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
Journal of Translational Medicine
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
Johannes Fleckenstein 0
Ruth Sittl 0 2
Beate Averbeck 2
Philip M Lang 0
Dominik Irnich 0
Richard W Carr 1 2
0 Department of Anaesthesiology, Multidisciplinary Pain Center, University of Munich , Pettenkoferstr. 8a, Munich 80336 , Germany
1 Department of Anaesthesiology and Intensive Care Medicine, Medical Faculty Mannheim, Heidelberg University , Mannheim , Germany
2 Department of Physiology, Ludwig-Maximilians University , Munich , Germany
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 M) 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.
Kv7 potassium channel; Flupirtine; Human myelinated axon; A fibre; Randomised controlled trial
Blockade of voltage-dependent sodium channels has been
successful in the treatment of some forms of clinical pain,
e.g. diabetic polyneuropathy, lumbar radiculopathies,
complex regional pain syndrome, neuralgic pain and traumatic
peripheral nerve injuries for review: . A group of
compounds which might also influence axonal excitability are
synthetic activators of slow axonal Kv7 potassium channels,
such as flupirtine which has recently been used in the
treatment regimens of patients with peripheral neuropathies .
The target of such strategies is the KV7 (formerly KCNQ/
M) family of potassium channels.
Five genes (KCNQ1-5) encode Kv7 subunits, each with
6 trans-membrane spanning domains, a P-loop and a
conserved A-domain in the cytoplasmic C-terminal region.
Neurones express four Kv7 subunits (Kv7.2-Kv7.5) that
co-assemble to form either homo- or hetero-tetrameric
voltage-gated channels [3,4] and channel activity can be
readily inhibited by agonist action at Gq/11-coupled
receptors . In rodents, Kv7.2 is found at nodes of
Ranvier in peripheral nerve  and in motoneurones in
the spinal ventral horn , while Kv7.3 is expressed in
myelinating Schwann cells . In myelinated axons of
smaller calibre, both Kv7.2 and Kv7.3 subunits are found
nodally  Similarly, Kv7.2, Kv7.3 and Kv7.5 are found
along somatic unymelinated axons  visceral
unmyelinated axons  and in baroreceptor nerve terminals .
The presence of axonal Kv7 subunits has also been
confirmed functionally in peripheral nerve. The synthetic Kv7
channel activator retigabine shifts the activation of
myelinated axons in the hyperpolarizing direction  and reduces
the electrical threshold of unmyelinated axons in human
sural nerve . Similarly, flupirtine increases the electrical
threshold of myelinated axons in rat sural  nerve.
Kv7 channels activate in response to depolarization,
beginning at around -60 mV, and deactivate slowly but
do not inactivate . The slow time course of voltage
activation and lack of inactivation of Kv7 channels suggests
that one role of Kv7 in peripheral axons may be to limit
the extent of repetitive burst discharges. Consistent with
this, the phenotype accompanying dominant-negative
mutations in KCNQ2 (Kv7.2) and on occasion KCNQ3
(Kv7.3) is benign familial neonatal convulsions BFNC;
. In some patients with BFNC, myokymia symptoms
are reported and characterized by spontaneous
involuntary muscle fasciculations [7,15-17]. Myokymic
symptoms arise from hyperexcitability of motoneurones
expressing Kv7.2  and are associated with a point
mutation of arginine at position 207 in KCNQ2 that
neutralizes the third of six positive charges in the
voltage-sensing S4 segment, leading to a slowing and a
depolarizing shift of activation [7,17].
Pharmacological activation of Kv7 thus offers an
important therapeutic strategy for reducing
hyperexcitability. The current study has examined the profile of action
of the Kv7 channel activator flupirtine by examining its
effects on peripheral myelinated axons at clinically
Materials and methods
Protocol and consent forms were approved by the
Committee for Ethics of the University of Munich and
ethical approval to study drug effects in healthy
volunteers was provided by the German Federal Institute for
Drugs and Medical Devices. In accord with the
declaration of Helsinki (Seoul 2008) all participants provided
their written informed consent prior to participation in
the study. ClinicalTrials registration is NCT01450865.
In vitro studies
Procurement and preparation of human sural nerve
Segments of sural and peroneal nerve were obtained
from patients previously scheduled for biopsy (n=7; 1
female, 6 male, age range: 4880). The diagnosis
precipitating biopsy was typically polyneuropathy of
unknown aetiology. Patients were informed about the
procedure by an anaesthesiologist prior to surgery.
Upon surgical removal nerve segments were
immediately placed in physiological solution containing (in
mM): 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2
NaH2PO4, 25 NaHCO3, 11 D-glucose (bubbled to pH
7.4 with 95 % O2 5 % CO2).
