0022-3565/97/2821-0093$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 282:93–100, 1997
Vol. 282, No. 1
Printed in U.S.A.
Evaluation of the Antimyotonic Activity of Mexiletine and Some
New Analogs on Sodium Currents of Single Muscle Fibers and
on the Abnormal Excitability of the Myotonic ADR Mouse1
ANNAMARIA DE LUCA, SABATA PIERNO, FEDELE NATUZZI, CARLO FRANCHINI, ANDREA DURANTI,
GIOVANNI LENTINI, VINCENZO TORTORELLA, HARALD JOCKUSCH and DIANA CONTE CAMERINO
Unit of Pharmacology, Department of Pharmacobiology (A.D.L., S.P., F.N., D.C.C.), and Department of Medicinal Chemistry (C.F., A.D., G.L.,
V.T.), Faculty of Pharmacy, University of Bari, 70125 Italy, and Developmental Biology Unit (H.J.), W7, University of Bielefeld, D-33501,
Germany
ABSTRACT
To search for use-dependent sodium channel blockers to selectively solve skeletal muscle hyperexcitability in hereditary
myotonias, mexiletine (MEX; compound I) and its newly synthetized analogs, 2-(4-chloro-2-methylphenoxy)-benzenethanamine (compound II) and (2)-S-3-(2,6-dimethylphenoxy)-2methylpropanamine (compound III), were tested on intercostal
muscle fibers from the myotonic ADR mouse through use of the
standard current-clamp microelectrode technique. In parallel,
the effects of these compounds on the sodium channels were
measured on frog muscle fibers under voltage-clamp conditions. The tonic and use-dependent blocks of peak sodium
currents (INamax) produced by each compound were evaluated
by using a single depolarizing pulse and a pulse train at 10 Hz
frequency, respectively. At 10 and 50 mM, MEX decreased the
occurrence of spontaneous excitability in myotonic muscle fibers; 100 mM was required to decrease the amplitude of the
action potential and the stimulus-induced firing of the membrane as well as to increase the threshold for generation of
action potential. At 300 mM, MEX decreased the latency of the
action potential and increased the threshold current to elicit a
The clinical term of myotonia is used to identify a series of
dominant and recessive forms of genetic diseases of skeletal
muscle characterized by abnormal membrane excitability
and delayed muscle relaxation after voluntary contraction.
Sodium channel myotonias, paramyotonia congenita and hyperkalemic periodic paralysis are due to mutations of the
Received for publication October 22, 1996
1
The financial support of Telethon-Italy for the project Therapeutical Approach of Muscle Diseases Due to Gen Mutation-Induced Malfunctions of Ion
Channels: “In Vivo” and “In Vitro” Studies of Pharmacological Treatments of
Myotonia and Hypokalemic Periodic Paralysis“ (Project 579) is gratefully
acknowledged.
single action potential. MEX produced a tonic block of INamax
with an half-maximal concentration (IC50) of 83 mM, but the IC50
value for use-dependent block was 3-fold lower. Compound III,
which differs from MEX in that it has a longer alkyl chain,
similarly blocked first the spontaneous and then the stimulusevoked excitability of myotonic muscle fibers but at 2-fold
lower concentrations than MEX. Compound III was less potent
than MEX in producing a tonic block of INamax (IC50 5 108 mM)
but was a strong use-dependent blocker with an IC50 close to
15 mM. The more lipophylic compound II irreversibly blocked
both spontaneous and stimulus-evoked membrane excitability
at concentrations as low as 10 mM and shortened the latency of
the action potential in a concentration-dependent fashion.
Compound II produced a potent tonic block of INamax (IC50 5
30 mM), and its potency increased 2-fold during high-frequency
stimulation. Both of the new analogs (compound II in particular), but not MEX, were less effective on the excitability parameters of striated fibers of healthy vs. ADR mice, a characteristic
that increases their interest as potential antimyotonic agents.
gene coding for the skeletal muscle type of voltage-gated
sodium channels (SCN4). The mutated channels show different degrees of impairment of inactivation, which in turn
causes membrane depolarization (Cannon, 1996; LehmannHorn and Rüdel, 1996). Mutations producing mild depolarizations are responsible for the clinical phenotype of hyperexcitability, whereas those exerting sustained depolarizations lead
to paralytic attacks (Cannon, 1996). In the dominant myotonia
congenita and recessive generalized myotonia, the characteristic hyperexcitability and SpDs of action potentials are related to
an abnormally low resting GCl (Adrian and Bryant, 1974; Bryant and Morales-Aguilera, 1971). The protein ClC-1 has been
ABBREVIATIONS: MEX, mexiletine; GCl, resting chloride conductance; ADR, arrested development of righting response phenotype; Ith, threshold
current; AP, action potential amplitude; Lat, latency of the action potential; Th, threshold potential; SpD, spontaneous discharge; AD, afterdischarge; INa, sodium current; IC50, half-maximal blocking concentration; INamax, maximal peak sodium current; pKa, negative logarithm of acid
dissociation constant; KATP channels, ATP-dependent potassium channels.
