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Title Page

Differential block of sensory neuronal voltage-gated sodium channels by

lacosamide, lidocaine and carbamazepine.

Patrick L. Sheets, Cara Heers1, Thomas Stoehr, Theodore R. Cummins

Department of Pharmacology and Toxicology, Indiana University School of Medicine,

Indianapolis, Indiana (P.L.S., T.R.C.); Schwarz BioSciences, Department of

Pharmacology/Toxicology, GmbH, Monheim, Germany (C.H., T.S.).

JPET Fast Forward. Published on March 31, 2008 as DOI:10.1124/jpet.107.133413

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

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Running Title Page

Running Title: Inhibition of sensory sodium channels by lacosamide

Address correspondence to: Contact Information: Theodore Cummins, Ph.D.,

Department of Pharmacology and Toxicology, Indiana University School of Medicine,

950 West Walnut St., R2 468, Indianapolis, IN. Tel. (317) 278-9342, Fax (317) 278-

5849, E-mail: trcummin@iupui.edu

Document Statistics:

Number of text pages: 38

Number of tables: 2

Number of figures: 8

Number of references: 40

Number of words in Abstract: 246

Number of words in Introduction: 724

Number or words in Discussion: 1263

Abbreviations: TTX, tetrodotoxin; TTX-R, tetrodotoxin-resistant; TTX-S, tetrodotoxin-

sensitive; HEK, human embryonic kidney; DRG, dorsal root ganglion; DMSO, dimethyl

sulfoxide; HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);

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Abstract

Voltage-gated sodium channels play a critical role in excitability of nociceptors (pain-

sensing neurons). Several different sodium channels are thought to be potential targets

for pain therapeutics, including Nav1.7 which is highly expressed in nociceptors and

plays crucial roles in human pain and hereditary painful neuropathies, Nav1.3 which is

upregulated in sensory neurons following chronic inflammation and nerve injury, and

Nav1.8 which has been implicated in inflammatory and neuropathic pain mechanisms.

We compared the effects of lacosamide, a new pain therapeutic, with those of lidocaine

and carbamazepine on recombinant Nav1.7 and Nav1.3 currents and neuronal

tetrodotoxin-resistant (Nav1.8-type) sodium currents using whole-cell patch clamp

electrophysiology. Lacosamide is able to substantially reduce all three current types.

However, in contrast to lidocaine and carbamazepine, 1 mM lacosamide did not alter

steady-state fast-inactivation. Inhibition by lacosamide exhibited substantially slower

kinetics, consistent with the proposal that lacosamide interacts with slow inactivated

sodium channels. The estimated IC50 values for inhibition by lacosamide of Nav1.7,

Nav1.3 and Nav1.8-type channels following prolonged inactivation were 182, 415 and 16

µM, respectively. Nav1.7, Nav1.3, and Nav1.8-type channels in the resting state were

221, 123 and 257 fold less sensitive, respectively, to lacosamide than inactivated

channels. Interestingly, the ratio of resting to inactivated IC50’s for carbamazepine and

lidocaine were much smaller (ranging from 3 to 16). This suggests that lacosamide

should be more effective than carbamazepine and lidocaine at selectively blocking the

electrical activity of neurons that are chronically depolarized compared to those at more

normal resting potentials.

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Introduction

Neuropathic pain is a difficult type of pain to treat and it is estimated that as many

as half of all patients receive inadequate pain relief. More effective and better tolerated

treatments are needed. Lacosamide is a functionalized amino-acid that has shown

effectiveness as an anticonvulsant and analgesic and therefore might help fill this need

(Stohr et al., 2007; Beyreuther et al. 2007b). Lacosamide has shown efficacy in several

animal models of neuropathic pain (Beyreuther et al., 2006, 2007a) and is currently in

late stages of clinical development for epilepsy and diabetic neuropathic pain (Doty et al.

2007). Two modes of action have been identified for lacosamide, interaction with

collapsin-response mediator protein 2 and inhibition of voltage-gated sodium channel

activity by selective interaction with the slow inactivated conformation of the channel

(Beyreuther et al., 2007b, Errington et al. 2008). In this study we asked whether

lacosamide could inhibit voltage-gated sodium channels expressed in peripheral sensory

neurons and believed to be involved in pain mechanisms.