Immunohistochemistry Individual fascicles were
isolated from nerve segments and teased apart with fine
forceps. After incubating in PBS containing 0.3%
collagenase (30 min, room temp.) and washing twice in
PBS, preparations were mounted on gelatine-coated
slides, dried overnight and fixed in 4%
paraformaldehyde for 30 minutes. For immunostaining,
preparations were blocked in PBS containing 0.2% Triton
100 and 10% natural goat serum at room temperature
for 40 minutes, followed by overnight incubation at
4C with rabbit anti-KCNQ2-N antibodies diluted
1:200  raised and affinity-purified as previously
described [19,20]. KCNQ2 antibodies were directed
against residues 1337 of the conserved intracellular
N-terminal region [21,22]. Monoclonal antibodies
against peripherin (diluted 1:200) and the alpha
subunits of voltage-gated (NaV) sodium channels (panNaV,
diluted 1:200) were used. After incubation with the
primary antibody, slides were washed three times in
PBS and incubated with fluorescein- and
rhodamineconjugated donkey cross-affinity-purified secondary
antibodies at room temperature for two hours (all
diluted 1:400). Slides were then washed three times
with PBS and covered with Aqua-Poly/Mount
Coverslipping medium. Digital images were taken with a
laser-scanning confocal microscope (Bio-Rad MRC-1024,
Bio-Rad Laboratories GmbH, Munich, Germany). No
evidence of antibody cross-reactivity was observed in
multilabeling experiments with single label and secondary-only
Threshold tracking Nerve fascicles were mounted
between stimulating and recording electrodes in an organ
bath perfused continuously with physiological solution
tempered to 32C. Constant current was used for
stimulation (1 ms, A395, WPI, Sarasota, USA) and
extracellular signals were amplified (NPI, Tamm, Germany),
filtered (30 Hz - 1.3 kHz), sampled (BNC 2120, National
Instruments, USA) and stored to disk.
Electrical excitability of myelinated axons was assessed
using the TRONDXS4 protocol in QTRAC ( Prof. Bostock,
Institute of Neurology, London, UK).
Study design Volunteers were recruited for a
randomised, double-blind, placebo-controlled, two-way,
crossover clinical phase I trial (RCT) to investigate the effect
of oral flupirtine on the electrical excitability of
peripheral myelinated axons. Exclusion criteria were: ongoing
medication, prevailing organic disease, previous forearm
trauma, primary organ failure, pregnancy or
breastfeeding. All female participants were subject to a
pregnancy test (Cyclotest, UEBE, Wertheim, Germany) prior
Subjects were assigned randomly to a study arm in
accordance with an algorithm developed by the Institute
of Medical Information Technology, Biometry and
Epidemiology, University of Munich, Germany (IBE),
stratifying for gender. Subjects received a set of sealed
sequentially numbered pill-boxes each containing either
flupirtine or placebo tablets to be taken prior to the
recording sessions. Both the subjects and study
physicians were blind to the group allocation.
Preliminary results indicated that a sample size of 12
would be needed to detect a decrease in the primary
outcome measure (RRP) with 80% power at a
significance level of 5%. With provision for drop-outs,
20 subjects were recruited, with an allocation ratio
Participants were evaluated on seven occasions in
which motor axon excitability parameters and ischemic
EMG signals were recorded (Additional file 1: Figure
S1). The first 3 recording sessions, scheduled not less
than 48 hours apart, were used to establish baseline
values. The 4th recording session comprised 2 sequential
recordings of axonal excitability, one immediately prior
to pharmacologic intervention and a second two hours
after. EMG during ischemia was only recorded after
medication. The 5th session was a control recording
performed 48 hours after the 4th. The 6th recording session
was scheduled 7 1 days after the 5th and comprised 2
sequential recordings immediately before and 2 hours
after medication. The final (7th) recording session was a
control recording performed 48 hours after the 6th. A
pain questionnaire was assessed after the 3rd, the 4th and
the 6th session.
At the time of examination, subjects lay comfortably
in a temperature controlled room.
Outcome measures The primary outcome measure was
the relative refractory period (RRP) as determined with
threshold tracking. All other indices were considered
secondary outcome measures.
Threshold tracking All experiments were performed by
the same examiner. Skin temperature was determined at
the wrist at the end of each recording session using a
thermocouple (Voltcraft, Hirschau, Germany).
The median nerve of the right hand was used for
nerve excitability studies. A gel disc electrode
(H92SG, Kendall-Arbo, Neustadt, Germany)
positioned at the wrist between palmaris longus and
flexor carpi radialis tendons served as the cathode.
The anode was placed over the course of the median
nerve approximately 2 cm proximal to the cathode.
EMG from abductor pollicis brevis (APB) was
recorded from one electrode situated over the motor point
and a second electrode over the proximal interphalangeal
Constant current pulses (DS5, Digitimer, UK) were
used to determine electrical excitability in QTRAC using
the TRONDXM4 protocol with same measures as for
in vitro experiments (see above). The EMG signal was
amplified (gain 200-500x), filtered (bandpass 0.3 Hz to
10 kHz) and sampled at 50 kHz (BNC-2120, National
Instruments, Austin, USA).
Ischemia A cuff around the upper arm inflated
to >200mmHg was used to examine flupirtine and
placebo effects on ischemic and post-ischemic EMG from
APB. Muscle fasciculations are known to appear in some
subjects upon recovery from a 10 minute period of
ischemia of the lower arm with the intensity and
incidence increasing with the period of ischemia . EMG
signals were filtered, amplified and sampled at 20
kHz (ADInstruments GmbH, Spechbach, Germany).