93
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Accepted for publication March 5, 1997
94
De Luca et al.
myotonic activity and, in particular, (1) the role of the distance between the aromatic and amino-terminal groups
(compound III, fig. 1) and (2) the increase in lipophilicity
produced by the insertion of a chlorine atom in the para
position of the aromatic ring and of a phenyl group on the
carbon atom linked to the amino group (compound II, fig. 1).
These two compounds were chosen on the basis of the effects
they produced on skeletal muscle INas during preliminary
voltage-clamp experiments and further verified in the
present study. In fact, compound III showed noticeable usedependent behavior, whereas compound II was a very potent
INa blocker, two characteristics that may be of importance in
obtaining selective antimyotonic agents.
Methods
Current-clamp recordings of macroscopic membrane excitability of ADR myotonic and healthy mice. Myotonic ADR (genotype adr/adr) mice and wild-type healthy littermates (genotype
1/1 or adr/1) that were 3 months old were used for all the experiments. Myotonia in ADR mice is a recessive disease due to the
insertion of a transposon into the ClC-1 gene (Gronemeier et al.,
1994; Steinmeyer et al., 1991). The diseased homozygous animals
develop a severe and clearly distinguishable myotonic state, whereas
the genotypic adr/1 animals do not show recognizable signs of
myotonia and do not have an alteration of the macroscopic GCl with
Fig. 1. Chemical structure of MEX and its newly synthetized analogs.
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claimed to be the putative channel responsible for the macroscopic GCl; this hypothesis was supported by the finding of
several mutations of the ClC-1 genes in patients and myotonic
animals (Gronemeier et al., 1994; Lehmann-Horn and Rüdel,
1996; Steinmeyer et al., 1991). The attempts to restore the
macroscopic GCl of genetically myotonic goats with various
pharmaceuticals known to modulate this parameter in normal
subjects have been unsuccessful (Bryant and Conte Camerino,
1991).
This result is in line with the recent findings that in the
goat phenotype, the genetic mutation seriously impairs the
function of the chloride channel in its physiological range
(Beck et al., 1996). In fact, severe malfunctions up to a total
loss of function of muscular chloride channel gene product
(ClC-1) can result from different mutations (Fahlke et al.,
1995; Gronemeier et al., 1994). Thus, the therapy for all the
myotonic syndromes, whether related to sodium or chloride
channels, is mainly symptomatic and addressed at relief of
membrane hyperexcitability. Actually, the antimyotonic
drugs that are used clinically are orally effective lidocaine
derivatives such as MEX and tocainide (Jackson et al., 1994;
Lehmann-Horn and Rüdel, 1996; Rüdel et al., 1980), which
are able to block the generation and propagation of the action
potential in skeletal muscle by blocking the voltage-gated
sodium channels. Nevertheless, their use is restricted by
their possible side effects, especially those produced at the
hematopoietic and central nervous system levels (Roden and
Woosley, 1986). In addition, effective antimyotonic doses are
as large as those used to obtain antiarrhythmic action, with
a possible effect on cardiac function as well.
In the attempt to search for selective antimyotonic agents,
two characteristics should be taken into account. First, the
drugs should block the INas in a use-dependent manner. The
use-dependent blockers stabilize the bound channels in the
inactivated state from which the recovery is slowed down
(Catterall, 1987; De Luca et al., 1991; Grant and Wendt,
1992). This mechanism ensures a stronger potency of the
compound on tissues with excessive firing of action potentials
and/or permanent depolarization, such as the muscles affected by sodium or chloride channel phenotypes of myotonia,
than on tissues with a physiological excitability. Second,
sodium channels of the various tissues are genetically distinct and show different kinetic and pharmacological properties. It was recently reported that in addition to having
different sensitivities toward tetrodotoxin, cardiac and skeletal muscle types of sodium channels are affected differently
by local anesthetic-like drugs; the pharmacological profile is
further exacerbated by the different inactivation properties
of the two channel types (Wang et al., 1996). These observations support the possibility of designing use-dependent
blockers of INas to be more selective on skeletal muscle than
on the heart and to be able to relieve sarcolemmal hyperexcitability in myotonic subjects with fewer side effects.
