Activation of axonal Kv7 channels in human peripheral nerve by flupirtine but not placebo - therapeutic potential for peripheral neuropathies: results of a randomised controlled trial.

BACKGROUND: Flupirtine is an analgesic with muscle-relaxing properties that activates Kv7 potassium channels. Kv7 channels are expressed along myelinated and unmyelinated peripheral axons where their activation is expected to reduce axonal excitability and potentially contribute to flupirtine's clinical profile. TRIAL DESIGN: To investigate the electrical excitability of peripheral myelinated axons following orally administered flupirtine, in-vitro experiments on isolated peripheral nerve segments were combined with a randomised, double-blind, placebo-controlled, phase I clinical trial (RCT). METHODS: Threshold tracking was used to assess the electrical excitability of myelinated axons in isolated segments of human sural nerve in vitro and motoneurones to abductor pollicis brevis (APB) in situ in healthy subjects. In addition, the effect of flupirtine on ectopic action potential generation in myelinated axons was examined using ischemia of the lower arm. RESULTS: Flupirtine (3-30 µM) shortened the relative refractory period and increased post-conditioned superexcitability in human myelinated axons in vitro. Similarly, in healthy subjects the relative refractory period of motoneurones to APB was reduced 2 hours after oral flupirtine but not following placebo. Whether this effect was due to a direct action of flupirtine on peripheral axons or temperature could not be resolved. Flupirtine (200 mg p.o.) also reduced ectopic axonal activity induced by 10 minutes of lower arm ischemia. In particular, high frequency (ca. 200 Hz) components of EMG were reduced in the post-ischemic period. Finally, visual analogue scale ratings of sensations perceived during the post-ischemic period were reduced following flupirtine (200 mg p.o.). CONCLUSIONS: Clinical doses of flupirtine reduce the excitability of peripheral myelinated axons. TRIAL REGISTRATION: ClinicalTrials registration is NCT01450865.

Sea-anemone toxin ATX-II elicits A-fiber-dependent pain and enhances resurgent and persistent sodium currents in large sensory neurons.

BACKGROUND: Gain-of-function mutations of the nociceptive voltage-gated sodium channel Nav1.7 lead to inherited pain syndromes, such as paroxysmal extreme pain disorder (PEPD). One characteristic of these mutations is slowed fast-inactivation kinetics, which may give rise to resurgent sodium currents. It is long known that toxins from Anemonia sulcata, such as ATX-II, slow fast inactivation and skin contact for example during diving leads to various symptoms such as pain and itch. Here, we investigated if ATX-II induces resurgent currents in sensory neurons of the dorsal root ganglion (DRGs) and how this may translate into human sensations. RESULTS: In large A-fiber related DRGs ATX-II (5 nM) enhances persistent and resurgent sodium currents, but failed to do so in small C-fiber linked DRGs when investigated using the whole-cell patch-clamp technique. Resurgent currents are thought to depend on the presence of the sodium channel ß4-subunit. Using RT-qPCR experiments, we show that small DRGs express significantly less ß4 mRNA than large sensory neurons. With the ß4-C-terminus peptide in the pipette solution, it was possible to evoke resurgent currents in small DRGs and in Nav1.7 or Nav1.6 expressing HEK293/N1E115 cells, which were enhanced by the presence of extracellular ATX-II. When injected into the skin of healthy volunteers, ATX-II induces painful and itch-like sensations which were abolished by mechanical nerve block. Increase in superficial blood flow of the skin, measured by Laser doppler imaging is limited to the injection site, so no axon reflex erythema as a correlate for C-fiber activation was detected. CONCLUSION: ATX-II enhances persistent and resurgent sodium currents in large diameter DRGs, whereas small DRGs depend on the addition of ß4-peptide to the pipette recording solution for ATX-II to affect resurgent currents. Mechanical A-fiber blockade abolishes all ATX-II effects in human skin (e.g. painful and itch-like paraesthesias), suggesting that it mediates its effects mainly via activation of A-fibers.

Anticancer drug oxaliplatin induces acute cooling-aggravated neuropathy via sodium channel subtype Na(V)1.6-resurgent and persistent current.