EMG was recorded continuously over a 21 minute
period comprising one minute baseline followed by
10 minutes of ischemia and a further 10 minutes
Volunteers rated the intensity of sensations before,
during and after ischemia on a visual analogue scale
(0100 mm with 0 being no pain and 100 being maximal
pain) and completed the Short Form McGill Pain
Questionnaire (SF-MPQ) for sensations experienced
during the first 5 minutes following release of the
pressure cuff, i.e. post-ischemia. The SF-MPQ comprises
eleven sensory and four affective pain descriptors that
can be ranked in intensity from 0 = none to 3 = severe
. The sum of ranked values provides a sensory
(SPRI; 033), an affective (APRI; 012) and a total
pain score (TPRI; 045).
Axonal excitability Electrical excitability of myelinated
axons determined both in vitro and in vivo were
analyzed off-line with the MEM routine in QTRAC. Six
parameters were taken directly from the QTRAC
analysis, namely the strength-duration time constant,
rheobase current, refractoriness at 2 and 2.5 ms,
superexcitability at 5 and 7 ms and during the 90-100 ms
hyperpolarizing and depolarizing current pulses of the
threshold electrotonus. The relative refractory period
was determined using custom written software in Igor
Pro (WaveMetrics, Lake Oswego, USA) by determining
the first zero crossing of an exponential fit to recovery
cycle data points less than 5 ms.
Electromyography Rectified and integrated EMG was
used to quantify continuous signals. EMG power was
calculated by discrete fast Fourier transform (FFT) of
EMG recorded in the time domain.
Mean and standard deviation are used for population
descriptors while mean and standard error of the mean
are indicated for comparisons between groups.
Normality was tested using Kolmogorov-Smirnov analysis. For
parametric datasets Students paired t-test was used for
pairwise comparisons. Unpaired datasets were compared
using one-way ANOVA with post hoc Tukey-Kramer.
Non-parametric datasets were analysed using Wilcoxon
ranked sign test for paired data, and Kruskal-Wallis
ANOVA with post hoc MannWhitney-U tests for
non-paired data. The level of statistical significance is
indicated throughout with * for p < 0.05 and ** for
p < 0.01.
Chemicals and drugs
Triton 100, natural goat serum and the monoclonal
antibody against panNaV were purchased from
SigmaAldrich (Munich, Germany), the monoclonal antibody
against peripherin was obtained from Chemicon (EMD
Millipore Corporation, Billerica, USA). Anti-KCNQ2/
Kv7.2 was kindly provided by M. Schwake (Kiel, Germany).
Fluorescein-and rhodamine-conjugated donkey
crossaffinity-purified secondary antibodies were purchased
from Invitrogen (Paisley, UK). Aqua-Poly/Mount
Coverslipping Medium was purchased from Polysciences
Europe (Eppelheim, Germany). Flupirtine (in vitro)
was purchased from Sigma (Taufkirchen, Germany)
and XE991 from Biotrend (Wangen, Switzerland). The
final concentration of test substances used in vitro was
achieved by diluting stock solutions in the perfusing
solution on the day of the experiment.
Blinded oral preparations of flupirtine 100 mg (AWD.
pharma GmbH & Co.KG, Radebeul, Germany) and
placebo tablets (7 mm capsules (Winthrop Arzneimittel
GmbH, Frankfurt, Germany) were performed by the
Pharmaceutical Department, University of Munich
encapsulating both in gelatine (size 00, white opaque;
Shionogl Qualicaps S.A., Alcobendas / Madrid, Spain)
containing lactose, ferric oxide and food colouring
E172 (all Fagron GmbH, Barsbttel, Germany).
Immunohistochemistry of human A-fibre
Using polyclonal antibodies, Kv7.2 subunits could be
identified at nodes of Ranvier in teased fibre
preparations of human sural nerve (Figure 1). Nodes of Ranvier
were identified using DIC images to delineate
myelinating Schwann cells and co-labeling with mouse PanNaV
(voltage-gated sodium channels, Figure 1 vi-viii).
Notably, Kv7.2 was also detected along unmyelinated fibres
co-labeled with peripherin (Figure 1 iv, v).
In vitro assessment of human A-fibre excitability
Recordings were made from twenty-one individual fascicles
of human sural nerve. The average compound A-fibre
response to supra-maximal electrical stimulation (0.1 ms,
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
nonspecific voltage-gated sodium channel marker PanNav (vi) colocalise at a node of Ranvier (merge viii). Scale bars indicate 10 m.
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baseline (n = 21)
flupirtine 3 M (n = 7)
flupirtine 10 M (n = 8)
flupirtine 30 M (n = 16)
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76.61 50.58 42.74 45.44 44.03
2 3 4 5 6 7 8 910 2 3 4 5 6 7 8 9100 2
Interstimulus Interval (ms)
Figure 2 Effects of Kv7 channel activation and blockade on the recovery cycle of electrical excitability. Stimulusresponse 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 M) and their
reversal following selective Kv7 channel blockade with XE991 (10 M). 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 M) 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 M and
30 M) and at 7 ms (G; p < 0.01; for flupirtine 30 M).
11.03 5.43 mA) was 4.97 1.65 mV and had a latency to
half-maximum of 0.82 0.05 ms.