The aim of the present study was to screen the effects of
newly synthetized analogs of MEX as potential antimyotonic
agents by evaluating in vitro their ability to suppress the
pathological hyperexcitability of skeletal muscle isolated
from myotonic ADR mouse, a phenotype with a severe recessive form of low GCl myotonia (Gronemeier et al., 1994;
Mehrke et al., 1988; Steinmeyer et al., 1991). The newly
synthetized compounds were used to investigate the influence of some structural properties of the molecule on anti-
Vol. 282
1997
95
gain amplifier. Then, the solution in one of the central pools, called
the A pool (70–100 mm wide), was replaced by an external solution,
and recordings were performed at 10°C. The circuit of the voltageclamp amplifier was based on that described by Hille and Campbell
(1976). Briefly, a first operational amplifier was used to compare the
interior of the fiber with the ground and applied a negative feedback
in pool A to force the membrane potential to 0 mV. A second operational amplifier clamped the membrane potential to the command
potential via a negative feedback. The measurement of the membrane current in response to a voltage-clamp step was made by
measuring the voltage drop across a resistor of a known value. The
holding potential was 2100 mV. The voltage-clamp amplifier was
connected via a 12-bit AD/DA interface (Digidata 1200, Axon instruments, Forster City, CA) to a 80486 DX2/66 PC. The stimulation
protocols and data acquisition were driven by Clampex (pClamp 6
software package, Axon Instruments). The INas flowing in response
to depolarizing command voltages were low-pass filtered at 10 kHz
(Frequency Devices), visualized on an oscilloscope, sampled at 20
kHz and stored on the hard disk. When necessary, leak and capacities currents were subtracted using the P/4 method. The acquired
traces were later analyzed with Clampfit (pClamp 6 software package, Axon Instruments). Maximal INamaxs were elicited with test
pulses from the holding potential to 220 mV for 10 msec. The tonic
block exerted by the test compounds was evaluated as percent reduction of the peak INa elicited by single test pulses. The evaluation
of the use-dependent block by the drugs was made by using a 10-Hz
train of test pulses for a period of 30 sec and normalizing the residual
current at the end of this stimulation protocol with respect to that in
the absence of the drug (De Luca et al., 1995).
Solutions and drugs. The mouse intercostal muscle preparations were perfused with a physiological salt solution with or without
the test compounds: 148 mM NaCl, 4.5 mM KCl, 2.0 mM CaCl2, 1.0
mM MgCl2, 12 mM NaHCO3, 0.44 mM NaH2PO4 and 5.55 mM
glucose. The solutions were continuously gassed with 95% O2/5%
CO2 (pH 7.2, 7.4). For INa recordings, the semitendinosus muscle
fibers were perfused with an external solution consisting of 77 mM
NaCl, 38 mM coline-Cl, 1.8 mM CaCl2, 2.15 mM Na2HPO4 and 0.85
mM NaH2PO4 and dialyzed with an internal solution consisting of
105 mM CsF, 5 mM 3-(N-morpholino)propanesulfonic acid, 2 mM
MgSO4, 5 mM ethylene glycol bis(a-aminoethyl ether)-N,N,N9,N9tetraacetic acid and 0.55 mM Na2ATP (pH 7.2 with NaOH concentrated solution). The compounds tested were synthetized in our
laboratories. Briefly, MEX (compound I) was synthetized according
to procedures previously detailed (Franchini et al., 1994); compound
II [2-(4-chloro-2-methylphenoxy)-benzenethanamine] was obtained
through condensation of bromoketon with the opportune phenolic
derivative and transformation of the product to oxime through reaction with hydroxylamine and successive reduction to amine II2 and
compound III [(2)-S-3-(2,6-dimethylphenoxy)-2-methylpropanamine] was obtained in the optically active form through condensation
of the chiral alcohol with the opportune phenolic derivative after
protection of the nitrogen atom (Duranti et al., 1995) (fig. 1). All
compounds were fully characterized by routine spectroscopic analyses; analytical results for C, H and N were within 60.4% of the
theoretical values. Stock solutions of MEX and compound III were
prepared in physiological or external solutions for current-clamp and
voltage-clamp experiments, respectively, whereas stock solutions in
dimethylsulfoxide (100 ml/mg) were used for compound II. All the
stock solutions were prepared daily, and the final concentrations to
be tested in vitro on isolated muscle were obtained by further dilution of the stock solution as needed. Dimethylsulfoxide at the highest
concentration used (0.2%) was without effect on any of the parameters recorded. On both intercostal muscle and frog muscle fibers, no
more than three concentrations of the same compound were tested,
2
C. Franchini, A. Duranti, G. Lentini, V. Tortorella, manuscript in preparation.
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respect to wild-type (1/1) despite the defect in one allele (Gronemeier et al., 1994; Mehrke et al., 1988).
The intercostal muscle was dissected with the animals under
urethane anesthesia (1.2 g/kg i.p.) and immediately placed in a
muscle bath at 30°C. Soon after the removal of the muscle, the
still-anesthetized animals were killed with an overdose injection of
urethane. The two-microelectrode technique was used for the electrophysiological recordings (Bryant and Conte Camerino, 1991; De
Luca et al., 1992, 1995). A voltage-sensing electrode and a currentpassing electrode were placed 50 mm apart in the central region of
randomly selected superficial fibers. After recording of the resting
membrane potential, the fibers were current-clamped at 280 mV for
evaluation of the membrane excitability parameters. The excitability
characteristics of the sampled fibers were determined by recording
the intracellular membrane potential response to square-wave depolarizing constant current pulses of 100 msec at a frequency of 1 Hz.
The current intensity was gradually increased until the depolarization was just sufficient to elicit a single action potential. The current
intensity was then further increased in an attempt to generate two or
more action potentials. If these could not be elicited by a current
pulse that was $3-fold the pulse that elicited a single action potential, we considered this to be a failure to fire repetitively. In this way,
it was possible to record and measure the parameters of minimum
current intensity that would elicit a single action potential (Ith), the
membrane potential at which a single action potential could be
elicited (Th), the AP, the Lat (i.e., the maximal delay from the
beginning of the current pulse to the onset of the spike) and the
maximum number of action potentials that the muscle fibers could
generate (N spikes) (De Luca et al., 1992, 1995).