Voltage-gated sodium channels underlie the rapid action potentials characteristic

of neurons and muscle. They are transmembrane proteins with gated pores whose

opening and closing is controlled by changes in the voltage gradient across the membrane

(Catterall, 2000). The primary functional unit of the voltage-gated sodium channel is a

~240kD polypeptide α subunit. Nine distinct voltage-gated sodium channel α subunits

(Nav1.1-1.9) have been cloned from mammals (Goldin, 2002). Adult dorsal root ganglion

(DRG) sensory neurons can express multiple α subunits, including tetrodotoxin-sensitive

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(TTX-S) sodium channels (Nav1.1, Nav1.6 and Nav1.7) and tetrodotoxin-resistant

(TTX-R) sodium channels (Nav1.8 and Nav1.9).

Several sensory neuronal channels have been implicated in pain mechanisms. A

“nociceptor-specific” Nav1.7 knockout mouse showed significant deficits in responses to

inflammatory stimuli (Nassar et al., 2004) and humans that lack functional Nav1.7

channels exhibit a complete insensitivity to pain (Cox et al. 2006) with apparently no

other deficits, indicating that the loss of Nav1.7 activity selectively affects pain.

Additionally gain of function mutations in Nav1.7 cause two distinct inherited painful

neuropathies (Cummins et al., 2004; Drenth et al., 2005; Fertleman et al. 2006) and cause

hyperexcitability of DRG neurons (Rush et al., 2006; Sheets et al., 2007). These data

clearly indicate that Nav1.7 plays a crucial role in nociception. Nav1.3 might also play a

role in pain mechanisms. Nav1.3 is not normally expressed in adult sensory neurons but is

expressed following either chronic inflammation or nerve injury (Waxman et al. 1994;

Black et al. 2004) and has been connected to central neuropathic pain (Hains et al. 2003).

Based on these data, and because of its unique functional properties (Cummins et al.,

2001), Nav1.3 is hypothesized to play an important role in some types of pain. Two

distinct types of TTX-R currents (Nav1.8-type and Nav1.9-type) have been identified in

DRG neurons (Dib-Hajj et al., 1999; Cummins et al., 1999). Nav1.8 channels have been

implicated in both inflammatory (Akopian et al., 1999) and neuropathic pain (Lai et al.

2002) and selective block of Nav1.8 can alleviate both of these types of pain (Jarvis et al.,

2007). Nav1.8 currents are therefore also clearly of interest when considering the

mechanism of action of new pain therapeutics.

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All mammalian voltage-gated sodium channel α subunits have 4 domains, each

consisting of 6 transmembrane segments (S1-S6). The S6 segments are thought to be

important in binding local anesthetics such as lidocaine and anti-convulsants (Ragsdale

and Avoli, 1998; Clare et al., 2000). Many of the clinically relevant modulators exhibit a

pronounced state-dependent binding, where sodium channels that are rapidly and

repeatedly activated and inactivated are more readily blocked. This state-dependence is

thought to be very important in limiting the activity of cells exhibiting abnormal activity.

In a simplified scheme, voltage-gated sodium channels have four distinct states, open,

closed, fast-inactivated and slow-inactivated. The classic sodium channel modulators

such as lidocaine are believed to exhibit the highest affinity for the fast-inactivated state.

However, some studies have suggested that alteration of slow inactivation could be

clinically relevant too (Fukuda et al., 2005; Haeseler et al., 2006).

In this study we used whole-cell patch clamp electrophysiology to investigate the

effects of lacosamide on Nav1.7 and Nav1.3 voltage-gated sodium channels expressed in

HEK293 cells. In addition we studied the effects of lacosamide on Nav1.8-type TTX-R

currents from DRG neurons. We also compared the effects of lacosamide with those of

two common pain therapeutics, carbamazepine and lidocaine.

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Methods

Cell Culture

Human embryonic kidney 293 (HEK293) cells were grown under standard tissue

culture conditions (5% CO2; 37o C) in high-glucose DMEM (Invitrogen) supplemented

with 10% fetal bovine serum (Hyclone), 100 U/ml Penicillin and 100 µg/ml

Streptomycin. Two HEK293 cells lines were used. One line stably expressed the rat

Nav1.3 voltage-gated sodium channel (Cummins et al., 2001) and the other cell line

stably expressed the human Nav1.7 voltage-gated sodium channel (Cummins et al.,

1998). Stable cell lines were originally selected for using the antibiotic G418 (Geneticin;

Cellgro, Herndon, VA). No β subunits were transfected with the channels. At least 16

hours prior to electrophysiological recording experiments, the cells were harvested using

mechanical trituration and replated on 12mm glass covers slips.