Pooled data for each electrical excitability parameter
determined using the TRONDXS4 protocol in QTRAC is
shown in Table 1. Under control conditions, the
strengthduration time constant was 0.11 0.02 s and the
rheobase current 8.53 2.55 A. ANOVA indicated that
neither flupirtine (3-30 M; strength-duration time constant
F (3:43) 2.00, p = 0.13; rheobase F (3:43) 0.22, p = 0.88)
nor XE991 (10 M; strength-duration time constant
F (1:26) 1.45, p = 0.24; rheobase F (1:26) 0.27, p = 0.87)
had a significant effect on these indices of excitability.
Similarly, threshold changes observed during application
of depolarizing and hyperpolarizing conditioning currents
were also not significantly affected by flupirtine (<30M,
see Table 1).
Flupirtine (10-30 M) did however reduce the RRP
(ANOVA F (3:48) 8.61, p < 0.01) determined in human
A-fibres in vitro and this in a concentration-dependent
manner (see Figure 2B&C, Table 1; post-hoc
Tukey-Kramer p < 0.01for 10 M and 3 M). Importantly, these
effects could be reversed by bath application of the
specific Kv7 channel blocker XE911 (10 M; Figure 2 B&C
and Table 1; ANOVA F (3:36) 7.46, p < 0.01; post-hoc
Tukey-Kramer p < 0.05 for flupirtine 30 M). Flupirtine
(30 M) also reduced refractoriness at 2.5 ms
(Figure 2D; ANOVA F (3:48) 5.39, p < 0.01; post-hoc
Tukey-Kramer p < 0.01). Similarly, flupirtine produced a
concentration-dependent increase in the magnitude of
post-spike superexcitability at 5 ms (Figure 2F and Table 1;
ANOVA F (3:48) 11.56, p < 0.01; post-hoc Tukey-Kramer
p < 0.01 for flupirtine 10 M and 30 M) and at 7 ms
(Figure 2G; ANOVA F (3:48) 7.31, p < 0.01; post-hoc
Tukey-Kramer p < 0.01 for flupirtine 30 M). Since the
RRP is determined from a fit to 3 or more data points, we
consider this the most reliable index of changes in
In vivo assessment of human A-fibre excitability
Twenty volunteers (10 male, 10 female; age 28.3 4.4
years) were randomly allocated to a study arm and
received both placebo and flupirtine (200 mg p.o.)
capsules to be taken in a cross-over design. The trial was
enroled between October 2009 and August 2010. None
of the subjects dropped out, all subjects received the
allocated interventions and the follow-up was complete.
Statistical analysis of electrical excitability datasets
from the initial three recording sessions revealed no
time dependent differences in any of the excitability
parameters and further comparisons between this
baseline dataset and each of the two pre interventional
sessions also showed no differences amongst excitability
parameters. Consequently, all five baseline datasets were
pooled and used as the baseline values for statistical
Compound muscle action potential (CMAP) responses
were recorded from APB in response to supra-maximal
electrical stimulation (4.40 0.22 mA) in 20 volunteers.
CMAPs had an amplitude of 8.59 0.54 mV and a
latency to half-maximum of 7.40 0.22 ms. The
strengthduration time constant was 0.48 0.01 s and the
rheobase current 2.87 0.14 mA. Neither flupirtine (200
mg p.o.) nor placebo affected these measures of
excitability (Table 2).
Consistent with observations made in vitro, RRP in
motoaxons to APB was reduced 2 hours after oral
flupirtine (200 mg p.o.; baseline 3.40 0.07 ms versus
flupirtine 3.23 0.09 ms; p < 0.01 paired t-test; see
Figure 3B&C and Table 2) but not placebo (3.45 0.12
ms, p = 0.53). Oral flupirtine also reduced refractoriness
at 2 ms and 2.5 ms (Figure 3D&E and Table 2). 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 (200 mg p.o; Figure 3F&G).
Flupirtine and placebo were also both without effect on
deand hyperpolarizing threshold electrotonus in vivo
Recordings from APB during ischemia
Cuff-induced (200 mmHg) ischemia, induced a
substantial increase in surface EMG activity in all 20 subjects
(Figure 4A). EMG activity began on average 68.25 8.72
s after cuff inflation and persisted for 288.65 22.93 s.
The total rectified integrated EMG during the baseline
period of ischemia was 1.08 0.12 V.s and this was
reduced to 0.80 0.08 V.s (p < 0.01, paired t-test) 2 hours
after flupirtine (200 mg) but not placebo (0.98 0.13 V.s;
p = 0.29).
Interestingly, in eleven subjects an increase in surface
EMG was also observed in the post-ischemic period
(Figure 4A). Post-ischemic EMG activity began on
average 125.80 21.11 s after release of the cuff,
persisted for 295.20 27.85 s and the total rectified
integrated EMG in the 10 minute period following release
of the cuff was 0.85 0.10 V.s. Consistent with its
effect on EMG activity in the peri-ischemic period,
165.00 105.83 169.77
66.27 40.09 60.28
-24.57 -24.73 -24.11
-27.12 -25.69 -27.05
Figure 3 Effect of oral flupirtine and placebo on the recovery cycle of electrical excitability in motoneurones to abductor pollicis
brevis in healthy subjects. Stimulusresponse 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).
flupirtine also reduced total integrated EMG activity in
the post-ischemic period (200 mg p.o.; 0.58 0.05 V.s;
p = 0.023 paired t-test, see Figure 4D&E) and this
effect was not significant following placebo (0.72 0.09
V.s; p = 0.24).