Most of the myotonic fibers showed characteristic SpD of action
potentials on the insertion of the voltage-sensing microelectrode and
self-sustained ADs (i.e., discharges of action potential after the end of
the application of the depolarizing stimulus) (Adrian and Bryant,
1974; Mehrke et al., 1988). The high electrical activity of myotonic
muscle fibers was often accompanied by visible contraction (never
observed in muscle fibers of healthy animals), which could cause
either the dislodgement of the electrode from the fibers or a postcontraction depolarization affecting the quality of the current-clamp
procedure. To avoid such mechanically induced artifacts, most of the
myotonic preparations were pretreated in vitro with dantrolene sodium (2 mg/liter). As already described (Bryant and Conte Camerino,
1991; Morgan and Bryant, 1977), this compound avoided fiber contraction due to inhibition of calcium release from intracellular stores
but did not affect either the occurrence of spontaneous electrical
activity of sarcolemma or the characteristics of the stimulus-induced
action potential. Thus, in each preparation, the number of fibers
showing either SpDs or ADs was calculated as a percentage of the
total number of fibers sampled both in the absence and presence of
the drugs to evaluate their ability to suppress these pathological
manifestations in each individual muscle preparation.
Recordings of INa using the three vaseline gap voltageclamp technique. Voltage-clamp recordings of INa were performed
on 1–2-cm-long segment of single muscle fibers obtained by microsurgery from the ventral branch of semitendinosus muscle of rana
esculenta. The cut end fiber was superfused with an internal solution
and transferred into the recording chamber, filled with the same
solution. The muscle chamber used was based on that of Hille and
Campbell (1976). The fiber was mounted across three chamber partitions with edges that had been previously covered with strings of
commercial grease (Glisseal, Borer Chemie AG, Zuchwil, Switzerland), which delineated four pools. With the fiber in position, three
additional strips of grease were applied over the fiber and carefully
sealed to the fiber to reduce leakage. The length of the cut ends of the
fiber was ;300 mm. Four KCl/agar bridges electrodes (one for each
pool) connected the recording chamber to the voltage-clamp amplifier. When the solution was lowered below the glisseal grease strips,
the four pools were physically and electrically independent of each
other, as established by the absence of leak current on increase of the
Mexiletine Analogs as Antimyotonic
96
De Luca et al.
Vol. 282
and the preparations were exposed to each concentration for $10
min before recording to allow the maximum effect of the drug to be
reached.
Statistical analysis. Data are expressed as mean 6 S.E.M. The
statistical significances of the differences between groups of mean
values were calculated by unpaired Student’s t test. The molar concentrations of the drugs producing a 50% block of firing or of INamax
(IC50) were determined by using a nonlinear least-squares fit of the
concentration-response curves to the following logistic equation: Effect 5 2100/1 1 {K/[drug]}n, where Effect is percent change of the
parameter, 2100 is the maximal effect, K is the IC50 value of the
tested drug, n is the logistic slope factor and [drug] is the molar
concentration of the tested drug (De Luca et al., 1992, 1995). The
estimates of S.E.M. and n for normalized percent values were obtained as described by Green and Margerison (1978).
Results
Fig. 2. Typical electrophysiological recordings of excitability characteristics of mouse intercostal muscle fibers using the intracellular microelectrode current-clamp technique as described in the text. Recordings from (A) healthy and (B) myotonic ADR mice and recordings
showing effects of (C) 100 mM MEX, (D) 50 mM compound III and (E) 10
mM compound II on the excitability of myotonic ADR mice are shown.
Each row shows a recording from the same fiber in which the intensity
of the depolarizing pulse was slightly increased (from left to right) to
reach the Ith for generating a single and a train of action potentials,
respectively. B, The myotonic fibers showed self-sustained ADs of
action potentials at the end of the current pulse that elicited a membrane firing and SpDs on insertion of the voltage-sensitive microelectrode.
was noted during recordings on the basis of a more stable
membrane potential during impalement of the fibers. In fact,
a significant reduction of the SpDs was observed at 10 mM; at
50 mM, these were completely suppressed along with a significant reduction in the occurrence of the ADs, although the
stimulus-evoked firing of the membrane was not modified.
This latter was appreciably reduced by 100 mM MEX (fig. 3),
a concentration at which a reduction in AP and a significant
5-mV increase in Th (P , .05) were also observed (table 2 and
fig. 2). The above effects of MEX were more remarkable at
300 mM, which further produced a significant shortening of
TABLE 1
Excitability parameters of intercostal muscle fibers of myotonic and healthy mice
Membrane excitability parameters were recorded from intercostal muscle fibers of healthy and myotonic ADR mice.
