentration dependent, with a similarly slow recovery from block. Furthermore, the onset of block by PF-05089771 develops with similar rates using protocols that bias channels into predominantly fast- or slow-inactivated states, suggesting that channel inhibition is less dependent on the availability of a particular inactivated state than the relative time that the channel is depolarized. Taken together, the inhibitory profile of PF-05089771 suggests that a conformational change in the domain IV VSD after depolarization is necessary and sufficient to reveal a high-affinity binding site with which PF-05089771 interacts, stabilizing the channel in a nonconducting conformation from which recovery is slow.

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release.

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.

A novel selective and orally bioavailable Nav 1.8 channel blocker, PF-01247324, attenuates nociception and sensory neuron excitability.

BACKGROUND AND PURPOSE: NaV 1.8 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 Nav 1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACH: The inhibition of Nav 1.8 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 RESULTS: PF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50 : 331 nM) and in recombinantly expressed h Nav 1.8 channels (IC50 : 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav 1.5 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 Nav 1.8 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 IMPLICATIONS: Using PF-01247324, we have confirmed a role for Nav 1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav 1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.

Recent progress in sodium channel modulators for pain.

Voltage-gated sodium channels (Navs) are an important family of transmembrane ion channel proteins and Nav drug discovery is an exciting field. Pharmaceutical investment in Navs for pain therapeutics has expanded exponentially due to genetic data such as SCN10A mutations and an improved ability to establish an effective screen sequence for example IonWorks Barracuda®, Synchropatch® and Qube®. Moreover, emerging clinical data (AZD-3161, XEN402, CNV1014802, PF-05089771, PF-04531083) combined with recent breakthroughs in Nav structural biology pave the way for a future of fruitful prospective Nav drug discovery.

Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels.

Voltage-gated sodium (Nav) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Nav channel inhibitors that exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) or Nav1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1-S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the "extracellular" facing regions of the S2 and S3 transmembrane segments of Nav1.3 and Nav1.7 seem to be major determinants of Nav subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Nav1.3)/D1586 (Nav1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Nav channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.

Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Na(v)1.8 sodium channel with efficacy in a model of neuropathic pain.

Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.

Subtype-selective Na(v)1.8 sodium channel blockers: identification of potent, orally active nicotinamide derivatives.

A series of aryl-substituted nicotinamide derivatives with selective inhibitory activity against the Na(v)1.8 sodium channel is reported. Replacement of the furan nucleus and homologation of the anilide linker in subtype-selective blocker A-803467 (1) provided potent, selective derivatives with improved aqueous solubility and oral bioavailability. Representative compounds from this series displayed efficacy in rat models of inflammatory and neuropathic pain.

A-887826 is a structurally novel, potent and voltage-dependent Na(v)1.8 sodium channel blocker that attenuates neuropathic tactile allodynia in rats.

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na(v)1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na(v)1.8 channel blockers. A-887826 exemplifies this series and potently (IC(50)=11nM) blocked recombinant human Na(v)1.8 channels. A-887826 was approximately 3 fold less potent to block Na(v)1.2, approximately 10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na(+)) currents and was >30 fold less potent to block Na(V)1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na(+)) currents (IC(50)=8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund's adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by approximately 4mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na(v)1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na(v)1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na(v)1.8 sodium channels in pathological pain states.

The effect of kappa-opioid receptor agonists on tetrodotoxin-resistant sodium channels in primary sensory neurons.