Infusion of the chemotherapeutic agent oxaliplatin leads to an acute and a chronic form of peripheral neuropathy. Acute oxaliplatin neuropathy is characterized by sensory paresthesias and muscle cramps that are notably exacerbated by cooling. Painful dysesthesias are rarely reported for acute oxaliplatin neuropathy, whereas a common symptom of chronic oxaliplatin neuropathy is pain. Here we examine the role of the sodium channel isoform Na(V)1.6 in mediating the symptoms of acute oxaliplatin neuropathy. Compound and single-action potential recordings from human and mouse peripheral axons showed that cooling in the presence of oxaliplatin (30-100 µM; 90 min) induced bursts of action potentials in myelinated A, but not unmyelinated C-fibers. Whole-cell patch-clamp recordings from dissociated dorsal root ganglion (DRG) neurons revealed enhanced tetrodotoxin-sensitive resurgent and persistent current amplitudes in large, but not small, diameter DRG neurons when cooled (22 °C) in the presence of oxaliplatin. In DRG neurons and peripheral myelinated axons from Scn8a(med/med) mice, which lack functional Na(V)1.6, no effect of oxaliplatin and cooling was observed. Oxaliplatin significantly slows the rate of fast inactivation at negative potentials in heterologously expressed mNa(V)1.6r in ND7 cells, an effect consistent with prolonged Na(V) open times and increased resurgent and persistent current in native DRG neurons. This finding suggests that Na(V)1.6 plays a central role in mediating acute cooling-exacerbated symptoms following oxaliplatin, and that enhanced resurgent and persistent sodium currents may provide a general mechanistic basis for cold-aggravated symptoms of neuropathy.

Sustained increase in the excitability of myelinated peripheral axons to depolarizing current is mediated by Nav1.6.

Changes in the excitability of peripheral myelinated axons in response to long-lasting subthreshold depolarizing or hyperpolarizing currents (threshold electrotonus) are used as a complementary electrophysiological parameter in the study of peripheral nerve diseases in people. However, the contribution made by various axonal ion channels to specific components of threshold electrotonus remains incompletely understood. In this study, we have recorded threshold electrotonus responses from isolated nerve segments of sural nerve from control and Scn8amed mice, which lack functional Nav1.6 voltage-gated sodium channel. In med mice, the increase in axonal excitability produced by application of subthreshold depolarizing currents for 100-200ms was not sustained. In contrast, there was no difference in threshold electrotonus responses to subthreshold hyperpolarizing current application between Scn8amed and control mice. These data reveal the specific functional role of an identified subtype of voltage-gated sodium channel (Nav1.6) in mediating the depolarizing threshold electrotonus response of peripheral myelinated nerve fibers.

Enhancement of axonal potassium conductance reduces nerve hyperexcitability in an in vitro model of oxaliplatin-induced acute neuropathy.

Oxaliplatin is used in the chemotherapeutic treatment of malignant tumours. A common side effect of oxaliplatin is an acute peripheral neuropathy characterized by axonal hyperexcitability, which can be painful and is aggravated by exposure to cold. Electrophysiological studies on isolated segments of peripheral rodent nerve have been able to replicate oxaliplatin's effect on axonal hyperexcitability in vitro. In the present study we have used this in vitro model to examine whether flupirtine, a clinically available analgesic, which activates slow axonal potassium (Kv7) channels, can suppress axonal hyperexcitability resulting from exposure of peripheral nerve to oxaliplatin. In the presence of oxaliplatin (30µM), the A-fibre compound action potential response of isolated rat nerve segments to a brief electrical stimulus (0.1ms) changed considerably with the emergence of after-activity that persisted for a period of tens of milliseconds after the electrical stimulus. Lowering the bath temperature by 4°C enhanced the magnitude and prolonged the time course of this axonal after-activity. Application of flupirtine (10µM) reduced both the magnitude and duration of oxaliplatin-induced axonal after-activity in myelinated axons. These findings were also confirmed in isolated human sural nerve segments. The data indicate that activation of slow potassium channels in the A-fibres of peripheral nerve may attenuate the acute neuropathy associated with oxaliplatin in humans.

The Kv7 potassium channel activator flupirtine affects clinical excitability parameters of myelinated axons in isolated rat sural nerve.