Experimental
conditions
Healthy
ADR
Preparations/
fibers
AP
Ith
Lat
Th
N spikes
n
mV
nA
msec
mV
n
4/20
4/25
98.1 6 2.3
95.3 6 4.7
102.0 6 5.8
22.4 6 2.7a
12.8 6 0.7
92.3 6 2.4a
27.0 6 1.2
25.7 6 1.1
5.1 6 0.5
11.7 6 0.7a
Fibers with
SpD
fibers
with AD
%
0
74 6 2
0
87 6 7
Ith, current intensity at rheobase to elicit one action potential. N spikes, maximum number of action potentials elicitable. The percent of fibers showing SpDs just
on the insertion of the voltage-sensitive microelectrode was obtained by calculating in each preparation the fibers with SpDs divided by the total number of fibers
sampled (from 20 to 30 fibers) and then averaging the individual values. The percent of fibers with ADs after the stimulus-induced firing was calculated in a similar
manner. Values are mean 6 S.E.M. from n fibers.
a
Significantly different from the same parameter of control animals by Student’s t test (P , .001).
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Excitability characteristics of muscle fibers of myotonic ADR mice. The excitability characteristics of intercostal muscle of myotonic ADR mice are detailed in table 1, and
representative recordings are shown in figure 2. Spontaneous
myotonic activity, such as high-frequency discharges of action potentials (.10 Hz) on insertion of the recording electrode, was detected in ;60% to 80% of fibers in each ADR
muscle preparation (table 1 and fig. 2). The myotonic muscle
fibers needed very little depolarizing current to generate one
action potential and, as expected on the basis of the low GCl
myotonic phenotype, the action potential showed a significantly longer Lat than muscle fibers from healthy mice. However, no significant differences with respect to normal animals were observed in AP or values of Th. As is typical of
myotonic state, the muscle fibers of ADR mice were able to
generate a significantly higher number of action potentials
than healthy ones when the depolarizing stimulus was just
slightly increased over the Ith (table 1). In myotonic muscle
fibers, the stimulus-evoked train of action potentials was
followed by self-sustained ADs at the end of the depolarizing
current pulse in ;75% to 100% of the fibers sampled (table 1
and fig. 2).
Effects of MEX and its analogs on excitability characteristics of muscle fibers of myotonic ADR mouse. All
of the tested compounds affected the excitability characteristics of myotonic muscle fibers, and we took care to evaluate
the ability of each compound to relieve at low concentrations
the typical myotonic manifestations without changing the
parameters unaffected by the disease (table 2). The ability of
each compound to reduce in a concentration-dependent manner the excessive stimulus-induced firing of action potentials
of ADR muscle sarcolemma was also evaluated (fig. 3). MEX
at 10 mM started to have a membrane-stabilizing effect that
1997
97
Mexiletine Analogs as Antimyotonic
TABLE 2
Effects of MEX and its analogs on excitability characteristics of myotonic and healthy mice
Shown are the effects of MEX, compound III and compound II on the excitability parameters of intercostal muscle fibers of myotonic (ADR) and healthy (Control) mice.
The drug effects are illustrated as the percent modification of each parameter (1, percent increase; 2, percent decrease, N.C., no change) with respect to the relative
value in the absence of drug 6 S.E.M. from 6 to 28 normalized number of fibers.
Drug
ADR mice
MEX
Concentration
AP
Ith
Lat
Th
SpD
AD
mM
mV
nA
msec
mV
n
n
N.C.
19.0 6 7
122.7 6 8
133.9 6 9
235 6 8
290 6 10
2100
2100
N.C.
268 6 9
2100
2100
N.C.
N.C.
113 6 4
147.2 6 7
27.0 6 6
234 6 9
2100
2100
225 6 9
280 6 6
2100
2100
15.2 6 9
129.4 6 12
225 6 6
2100
280 6 8
2100
N.C.
N.C.
211.0 6 7
213.0 6 6
N.C.
N.C.
N.C.
1100 6 37
Compound III
5
10
50
100
N.C.
29.1 6 11
218.8 6 6
228.8 6 3
N.C.
N.C.
137.8 6 21
1235 6 80
Compound II
10
50
N.C.
212.6 6 8
1156 6 22
178 6 14
300
213.8 6 4
100
213.6 6 4
50
100
211.4 6 3
220.6 6 3
Healthy mice
MEX
Compound III
Compound II
a–c
.01,
b
N.C.
N.C.
N.C.
264.1 6 7
N.C.
N.C.
N.C.
N.C.
228.6 6 17
263.5 6 8
153.8 6 9
a
217.0 6 13
23.5 6 12
213.0 6 10
b
c
N.C.
130.0 6 11
N.C.
18.5 6 6c
N.C.