BACKGROUND: A non-opioid receptor-mediated inhibition of sodium channels in dorsal root ganglia (DRGs) by kappa-opioid receptor agonists (kappa-ORAs) has been reported to contribute to the antinociceptive actions in animals and humans. In this study, we examined structurally diverse kappa-ORAs for their abilities to inhibit tetrodotoxin-resistant (TTX-r) sodium channels in adult rat DRGs. METHODS: Whole-cell recordings of TTX-r sodium currents were performed on cultured adult rat DRGs. Structurally diverse kappa-ORAs were studied for their abilities to inhibit TTX-r sodium channels. RESULTS: The racemic kappa-ORA, (+/-)U50,488, inhibited TTX-r sodium currents in a voltage-dependent manner, yielding IC(50) values of 49 and 8 muM, at prepulse potentials of -100 and -40 mV, respectively. Furthermore, we found that both the kappa-ORA U50,488 active enantiomer 1S,2S U50,488 and the inactive enantiomer 1R,2R U50,488 were equally potent inhibitors of TTX-r sodium currents. Structurally related kappa-ORAs, such as BRL 52537 and ICI 199,441 also inhibited TTX-r sodium currents. However, sodium channel inhibition and kappa-opioid receptor agonism have a distinct structure-activity relationship because another kappa-ORA (ICI 204,488) was inactive versus TTX-r sodium channels. We further investigated the sodium channel block of this class of compounds by studying (+/-)U50,488. (+/-)U50,488 was found to preferentially interact with the slow inactivated state of TTX-r sodium channels and to retard recovery from inactivation. CONCLUSION: Our results suggest that TTX-r sodium channels can be inhibited by many kappa-ORAs via an opioid receptor-independent mechanism. Although the potency for sodium channel inhibition is typically much less than apparent affinity for opioid receptors, sodium channel block may still contribute to the antinociceptive effects of this class of compounds.

Discovery of potent furan piperazine sodium channel blockers for treatment of neuropathic pain.

The synthesis and pharmacological characterization of a novel furan-based class of voltage-gated sodium channel blockers is reported. Compounds were evaluated for their ability to block the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3) as well as the Na(v)1.2 and Na(v)1.5 subtypes. Benchmark compounds from this series possessed enhanced potency, oral bioavailability, and robust efficacy in a rodent model of neuropathic pain, together with improved CNS and cardiovascular safety profiles compared to the clinically used sodium channel blockers mexiletine and lamotrigine.

Discovery and biological evaluation of 5-aryl-2-furfuramides, potent and selective blockers of the Nav1.8 sodium channel with efficacy in models of neuropathic and inflammatory pain.

Nav1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons and has been implicated in the pathophysiology of inflammatory and neuropathic pain. Recent studies using an Nav1.8 antisense oligonucleotide in an animal model of chronic pain indicated that selective blockade of Nav1.8 was analgesic and could provide effective analgesia with a reduction in the adverse events associated with nonselective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 5-substituted 2-furfuramides, which are potent, voltage-dependent blockers (IC50 < 10 nM) of the human Nav1.8 channel. Selected derivatives, such as 7 and 27, also blocked TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons with comparable potency and displayed >100-fold selectivity versus human sodium (Nav1.2, Nav1.5, Nav1.7) and human ether-a-go-go (hERG) channels. Following systemic administration, compounds 7 and 27 dose-dependently reduced neuropathic and inflammatory pain in experimental rodent models.

The influence of crank configuration on muscle activity and torque production during arm crank ergometry.

This study investigated the effect of crank configuration on muscle activity and torque production during submaximal arm crank ergometry. Thirteen non-specifically trained male participants volunteered. During the research trials they completed a warm-up at 15W before two 3-min exercise stages were completed at 50 and 100W; subjects used either a synchronous or asynchronous pattern of cranking. During the final 30-s of each submaximal exercise stage electromyographic and torque production data were collected. After the data had been processed each parameter was analysed using separate 2-way ANOVA tests with repeated measures. The activity of all muscles increased in line with external workload, although a shift in the temporal pattern of muscle activity was noted between crank configurations. Patterns of torque production during asynchronous and synchronous cranking were distinct. Furthermore, peak, minimum and delta (peak-minimum) torque values were different (P<0.05) between crank configurations at both workloads. For example, at 100W, peak torque using synchronous [19.6 (4.3) Nm] cranking was higher (P<0.05) compared to asynchronous [16.8 (1.6) Nm] cranking. In contrast minimum torque was lower (P<0.05) at 100 W using synchronous [4.8 (1.7)Nm] compared to asynchronous [7.3 (1.2)Nm] cranking. There was a distinct bilateral asymmetry in torque production during asynchronous cranking with the dominant transmitting significantly more force to the crank arm. Taken together, these preliminary data demonstrate the complex nature of muscle activity during arm crank ergometry performed with an asynchronous or synchronous crank set-up. Further work is required to determine how muscle activity (EMG activity) and associated patterns of torque production influence physiological responses and functional capacity during arm crank ergometry.