Under baseline conditions, the surface EMG signal
recorded in the post-ischemic period exhibited
pronounced high frequency motor unit action potential
discharges (MUAP; see insets in Figure 4A&D). The
uniformity of MUAP shape and grouping of these
discharges suggest that they arose from repetitive
discharges in single motor units. The incidence of such
high frequency burst discharges was higher in the
postischemic period than during ischemia. This is evident
in the running power spectral density plots shown
below the raw EMG traces in Figure 4A&D. Following
flupirtine, post-ischemic high frequency repetitive
discharges are also evident in surface EMG recordings
however the length of each burst was curtailed
(Figure 4D, inset). The tendency for flupirtine to
curtail high frequency motor unit discharge is shown in
the normalised power spectral density plots (PSD;
Period of ischemia
Period of ischemia
Flupirtine - Raw Power
Figure 4C&F) where the relative power in the
150200Hz band for post-ischemic discharge is reduced
following flupirtine (Figure 4F).
Assessment of sensations during and after ischemia
Cuff-induced ischemia affects both motor and sensory
axons. To assess the sensory component, a visual
analogue scale (VAS) was used to rate the intensity of
painful sensations during and after ischemia. The VAS
comprised a 0 point anchor indicating no pain and an
anchor at 100 as the most intense pain imaginable.
Averaged across three baseline recordings, pain was rated at
31.1 20.9 mm during ischemia and 33.2 25.4 mm in
the postischemic period (Figure 5A). Two hours after
oral flupirtine (200 mg) the average ischemic pain rating
was 23.2 16.8 mm VAS, (p = 0.12 paired t-test) and
19.6 20.3 mm in the post-ischemic period (-41%, p =
0.03). Oral placebo did not change either the ischemic
(30.5 20.6 mm VAS, -1.7%, p = 0.70 paired t-test) nor
the post-ischemic pain rating (32.8 27.1 mm VAS, -1.2%,
p = 0.69; Figure 5A).
The SF-MPQ was used to assess the intensity and
quality of sensations arising in the 10 minute period
following release of the cuff. Under baseline conditions,
total rating index in the postischemic period was 14.1
1.5 with a sensory and affective component scores of
11.0 1.2 and 3.1 3.0 respectively (Figure 5B). Two
hours after flupirtine (200 mg p.o.) the total intensity
rating (45% to 7.8 1.0, p < 0.01, paired t-test) and
sensory component scores were reduced (-48%, 5.8
0.7, p < 0.01, paired t-test). In contrast, total intensity
ratings were not affected 2 hours after oral placebo
80x10-3 Flupirtine - Normalized
(12.0 1.3, p = 0.25, Figure 5B). The SF-MPQ offers
descriptors to qualitatively describe sensations. Under
baseline conditions, the average rating of throbbing,
shooting, stabbing, sharp and hot-burning during
ischemia was mild-moderate (Figure 5C). For the affective
descriptors, only sickening was rated as moderate. Two
hours after oral flupirtine (200 mg p.o.) the ratings of
hot-burning (p < 0.01 Kruskal-Wallis) and throbbing
(p = 0.04) were reduced from moderate to mild.
Strength and limitations
The primary strength of this study examining drug-induced
effects on axonal excitability in vivo and in vitro is the
ability to compare and thus judge target engagement and the
efficacy of pharmacological compounds on peripheral
myelinated axons. In particular, the ability to expose nerve
segments to high concentrations provides a basis for the
interpretation of subtle changes in excitability detected in
people in situ, where dose is limited.
Segments of human nerve material were obtained at biopsy
and are thus potentially pathological. Previous experiments
indicate however that the electrical excitability of nerve
fascicles obtained from patients at biopsy is not correlated in a
systematic way with the underlying pathology [25,26].
A common deficit encountered during the clinical and
pre-clinical development phases of pharmacological
compounds targeting peripheral nerve is the inability to
determine directly the axonal effects in people. To address
this, standardized measures of axonal excitability were
used to examine the effect of flupritine on human
peripheral nerve in vitro and in situ. Demonstrable changes in
the excitability of myelinated axons were observed in vitro
(Figure 2) and in motoraxons in situ (Figure 3). This
confirmation of target engagement was shown to be
therapeutically relevant with flupirtine suppressing ectopic
axonal activity evoked by ischemia (Figure 4).
Kv7.2 subunits were identified nodally in large diameter
myelinated axons in human sural nerve (Figure 1) consistent
with previous reports using myelinated rat axons [6,8,9].