212.3 6 9
115.3 6 6
19.4 6 6
Significant difference between the effects produced by the same drug at the same concentration in ADR and control mouse muscles by Student’s t test (a P ,
P , .005 and c P , .001).
the Lat to 33.5 6 7.1 msec (n 5 9) (P , .001) and a 40-nA
increase in Ith (table 2). Compound III was able to decrease
dose-dependently both SpDs and ADs in the range of 5 to 10
mM, being more potent than MEX on the ADs. At the above
concentrations, the firing capability of the membrane was
also slightly reduced (fig. 3). Compound III at 50 mM completely suppressed spontaneous myotonic manifestations and
significantly reduced the maximum number of spikes that
the membrane could generate from 13 6 0.8 (n 5 6) to 8.3 6
0.8 (n 5 9) (P , .005). In fact, compound III inhibited the
firing capability of the membrane more effectively than MEX,
with an IC50 value of 66 vs. 214 mM (fig. 3). Compound III
was also more potent than MEX in reducing the AP and in
increasing both Ith and Th (fig. 2 and table 2). The effects of
this compound increased at 100 mM, and at 180 mM we
observed a complete block of the excitability in that none of
Fig. 3. Concentration-response curves showing the percent inhibition
of the maximum number of spikes elicitable (firing) produced by MEX,
compound III and compound II on the intercostal muscle of myotonic
ADR mice. Each value is the mean 6 S.E.M. from 8 to 12 normalized
fibers. The curves fitting the experimental points were obtained using
the logistic function detailed in the text. The IC50 value for each compound calculated from the fit was 214 6 26, 66 6 6 and 24 6 4 mM for
MEX, compound III and compound II, respectively.
the sampled fibers were able to generate one action potential
(data not shown). Compound II was very potent in depressing myotonic hyperexcitability but showed a different pattern of effects compared with MEX and compound III. In fact,
at concentrations as low as 10 mM, this compound not only
depressed the occurrence of SpD and ADs but also produced
a reduction of the firing capability comparable to that observed with 100 mM MEX, with the half-maximal concentration on this parameter being 24 mM (fig. 3 and table 2).
Furthermore, at 10 mM it decreased the latency of the action
potential to 66 6 16 msec (n 5 6), increased significantly Ith
from 19 6 1.5 (n 5 7) to 48.7 6 4.5 (n 5 6) nA (P , .001) and
slightly shifted Th toward more positive potentials (fig. 2).
All these effects were concentration dependent, being more
pronounced at 50 mM. At this concentration, compound II
significantly shortened the Lat to 33.7 6 7.4 msec (n 5 7)
(P , .001) and almost completely blocked the ability of the
fiber to generate more than two action potentials. A decrease
in the amplitude of the action potential was also observed
(table 2). At 100 mM, compound II completely blocked the
ability of the fibers to generate even one action potential. In
contrast to MEX and compound III, the effects of compound
II were not reversible on washout. None of the compounds
tested significantly modified the resting membrane potential.
Effects of MEX and its analogs on excitability parameters of muscle fibers of healthy mice. The effects of the
test compound on the excitability of intercostal muscles of
healthy mice were studied to better evaluate the specificity of
the antimyotonic activity. As shown in table 2, MEX modified
the depolarization-induced excitability parameters of
healthy muscle fibers at concentrations similar to those effective on the same parameters in myotonic ones. Different
effects were observed with the two analogs. Compound III at
100 mM was much less effective on the excitability parameters of healthy muscle fibers than on those of ADR muscle
(table 2), with the exception of the membrane firing capability, which was similarly reduced in both cases (by 70 6 3% in
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10
50
100
300
98
De Luca et al.
dependent block (i.e., a block of the INa in a situation of
high-frequency stimulation such as that occurring in the
pathological myotonic state). MEX produced a tonic block of
the INa in a concentration-dependent fashion with a calculated IC50 value of 83 mM. As expected, in the presence of
MEX, the repetitive stimulation at 10-Hz frequency produced
a further cumulative reduction of the INa due to use-dependent block. After 30 sec of such a stimulation, the use-dependent block by MEX fully attained the equilibrium, and the
residual current normalized with respect to that in the absence of drug allowed the calculation of the amount of usedependent blockade. The IC50 value for use-dependent block
was three times lower than that for tonic block. Compound
III was less potent in producing a tonic block of the currents
with respect to MEX; in figure 4A, the effects of 50 mM MEX
and compound III on inward sodium transients are compared. The calculated IC50 value of compound III for tonic
block was 108 mM. However, the 10-Hz stimulation showed a
remarkable use-dependent blockade by this compound so
that 50 mM compound III or MEX produced a comparable
block of INa. Thus, the IC50 value of compound III, calculated
with the high-frequency stimulation protocol, was almost six
times lower than that for obtaining the tonic block. Different
behavior was observed with compound II; this compound
produced a remarkable and irreversible tonic block of the
INas, with IC50 values as low as 30 mM. Nevertheless, little
cumulative block was observed during the 10-Hz stimulation
Fig. 4. A, INa transients recorded by the three vaseline gap voltage-clamp method from single muscle fibers of frog semitendinosus muscle and
(from left to right) effects of MEX (50 mM), compound III (50 mM) and compound II (10 mM), respectively. After recording the control trace elicited
by a single test pulse from 2100 to 220 mV (a), the test compounds were applied. After 10 min of incubation, a single test pulse was again applied.