Flupirtine is an activator of Kv7 (KCNQ/M) potassium channels that has found clinical use as an analgesic with muscle relaxant properties. Kv7 potassium channels are expressed in axonal membranes and pharmacological activation of these channels may restore abnormal nerve excitability. We have examined the effect of flupirtine on the electrical excitability of myelinated axons in isolated segments of rat sural nerve. Axonal excitability was studied in vitro with the same parameters used by clinical neurophysiologists to assess peripheral nerve excitability in situ. Application of flupirtine in low micromolar concentrations resulted in an increase in threshold current, a reduction of refractoriness and an increase in post-spike superexcitability. These effects are consistent with an increase in Kv7 conductance and membrane hyperpolarization. Flupirtine also enhanced and prolonged the late, long-lasting period of axonal subexcitability that follows a short burst of action potentials. This effect was blocked by XE 991 (10 microM), an antagonist of Kv7 channels. In summary, flupirtine affects measures of excitability that are altered in the myelinated axons of patients with peripheral nerve disorders. This indicates that neuropathies with abnormal nerve excitability parameters corresponding to those affected by flupirtine may benefit from activation of axonal Kv7 potassium channels.

GABA increases electrical excitability in a subset of human unmyelinated peripheral axons.

BACKGROUND: A proportion of small diameter primary sensory neurones innervating human skin are chemosensitive. They respond in a receptor dependent manner to chemical mediators of inflammation as well as naturally occurring algogens, thermogens and pruritogens. The neurotransmitter GABA is interesting in this respect because in animal models of neuropathic pain GABA pre-synaptically regulates nociceptive input to the spinal cord. However, the effect of GABA on human peripheral unmyelinated axons has not been established. METHODOLOGY/PRINCIPAL FINDINGS: Electrical stimulation was used to assess the effect of GABA on the electrical excitability of unmyelinated axons in isolated fascicles of human sural nerve. GABA (0.1-100 microM) increased electrical excitability in a subset (ca. 40%) of C-fibres in human sural nerve fascicles suggesting that axonal GABA sensitivity is selectively restricted to a sub-population of human unmyelinated axons. The effects of GABA were mediated by GABA(A) receptors, being mimicked by bath application of the GABA(A) agonist muscimol (0.1-30 microM) while the GABA(B) agonist baclofen (10-30 microM) was without effect. Increases in excitability produced by GABA (10-30 microM) were blocked by the GABA(A) antagonists gabazine (10-20 microM), bicuculline (10-20 microM) and picrotoxin (10-20 microM). CONCLUSIONS/SIGNIFICANCE: Functional GABA(A) receptors are present on a subset of unmyelinated primary afferents in humans and their activation depolarizes these axons, an effect likely due to an elevated intra-axonal chloride concentration. GABA(A) receptor modulation may therefore regulate segmental and peripheral components of nociception.

Modulation of remifentanil-induced analgesia and postinfusion hyperalgesia by parecoxib in humans.

BACKGROUND: Numerous experimental and clinical studies suggest that brief opioid exposure can enhance pain sensitivity. It is suggested that spinal cyclooxygenase activity may contribute to the development and expression of opioid tolerance. The aim of the investigation was to determine analgesic and antihyperalgesic properties of the cyclooxygenase-2 inhibitor parecoxib on remifentanil-induced hypersensitivity in humans. METHODS: Fifteen healthy male volunteers were enrolled in this randomized, double-blind, placebo-controlled study in a crossover design. Transcutaneous electrical stimulation at high current densities was used to induce spontaneous acute pain (numeric rating scale 6 of 10) and stable areas of pinprick hyperalgesia. Pain intensities and areas of hyperalgesia were assessed before, during, and after a 30-min intravenous infusion of remifentanil (0.1 microg x kg x min) or placebo (saline). Parecoxib (40 mg) was administered intravenously either with onset of electrical stimulation (preventive) or in parallel to the remifentanil infusion. RESULTS: Remifentanil reduced pain and mechanical hyperalgesia during the infusion, but upon withdrawal, pain and hyperalgesia increased significantly above control level. Preventive administration of parecoxib led to an amplification of remifentanil-induced antinociceptive effects during the infusion (71.3 +/- 7 vs. 46.4 +/- 17% of control) and significantly diminished the hyperalgesic response after withdrawal. In contrast, parallel administration of parecoxib did not show any modulatory effects on remifentanil-induced hyperalgesia. CONCLUSION: The results confirm clinically relevant interaction of mu opioids and prostaglandins in humans. Adequate timing seems to be of particular importance for the antihyperalgesic effect of cyclooxygenase-2 inhibitors.