Kv7.2 expression was also evident along unmyelinated axons
 where it is also postulated to regulate excitability (6)
and suppress axonal discharge . From voltage clamp
studies performed on single nodes of Ranvier of myelinated
axons from human nerve, it is possible that up to 30% of
the slow potassium current may be active at rest . Since,
flupirtine shifts the voltage-dependent activation of
cellexpressed Kv7.2 in the hyperpolarizing direction  it
would be expected to hyperpolarize axons by increasing the
number of open Kv7 channels at resting membrane
potentials around 60 to 70 mV. For the human myelinated
axons examined here, flupirtine (3-30M) produced a
concentration-dependent shortening of the relative
refractory period and an increase in post-spike superexcitability
in vitro, as well as a comparable reduction in the RRP
in vivo (2 hours after oral flupirtine).. The increase in
postspike superexcitability seen in vitro (Figure 2C) was
only apparent for flupirtine concentrations above 10
M (Figure 2F,G). Previous reports indicate that the
peak plasma concentration of flupirtine reaches
values in the range of 5 to 6.5 M, 2 hours after oral
flupirtine 200 mg; [30-32]. This concentration is
below that at which effects can be detected in vitro
and potentially accounts for the lack of effect of
flupirtine on the superexcitable phase in motor axons as
well as the modest effect of flupirtine on the RRP
in vivo (Figure 3B,C).
The modest reduction in the value of the relative
refractory period in motoraxons to APB may well be
secondary to a change in temperature, specifically a
warming of peripheral nerve. Propofol was recently
reported to shorten the relative refractory period in
motoaxons and this effect was attributed to a
nonspecific increase in skin temperature . Although no
global differences in skin temperature were detected
between baseline, placebo and flupirtine recording
sessions, flupirtine could potentially lead to a warming
of peripheral nerve either directly by relaxing vascular
smooth muscle  or indirectly through suppression of
activity in sympathetic neurones .
The only other study examining the effects of
flupirtine on peripheral axons in people used a 400 mg oral
dose and they reported no change in soleus H-reflex
latency but a reduction in medium latency flexor reflex
responses evoked with 5 pulses at 200 Hz . It is
possible that this effect of flupirtine reflects the ability of
slow axonal potassium channels to suppress high
Flupirtine is however also reported to act as an agonist
at GABAA receptors in DRG neurones . While an
agonist action of flupritine at GABAA receptors may
contribute to flupritines ability to alter spinal reflex
latency, peripheral axons maintain a high intra-axonal
chloride concentration and thus activation of GABAA
receptors alone cannot account for an increase in
postspike superexcitability or a shortening of the relative
refractory period in peripheral myelinated axons.
Interestingly, post-spike recovery cycles recorded from
myelinated sural nerve axons in vitro (Figure 1B) appear
to lack the late phase of sub-excitability (between ca. 7
and 100 ms) which is otherwise prominent in recovery
cycles from median nerve motor axons in vivo (Figure 2B).
Recordings in people indicate that late sub-normality is
typically smaller in sural nerve than it is in median nerve
 and that, sural axons thus have less slow nodal
potassium channels (Kv7) .
Post-ischemic EMG activity arises axonally [39,40] and
for a 10 minute period of ischemia affects predominantly
myelinated axons [41,42]. In myelinated sensory axons,
ectopic activity produces paraesthesias and in motor
axons fasciculations are observed , with the former
being more susceptible due to a more pronounced
persistent nodal sodium current . During the period of
ischemia, ectopic activity in myelinated axons is typically
attributed to a protracted axonal depolarization arising
chiefly from a reduction in electrogenic Na+/K+-ATPase
activity and possibly a resultant accumulation of
extracellular potassium, particularly in the adaxonal space
. Activation of slowly activating, non-inactivating
Kv7 channels by flupirtine should adequately counter
slow depolarization and indeed flupirtine effectively
reduced total EMG activity during ischemia (Figure 4).
Post-ischemic EMG activity comprises high frequency
components (Figure 4) and microneurographic
recordings from myelinated axons in people following ischemia
also show prominent high frequency burst discharges ca.
200 Hz; [41,42]. Although high frequency bursts still
occurred in the post-ischemic period following
flupirtine, there was a reduction in the burst duration, i.e.
number of impulses (Figure 4). Flupirtines (200 mg p.o)
curtailing of action potential burst length in human
motoneurones is consistent with previous observations
in rat peripheral myelinated axons using retigabine 
and hippocampal CA1 neurones  and suggests that
flupirtine may be beneficial in limiting the duration of
ectopic bursts in peripheral axons.
Changes in the excitability of peripheral myelinated
axons in people (Figures 2 and 3) were comparable with
those seen in vitro using human sural nerve segments,
suggesting that flupirtine affects Kv7 subunits expressed
in peripheral nerve.
Conclusion and clincial implications
The synthetic Kv7-channel activator flupirtine has been
used clinically in Europe for over two decades and its
analgesic effects have long been associated with an
action on neurones in the CNS. Using a unique
combination of in vitro and in vivo methodologies, the clinical
profile of flupirtine is shown here to extend to effects on
peripheral myelinated axons, reducing their excitability
and suppressing aberrant post-ischemic axonal
discharge. This highlights the potential benefit of Kv7
channel activators for clinical strategies aimed at reducing
hyperexcitability and ectopic discharge in peripheral
Additional file 1: Figure S1. Study Design.
The authors declare that they have no competing interests.
JF conceived of the study, performed the experiments, analyzed the data
and wrote the manuscript. RS conceived of the study, performed the
experiments, analyzed the data and wrote the manuscript. BA carried out the
immunohistochemical experiments, analyzed the data and and helped to
draft the manuscript. PML conceived of the study, and participated in its
design and coordination. DI conceived of the study, and participated in its
design and coordination. RWC conceived of the study, performed the
experiments, analyzed the data and wrote the manuscript. All authors read
and approved the final manuscript.