The reduction in the current under this condition was the result of the tonic block exerted by the drug (b), and its amount was quantified by percent
normalization with respect to the current in the absence of drug. A train of pulses at a frequency of 10 Hz was then applied for 30 sec, and a further
cumulative block of the INas over the tonic block was due to the use-dependent behavior of the compound (c). The amount of use-dependent block
was calculated from percent normalization of the residual current at the end of the 30-sec stimulation with respect to the relative current in the
absence of drug. As it can be seen, 50 mM MEX produced a greater tonic block of the current with respect to the same concentration of compound
III; however, this latter was more use dependent, so at the end of the stimulation protocol, the percent reduction by compound III was comparable
to that produced by MEX. In contrast, compound II was much more potent than both MEX and compound III in blocking INa, although this
compound showed less use-dependent behavior. This protocol was repeated with various concentrations of the test compounds to construct
concentration-response relationships (B). Each point is the mean 6 S.E.M. of percent block of INamax from three to five fibers. The curves fitting
the experimental points led to the calculated values of IC50 for both tonic and use-dependent block that were 83 6 14 and 30 6 5 mM for MEX;
108 6 13 and 17 6 4 mM for compound III and 30 6 3 and 15 6 2 mM for compound II, respectively.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
healthy and by 68.5 6 5% in ADR muscle fibers). Compound
II at 50 mM reduced the amplitude of the action potential of
healthy muscle fibers in a manner similar to the effect produced in myotonic fibers. However, the decrease in membrane firing capability was much less evident on normal
muscle fibers. At 50 and 100 mM, compound II reduced the
firing capability by 51 6 6% and 68.3 6 6%, respectively,
effects that were much less pronounced than those observed
with 50 mM in myotonic fibers. At 50 and 100 mM, a concentration-dependent decrease in Ith and a small increase in Th
were observed. Under this experimental condition, the Lat
was little modified by compound III; this parameter was only
slightly reduced at 100 mM (table 2). In addition, on healthy
muscle fibers, the test compounds did not produce any remarkable effect on resting membrane potential.
Effects of MEX and its analogs on INas of single muscle fibers. The effects of in vitro application of MEX and its
analogs on INas of frog semitendinosus muscle fibers are
illustrated in figure 4. As detailed in the text, INamaxs were
elicited with 10-msec test pulses from the holding potential of
2100 to 220 mV. Such a test pulse was applied as a single
stimulus in both the absence and presence of the test drugs to
evaluate the amount of tonic block (i.e., block exerted by the
drug during the resting state of the channel and the membrane). After evaluation of the tonic block, the test pulse was
repetitively applied at the frequency of 10 Hz for 30 sec to
evaluate the ability of the test compounds to produce a use-
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1997
Mexiletine Analogs as Antimyotonic
protocol, with the calculated IC50 being twice as low as that
for tonic block (fig. 4).
Discussion
mulation of channel block if the membrane remains depolarized or the intervals between successive depolarizing stimuli
are too short with respect to the dissociation constant of the
drug from the channel (Grant and Wendt, 1992). At the same
time, the physicochemical properties influence the possibility
of the molecule gaining access to the binding site.
A compound whose pKa favors an increase in the ratio
between charged/uncharged forms usually shows stronger
use-dependent behavior with respect to an uncharged or a
highly lipophilic compound because the former needs a channel opening to gain access to the receptor site (Hille, 1977).
The minimal chemical modification introduced with compound III can increase the pKa of the molecule (with an
increased amount of charged form at physiological pH) and
change the affinity of the drug/receptor interaction, since in a
series of lidocaine derivatives, the presence of an alkyl chain
of two carbons has been found to optimize the binding to the
receptor (Sheldon et al., 1991). In turn, the high use-dependent behavior can account for the ability of compound III to
act on high-frequency discharges of action potentials, either
spontaneously occurring or induced by the depolarizing stimuli, at lower concentrations with respect to those of MEX. On
the other hand, an increase in the lipophilicity of the molecule via the insertion of a chlorine atom in the para position
on the aromatic ring and of a phenyl group on the carbon
atom linked to the amino group led to compound II, which
proved to be almost 10 times more potent than MEX in
reducing the excitability characteristics of myotonic muscle
fibers although with less graduation of the effect on the
various myotonic manifestations (e.g., the SpDs). Concomitantly, compound II produced a remarkable and irreversible
tonic block of INas during voltage-clamp experiments, and the
additional use-dependent block was slight, likely due to its
high lipophilicity.
The weak use-dependent behavior, irreversible action and
lipophilicity made compound II appear to be of little interest
from a therapeutical point of view. One would predict a
toxicological potential due to poor discrimination between
pathological and physiological excitability patterns and longlasting accumulation in various tissues. Nevertheless, compound II shows interesting features; in particular, (1) it was
effective at low concentrations, (2) in contrast to MEX, it was
more effective on myotonic fibers than on healthy ones and
(3) it was able to shorten the Lat in a concentration-dependent manner in the myotonic but not the healthy fibers. This
latter effect, which was also observed with MEX at doses as
high as 300 mM, may be beneficial for therapeutic interventions on the low-GCl myotonic state, such as the ADR mouse.