DRG Culture

Adult male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) were

rendered unconscious by exposure to CO2 and decapitated. The L4 and L5 DRGs were

removed and incubated with a mixture of protease (12.5 mg/ml) and collagenase

(5 mg/ml) in bicarbonate-free DMEM for 40 minutes at 37 oC. The enzyme-treated

DRGs were then triturated using fire-polished glass pipettes, plated on poly-D-lysine

coated dishes, and maintained under standard tissue culture conditions (5% CO2; 37 oC)

in DMEM supplemented with 10% fetal bovine serum, 100 U/ml Penicillin and

100 µg/ml Streptomycin. TTX-R Nav1.8-type sodium currents were recorded from DRG

neurons 12-36 hours after plating.

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Whole-cell patch clamp recordings

Whole cell patch-clamp recordings were conducted at room temperature (~21oC)

using a HEKA EPC-10 amplifier. Data were acquired on a Windows-based Pentium IV

computer using the Pulse program (v 8.65, HEKA Electronic, Germany). Fire-polished

electrodes (0.9-1.3 MΩ) were fabricated from 1.7 mm capillary glass using a Sutter P-97

puller (Novato, CA, USA). The standard pipette solution contained (in mM): 140 CsF,

10 NaCl, 1.1 EGTA, 1 mM MgCl2 and 10 HEPES (4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid ), pH 7.3. The standard bathing solution contained (in

mM): 140 NaCl, 1 MgCl2, 3 KCl, 1 CaCl2, and 10 HEPES, pH 7.3 (adjusted with NaOH).

Series resistance errors were compensated to be less than 3 mV by using 80-85% series

resistance compensation and 1.0-1.5 MΩ pipettes. Cells on glass cover slips were

transferred into a recording chamber containing 250 µl of standard bathing solution. The

amplifier’s offset potential was zeroed with the electrode almost touching the cell of

interest. After obtaining a gigaseal, suction was used to establish the whole-cell

recording configuration. Cells were held at –80 mV for 3.5 minutes before initiating the

experimental protocols. Cells with less than 1 nA of peak sodium current were not used

for this study. Compounds were added to the bath compartment by first withdrawing 25

µl of bathing solution, then adding 25 µl of 10-fold concentrated compound and mixing

10-15 times with a 25 µl pipettor. Only one compound was tested on each cell, although

we typically tested two concentrations of a compound (without intervening washout) on

each cell. Cells were held at -80 mV for 2-3 minutes during initial application of a

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compound before running the specific voltage protocols described in the results section

and illustrated in the figures.

Chemicals and Solutions

Lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide) was

obtained from Schwarz Pharma (Monheim, Germany). A 500 mM solution was made up

in dimethyl sulfoxide (DMSO). A 25 mM stock solution was made from this by 20-fold

dilution into standard bathing solution (see above). This stock solution was aliquoted and

stored at –20 °C. Lidocaine hydrochloride was purchased from Sigma Aldrich Co., St.

Louis, MO. Lidocaine stock solutions were prepared fresh daily. A 100 mM stock

solution was prepared in standard bathing solution and stored on ice. Carbamazepine was

purchased from Sigma-Aldrich Co. (St. Louis, MO). A 250 mM solution was made up in

DMSO. Frozen aliquots of this stock solution were stored at –20 °C. Each of the stock

solutions were diluted into standard bathing solution to create10-fold concentrated

solutions of 30 mM, 10 mM, 3 mM, 1 mM, 300 µM, 100 µM, 30 µM and 10 µM . The

concentrated solutions were diluted 1:10 into the recording chamber and thoroughly

mixed during the experiments to provide final concentrations of 3 mM, 1 mM, 300 µM,

100 µM, 30 µM, 10 µM, 3 µM and 1 µM for each of the three compounds. The 10-fold

concentrated solutions were prepared fresh daily and stored on ice. In a separate set of

control experiments, DMSO at 0.5% (v/v) had negligible effects on the functional

properties of Nav1.3 (n=4) or Nav1.7 (n=5) currents (see supplemental Figures 1 and 2) .