We would like to thank Prof. Michael Schwake, PhD, Assistant Professor at
the Unit of Molecular Cell Biology and Transgenic Research, Institute of
Biochemistry, University of Kiel, for the primary Kv7.2 antibody. We want to
thank all the volunteers who participated in this trial.
1. Priestley T , Hunter JC : Voltage-gated sodium channels as molecular targets for neuropathic pain . Drug Dev Res 2006 , 67 : 360 - 375 .
2. Miceli F , Soldovieri MV , Martire M , Taglialatela M : Molecular pharmacology and therapeutic potential of neuronal Kv7-modulating drugs . Curr Opin Pharmacol 2008 , 8 : 65 - 74 .
3. Hadley JK , Passmore GM , Tatulian L , Al-Qatari M , Ye F , Wickenden AD , Brown DA : Stoichiometry of expressed KCNQ2/KCNQ3 potassium channels and subunit composition of native ganglionic M channels deduced from block by tetraethylammonium . J Neurosci 2003 , 23 : 5012 - 5019 .
4. Wang HS , Pan Z , Shi W , Brown BS , Wymore RS , Cohen IS , Dixon JE , McKinnon D : KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel . Science 1998 , 282 : 1890 - 1893 .
5. Hernandez CC , Zaika O , Tolstykh GP , Shapiro MS : Regulation of neural KCNQ channels: signalling pathways, structural motifs and functional implications . J Physiol 2008 , 586 : 1811 - 1821 .
6. Schwarz JR , Glassmeier G , Cooper EC , Kao TC , Nodera H , Tabuena D , Kaji R , Bostock H : KCNQ channels mediate IKs, a slow K+ current regulating excitability in the rat node of Ranvier . J Physiol 2006 , 573 : 17 - 34 .
7. Dedek K , Kunath B , Kananura C , Reuner U , Jentsch TJ , Steinlein OK : Myokymia and neonatal epilepsy caused by a mutation in the voltage sensor of the KCNQ2 K+ channel . Proc Natl Acad Sci USA 2001 , 98 : 12272 - 12277 .
8. Devaux JJ , Kleopa KA , Cooper EC , Scherer SS : KCNQ2 is a nodal K+ channel . J Neurosci 2004 , 24 : 1236 - 1244 .
9. Passmore GM , Selyanko AA , Mistry M , Al-Qatari M , Marsh SJ , Matthews EA , Dickenson AH , Brown TA , Burbidge SA , Main M , Brown DA : KCNQ/M currents in sensory neurons: significance for pain therapy . J Neurosci 2003 , 23 : 7227 - 7236 .
10. Wladyka CL , Kunze DL : KCNQ/M-currents contribute to the resting membrane potential in rat visceral sensory neurons . J Physiol 2006 , 575 : 175 - 189 .
11. Wladyka CL , Feng B , Glazebrook PA , Schild JH , Kunze DL : The KCNQ/Mcurrent modulates arterial baroreceptor function at the sensory terminal in rats . J Physiol 2008 , 586 : 795 - 802 .
12. Lang PM , Fleckenstein J , Passmore GM , Brown DA , Grafe P : Retigabine reduces the excitability of unmyelinated peripheral human axons . Neuropharmacology 2008 , 54 : 1271 - 1278 .
13. Sittl R , Carr RW , Fleckenstein J , Grafe P : Enhancement of axonal potassium conductance reduces nerve hyperexcitability in an in vitro model of oxaliplatin-induced acute neuropathy . Neurotoxicology 2010 , 31 : 694 - 700 .
14. Biervert C , Schroeder BC , Kubisch C , Berkovic SF , Propping P , Jentsch TJ , Steinlein OK : A potassium channel mutation in neonatal human epilepsy . Science 1998 , 279 : 403 - 406 .
15. Auger RG , Daube JR , Gomez MR , Lambert EH : Hereditary form of sustained muscle activity of peripheral nerve origin causing generalized myokymia and muscle stiffness . Ann Neurol 1984 , 15 : 13 - 21 .
16. Gancher ST , Nutt JG : Autosomal dominant episodic ataxia; a heterogeneous syndrome . Mov Dis 1986 , 1 : 239 - 253 .
17. Wuttke TV , Jurkat-Rott K , Paulus W , Garncarek M , Lehmann-Horn F , Lerche H : Peripheral nerve hyperexcitability due to dominant-negative KCNQ2 mutations . Neurology 2007 , 69 : 2045 - 2053 .
18. Castaldo P , del Giudice EM , Coppola G , Pascotto A , Annunziato L , Taglialatela M : Benign familial neonatal convulsions caused by altered gating of KCNQ2/KCNQ3 potassium channels . J Neurosci 2002 , 22 : RC199 .
19. Pongs O , Schwarz JR : Ancillary subunits associated with voltagedependent K+ channels . Physiol Rev 2010 , 90 : 755 - 796 .
20. Cooper EC , Harrington E , Jan YN , Jan LY : M channel KCNQ2 subunits are localized to key sites for control of neuronal network oscillations and synchronization in mouse brain . J Neurosci 2001 , 21 : 9529 - 9540 .