In fact the long latency, and the consequent prolongation of
action potential duration, is a characteristic feature of the
decrease in GCl (Adrian and Bryant, 1974). The Lat reflects
the electrotonic response of the fibers before reaching the
threshold, and it is mainly due to the resting ionic conductances to chloride and potassium ions. In the absence of GCl,
as in ADR fibers, a shortening of the latency can be mediated
by increasing potassium conductance through a modulation
of potassium channels active at resting potential. It has been
recently shown that MEX, at high concentrations, can open
KATP channels in the heart and thus shorten cardiac action
potential duration (Sato et al., 1995). It is tempting to speculate that a similar mechanism can account for the decrease
in latency observed with MEX and compound II in ADR
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The present study was aimed at evaluating the ability of
MEX and its newly synthetized analogs to abolish the abnormal membrane excitability of myotonic muscle fibers of ADR
mouse. In this phenotype, the pathological hyperexcitability
is due to an abnormally low macroscopic GCl, the parameter
that ensures the electrical stability of the membrane under
resting conditions and maintenance of this latter after the
voluntary excitation-contraction cycle. As a consequence of
their low resting GCl, myotonic muscles spontaneously generate trains of action potentials, which trigger involuntary
spasms; the self-sustained ADs of action potentials occurring
in myotonic fibers are responsible for the delayed relaxation
after voluntary movements and are caused by the low GClinduced potassium accumulation in the t-tubules (Adrian
and Bryant, 1974; Bryant and Morales-Aguilera, 1971;
Lehmann-Horn and Rüdel, 1996).
Particular attention has therefore been devoted to evaluating the effects of MEX and its derivatives on these peculiar
myotonic signs of the ADR phenotype and to correlate them
with the ability to block the INas in a use-dependent manner.
All the three compounds tested were effective, although appreciable differences were observed between them. As far as
MEX is concerned, we observed that it started to reduce the
abnormal SpDs of myotonic fibers at concentrations as low as
10 mM, a concentration close to that described to be clinically
effective for this compound as an antiarrhythmic (Sato et al.,
1995; Wu et al., 1992) and in good agreement with those able
to produce a use-dependent block of the INas (Sunami et al.,
1993). The increased distance between the aromatic ring and
the amino-terminal group by the insertion of a methylene
moiety in the alkyl chain, in compound III, doubled the
potency in reducing the firing capability of myotonic muscle
fibers and slightly decreased the ability to modify the excitability characteristics of healthy muscle fibers. The stronger
potency of compound III than MEX as an antimyotonic drug
could be explained taking into account the results on the
blocking mechanism of this compound on the INas. Compound III was less potent than MEX in producing a tonic
block of the INas (i.e., a block of the sodium channels during
the resting state), but it caused a stronger use-dependent
accumulation of INa block during high-frequency stimulation.
The ability of this class of compounds to produce a tonic and
use-dependent block depends in part on the molecular mechanism at the level of the receptor site on the channel protein
and in part on the physicochemical properties of the molecule
itself. It is known that the sodium channel blockers act on a
receptor on the alpha subunit of sodium channel complex
that has stringent conformational requirements (i.e., it discriminates between optically active compounds) (De Luca et
al., 1995; Ragsdale et al., 1994). According to the receptormodulated hypothesis, the use-dependent mechanism is due
to a change in conformation of the receptor during the voltage-dependent channel transition, and this leads to a highaffinity state when the channel is open and/or inactivated
(Catterall, 1987; Grant and Wendt, 1992). The drug-blocked
channels recover more slowly from the nonconductive inactivated state (Sunami et al., 1993), and this can lead to accu-
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De Luca et al.
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fibers. KATP channels are abundantly present on sarcolemma
of striated fibers and are predisposed to opening at resting
potential when the level of intracellular ATP falls or the
cytoplasmic pH decreases (Longman and Hamilton, 1992;
Tricarico and Conte Camerino, 1994), metabolic situations
likely to occur in the myotonic muscle fibers because of the
intensive muscle work.
The hypothesis that compound II can act on KATP channels
at concentrations close to those effective on sodium channels
has yet to be validated through more direct experimental
evidence. However, it is a particularly attractive hypothesis
because the classic openers of KATP channels have been proposed as alternative antimyotonic agents (Longman and
Hamilton, 1992). Furthermore, the above hypothesized
mechanism would be specifically active in myotonic fibers,
since the healthy fibers have a large GCl and lack the metabolic alterations that facilitate opening of the KATP channels.
Other different mechanisms can also account for the effectiveness of compound II on the myotonic fibers at lower
concentrations than on healthy ones. It is possible that in the
healthy fiber, the compound acts with additive mechanisms
able to counteract the block of INas or, instead, that compound II has a very high affinity for inactivated sodium
channels, which would predominate in the high discharging
low-GCl fibers. For instance, riluzole, a novel psychotropic
agent with anticonvulsant properties, has been found to have
a high affinity for inactivated sodium channels despite its
weak use dependency (Hebert et al., 1994). Further investigation into the mechanisms underlying the effects of compound II, such as verification of its ability to open KATP
channels, and on its structure-activity relationship is of interest to improve the therapeutical potential of this mexiletine derivative. Finally, our results show that the newly
synthetized analogs of MEX have different features from the
parent compound and are of interest in the development of
more potent blockers of the skeletal muscle sodium channels
and potential antimyotonic agents.
Vol. 282