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

Abstract

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.

Figures

Figure 1

Immunohistochemistry of Kv7 channels in human sural nerve fascicles in vitro. The presence of Kv7.2 subunits at peripheral nodes of Ranvier as well as in unmyelinated fibres of human sural nerve was verified immunohistochemically. A node of Ranvier evident under bright field illumination (i) shows immunoreactivity for Kv7.2 (ii and merge iii). In addition, unmyelinated fibres labelled with peripherin (iv) also show immunoreactivity for Kv7.2 (ii and merge v). In a separate preparation, Kv7.2 immunoreactivity (vii) and immunoreactivity for the non-specific voltage-gated sodium channel marker PanNav (vi) colocalise at a node of Ranvier (merge viii). Scale bars indicate 10 μm

Figure 2

Effects of Kv7 channel activation and blockade on the recovery cycle of electrical excitability. Stimulus–response curves were obtained using unconditioned test stimuli of 1 ms duration. These established the maximal CMAP to supramaximal nerve stimulation and the size of the submaximal target CMAP (~ 40% of maximum). The stimulus current necessary to produce the target potential using a 1 ms test stimulus is referred to as the “threshold” for that potential. Electrical excitability, i.e. the current required to maintain a 40% compound A-fibre action potential response, was determined at discrete interstimulus intervals following a supra-maximal stimulus (A). The representative example in panel B illustrates the concentration-dependent changes in the recovery cycle observed with bath application of flupirtine (3-30 μM) and their reversal following selective Kv7 channel blockade with XE991 (10 μM). Changes in the recovery cycle of A-fibres were quantified with the empirically determined values of refractoriness at 2 ms (D) and 2.5 ms (E) and excitability at 5 ms (F) and 7 ms (G). The relative refractory period (C) was determined respectively by linear interpolation and first-order exponential fits (see Methods). The RRP (p < 0.01) was reduced in a concentration-dependent manner (C). Flupirtine (30 μM) also reduced refractoriness at 2.5 ms (E; p < 0.01) but not at 2 ms (D). Similarly, flupirtine produced a concentration-dependent increase in the magnitude of post-spike superexcitability at 5 ms (F; p < 0.01 for flupirtine 10 μM and 30 μM) and at 7 ms (G; p < 0.01; for flupirtine 30 μM)

Figure 3

Effect of oral flupirtine and placebo on the recovery cycle of electrical excitability in motoneurones to abductor pollicis brevis in healthy subjects. Stimulus–response curves were obtained using unconditioned test stimuli of 1 ms duration. These established the maximal CMAP to supramaximal nerve stimulation and the size of the submaximal target CMAP (~ 40% of maximum). The stimulus current necessary to produce the target potential using a 1 ms test stimulus is referred to as the “threshold” for that potential. Electrical excitability, i.e. the current required to maintain a 40% compound muscle potential response, was determined at discrete interstimulus intervals following a supra-maximal stimulus (A). The representative example in panel B illustrates the changes in the recovery cycle observed after oral uptake of flupirtine (200 mg) or placebo compared to baseline measures. Changes in the recovery cycle of motor axons were quantified with the empirically determined values of refractoriness at 2 ms (D) and 2.5 ms (E) and excitability at 5 ms (F) and 7 ms (G). The relative refractory period (C) was determined respectively by linear interpolation and first-order exponential fits (see Methods). RRP in motoaxons to APB (C) was reduced 2 hours after oral flupirtine (3.40 ± 0.07 ms; p < 0.01) but not placebo (3.45 ± 0.12 ms, p = 0.53). Oral flupirtine also reduced refractoriness at 2 ms and 2.5 ms (D, E). In contrast to the effect of flupirtine in vitro, the magnitude of superexcitability at both 5 and 7 ms determined in vivo was not affected by flupirtine (F,G)

Figure 4

Effect of oral flupirtine and placebo on postischemic ectopic axonal discharge in motoneurones to abductor pollicis brevis in healthy subjects. Representative EMG activity recorded from APB in a single subject before during (grey shading) and after a period of ischemia of the lower arm before (A, Control) and after a single oral dose of flupirtine (200 mg, C). The 10 minute period of ischemia (grey) was produced by inflating a cuff around the upper arm at 200 mmHg for. The upper traces in panels A&D show raw EMG activity while the lower trace is a running power spectral density (PSD calculated from discrete 2048 point FFTs with 50% overlap and a Hamming window), with power indicated by colour. For comparisons, average PSDs during the 600s peri-ischemic (grey) and post-ischemic (black) periods are shown as absolute power (B&E) and normalised to total power (C&F). Under control conditions (A), in the post-ischemic period EMG activity comprises high frequency motor unit discharges (A, inset right) and this is reflected in the increased power in the frequency domain around 200 Hz (B&C). Two hours after a single oral dose (200 mg) of flupirtine (D) EMG activity in the peri-ischemic period is little affected, however EMG activity in the post-ischemic period is considerably reduced. The absolute reduction in post-ischemic EMG activity is reflected in the absolute PSD (E). Following flupirtine, high frequency motor unit discharges still occur post-ischemically but number of action potentials in these bursts is less (D, inset right) and this is reflected in the normalised PSD (F) with frequency components around 200 Hz being less prominent than before flupirtine (C)

Figure 5

Subjective rating of sensations perceived during and after ischemia of the arm. Visual analogue scale ratings (A) were determined at rest (Baseline), at the end of a 10 minute period of ischemia (peri-ischemic) and 10 minutes after releasing the cuff (post-ischemic). While at rest there was no significant difference between groups, in the peri- and postischemic phase treatment did have an effect. Especially in the post-ischemic period application of flupirtine (200 mg p.o.) 2 hours prior to the experiment led to a significant decrease in intensity of sensations (-41%, p = 0.03). Sensations perceived in the postischemic period were additionally assessed with the McGill Pain Questionnaire (SF-MPQ; B&C). The total SF-MPQ score (TPRI) comprises both sensory (SPRI) and affective (APRI) components (B) and each were determined under control (filled bars) conditions and 2 hours after a single oral dose of flupirtine (open bars) or placebo (grey bars). Flupirtine significantly reduced TPRI (−45%; p < 0.01) and SPRI (-48%; p < 0.01). The SF-MPQ associative verbal descriptors (C) were also determined under control conditions (filled) and 2 hours after a single oral dose of flupirtine (200 mg; open) or placebo (grey). Under baseline conditions, the average rating of ‘throbbing’, ‘shooting’, ’stabbing’, ‘sharp’ and ‘hot-burning’ during ischemia was mild-moderate (filled). For the affective descriptors, only ‘sickening’ was rated as moderate. Oral flupirtine (200 mg p.o.) reduced the ratings of ‘hot-burning’ (p < 0.01) and ‘throbbing’(p = 0.04) from moderate to mild. Accordingly to the effect on sensory components, application of flupirtine led to a reduction of several sensory descriptors