21. Singh NA , Charlier C , Stauffer D , DuPont BR , Leach RJ , Melis R , Ronen GM , Bjerre I , Quattlebaum T , Murphy JV , et al: A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns . Nat Genet 1998 , 18 : 25 - 29 .
22. Pan Z , Selyanko AA , Hadley JK , Brown DA , Dixon JE , McKinnon D : Alternative splicing of KCNQ2 potassium channel transcripts contributes to the functional diversity of M-currents . J Physiol 2001 , 531 : 347 - 358 .
23. Bostock H , Baker M , Reid G : Changes in excitability of human motor axons underlying post-ischaemic fasciculations: evidence for two stable states . J Physiol 1991 , 441 : 537 - 557 .
24. Melzack R : The short-form McGill Pain Questionnaire . Pain 1987 , 30 : 191 - 197 .
25. Lang PM , Grafe P : Chemosensitivity of unmyelinated axons in isolated human gastric vagus nerve . Auton Neurosci 2007 , 136 : 100 - 104 .
26. Carr RW , Sittl R , Fleckenstein J , Grafe P : GABA increases electrical excitability in a subset of human unmyelinated peripheral axons . PLoS One 2010 , 5 : e8780 .
27. Rose K , Ooi L , Dalle C , Robertson B , Wood IC , Gamper N : Transcriptional repression of the M channel subunit Kv7.2 in chronic nerve injury . Pain 2011 , 152 : 742 - 754 .
28. Roza C , Lopez-Garcia JA : Retigabine, the specific KCNQ channel opener, blocks ectopic discharges in axotomized sensory fibres . Pain 2008 , 138 : 537 - 545 .
29. Martire M , Castaldo P , D'Amico M , Preziosi P , Annunziato L , Taglialatela M : M channels containing KCNQ2 subunits modulate norepinephrine, aspartate, and GABA release from hippocampal nerve terminals . J Neurosci 2004 , 24 : 592 - 597 .
30. Hlavica P , Niebch G : Pharmacokinetics and biotransformation of the analgesic flupirtine in humans . Arzneimittelforschung 1985 , 35 : 67 - 74 .
31. Niebch G , Borbe HO , Hummel T , Kobal G : Dose-proportional plasma levels of the analgesic flupirtine maleate in man . Application of a new HPLC assay. Arzneimittelforschung 1992 , 42 : 1343 - 1345 .
32. Hummel T , Friedmann T , Pauli E , Niebch G , Borbe HO , Kobal G : Doserelated analgesic effects of flupirtine . Br J Clin Pharmacol 1991 , 32 : 69 - 76 .
33. Maurer K , Wacker J , Vastani N , Seifert B , Spahn DR : Changes in axonal excitability of primary sensory afferents with general anaesthesia in humans . Br J Anaesth 2010 , 105 : 648 - 656 .
34. Yeung S , Schwake M , Pucovsky V , Greenwood I : Bimodal effects of the Kv7 channel activator retigabine on vascular K+ currents . Br J Pharmacol 2008 , 155 : 62 - 72 .
35. Zaika O , Lara LS , Gamper N , Hilgemann DW , Jaffe DB , Shapiro MS : Angiotensin II regulates neuronal excitability via phosphatidylinositol 4,5-bisphosphate-dependent modulation of Kv7 (M-type) K+ channels . J Physiol 2006 , 575 : 49 - 67 .
36. Timmann D , Plummer C , Schwarz M , Diener HC : Influence of flupirtine on human lower limb reflexes . Electroencephalogr Clin Neurophysiol 1995 , 97 : 184 - 188 .
37. Klinger F , Geier P , Dorostkar MM , Chandaka GK , Yousuf A , Salzer I , Kubista H , Boehm S : Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine . Br J Pharmacol 2012 , 166 : 1631 - 1642 .
38. Lin CS , Mogyoros I , Burke D : Recovery of excitability of cutaneous afferents in the median and sural nerves following activity . Muscle Nerve 2000 , 23 : 763 - 770 .
39. Weddell G , Sinclair DC : Pins and needles; observations on some of the sensations aroused in a limb by the application of pressure . J Neurol Neurosurg Psychiatry 1947 , 10 : 26 - 46 .
40. Merrington WR , Nathan PW : A study of post-ischaemic paraesthesiae . J Neurol Neurosurg Psychiatry 1949 , 12 : 1 - 18 .
41. Ochoa JL , Torebjork HE : Paraesthesiae from ectopic impulse generation in human sensory nerves . Brain 1980 , 103 : 835 - 853 .
42. Culp WJ , Ochoa JL , Torebjrk HE : Ectopic impulse generation in myelinted sensory nerve fibres in man. In Abnormal nerves and muscles as impulse generators . Edited by Culp WJ, Ochoa JL. New York : Oxford University Press ; 1982 : 490 - 512 .
43. Bostock H , Rothwell JC : Latent addition in motor and sensory fibres of human peripheral nerve . J Physiol 1997 , 498 (Pt 1): 277 - 294 .
44. Bostock H , Burke D , Hales JP : Differences in behaviour of sensory and motor axons following release of ischaemia . Brain 1994 , 117 (Pt 2): 225 - 234 .
45. Yue C , Yaari Y : KCNQ/M channels control spike afterdepolarization and burst generation in hippocampal neurons . J Neurosci 2004 , 24 : 4614 - 4624 .