Allosteric effects of external K+ ions mediated by the aspartate of the GYGD signature sequence in the Kv2.1 K+ channel.

K+ channels achieve exquisite ion selectivity without jeopardizing efficient permeation by employing multiple, interacting K+-binding sites. Introduction ofa cadmium (Cd2+)-binding site in the external vestibule of Kv2.1 (drk1), allowed us to functionally characterize a binding site for external monovalent cations. Permeant ions displayed higher affinity for this site than non-permeant monovalent cations, although the selectivity profile was different from that of the channel. Point mutations identified the highly conserved aspartate residue immediately following the selectivity filter as a critical determinant of the antagonism between external K+ and Cd2+ ions. A conservative mutation at this position (D378E) significantly affected the open-state stability. Moreover, the mean open time was found to be modulated by external K+ concentration, suggesting a coupling between channel closing and the permeation process. Reducing the Rb+ conductance by mutating the selectivity filter to the sequence found inKv4.1, also significantly reduced the effectiveness ofRb+ ions to antagonize Cd2+ inhibition, thereby implicating the selectivity filter as the site at which K+ions exert their antagonistic effect on Cd2+ block. The equivalent of D378 in KcsA, D80, takes part in an inter-subunit hydrogen-bond network that allows D80to functionally interact with the selectivity filter. The results suggest that external K+ ions antagonize Cd2+inhibition (in I379C) and modulate the mean open time(in the wild-type Kv2.1) by altering the occupancy profile of the K+-binding sites in the selectivity filter.

K channel subconductance levels result from heteromeric pore conformations.

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (K(v)2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the "open" state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open-close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.

The effect on the fetus of medications used to treat pregnant inflammatory bowel-disease patients.

OBJECTIVES: We reviewed data to investigate the effect of 5-ASA drugs, metronidazole, ciprofloxacin, prednisone, 6-mercaptopurine, azathioprine, and cyclosporine on pregnancy outcomes in patients with inflammatory bowel disease (IBD). METHODS: One hundred and thirteen female patients with a total of 207 documented conceptions were studied. Treatment information included: smoking history (patient and spouse), dates of conception and termination, and outcome of pregnancy (spontaneous abortion, therapeutic abortion, maternal or fetal illness resulting in abortion, premature birth, healthy full-term birth, multiple births, ectopic pregnancy, congenital defects), weight of baby, type of delivery (cesarian section, vaginal), medication history during each trimester (mean dose, maximum dose, frequency). We analyzed the effect on pregnancy outcome of medication use during the first trimester or at any time during the pregnancy. RESULTS: Thirty-nine patients (34.5%) had ulcerative colitis (UC), 73 (64.5%) had crohn's disease (CD), and 1 patient (1%) had indeterminate colitis. For 100 of the 207 conceptions, the patients were on 5-ASA drugs at some time during the pregnancy, 49 on prednisone, 101 on an immunomodulator (6-MP/azathioprine), 27 on metronidazole, 18 on ciprofloxacin, and 2 on cyclosporine. In 85 (31%) of the conceptions, patients were on none of these medications. No significant differences were found among the groups in each pregnancy with respect to outcome (p values 0.091 to 0.9). In multivariate analyses controlling for age of mother, there was no evidence that 5-ASA type drugs or any type of drug influenced pregnancy outcome. CONCLUSIONS: In 113 female patients with 207 conceptions none of the drugs used to treat IBD is associated with poor pregnancy outcomes.