Data Analysis

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Voltage-clamp experimental data were analyzed using the Pulsefit (v 8.65, HEKA

Electronic, Germany) and Origin (OriginLab Corp., Northhampton, MA, USA) software

programs. Slope factors of steady-state inactivation curves were calculated using the

general Boltzmann function: I(V) = Offset + (Amplitude/1 + exp(-(V-Vhalf)/Slope). To

estimate inhibition potencies, dose response curves were fit using the Hill function:

fi = 1 – (fmax) / (1 + ([drug] / IC50)h) where fi is the fraction inhibited, fmax is the maximum

fraction that can be inhibited and h is the Hill coefficient. Error bars in the figures

represent the S.E.M. Data were assumed to be normally distributed and unpaired

Student’s t-test was used for statistical comparisons.

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Results

Inhibition of Nav1.7 and Nav1.3 currents by lacosamide

In the initial set of experiments, the ability of 30 and 100 µM lacosamide to

inhibit sodium currents carried by Nav1.7 and Nav1.3 channels was tested by holding the

cells at –80 mV, running a current-voltage (IV) protocol, applying the test concentration

of lacosamide, then running a second IV protocol to determine the effect of lacosamide

on peak current amplitude and on the voltage-dependence of current activation. The IV

protocol consisted of giving 50 ms step depolarizations to voltages ranging from –80 to

+40 mV in 5 mV increments and recording the sodium currents elicited during each step

depolarization. Figure 1A shows an IV family of Nav1.7 currents recorded before and

after application of 30 µM lacosamide. The peak current elicited by each step

depolarization is measured and can be plotted versus the voltage. Figure 1B shows the

IV data from Nav1.7 cells (n=3) before and after 30 µM lacosamide (normalized to the

peak current measured before lacosamide). Lacosamide reduced the peak Nav1.7

currents obtained from a –80 mV holding potential by 30%. 100 µM lacosamide reduced

peak Nav1.7 currents by almost 60% (Figure 1C; n=3). By contrast, Nav1.3 currents

elicited from a holding potential of -80 mV were less sensitive to lacosamide, exhibiting

only an 11% decrease with application of 30 µM lacosamide (Figure 1D; n=4) and 25%

decrease with application of 100 µM lacosamide (Figure 1E; n=3). Lacosamide (100

µM) did not significantly alter the voltage-dependence of activation for either Nav1.7 or

Nav1.3 currents.

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Comparison of lacosamide’s effects to lidocaine and carbamazepine effects

Inhibition of resting (closed) channels. Lidocaine and carbamazepine exhibit

differential binding to resting (or closed) channels and inactivated channels. We

examined the ability of lacosamide, lidocaine, and carbamazepine to inhibit resting

channels by holding cells expressing Nav1.7 and Nav1.3 currents at –120 mV for

10 seconds (to allow all channels to move to the resting state) and then eliciting currents

with a 20 ms step depolarization to 0 mV. Peak sodium current amplitude was measured

before and after application of lacosamide (1 to 3000 µM; n=3-7), lidocaine (1to 3000

µM; n=3-4), and carbamazepine (1 to 3000 µM; n=3-4). Fits to the concentration

response curves estimate that the IC50 of resting Nav1.7 channels is 0.7 mM for lidocaine,

1.6 mM for carbamazepine and roughly 40 mM for lacosamide (Figure 2A, Table 1). Fits

to the concentration response curves estimate that the IC50 of resting Nav1.3 channels is

1.5 mM for lidocaine, 2.5 mM for carbamazepine, and roughly 51 mM for lacosamide

(Figure 2B, Table 1). These data indicate that lacosamide has a much higher IC50 for

resting Nav1.3 and Nav1.7 channels than either carbamazepine or lidocaine. Although the

estimates of the affinity of resting Nav1.7 and Nav1.3 channels for lacosamide should

only be considered as very rough estimates due to lack of data at higher concentrations, at

1mM, lidocaine and carbamazepine inhibit Nav1.3 and Nav1.7 to a significantly (p <

0.0005) greater extent than 1 mM lacosamide.

Inhibition of inactivated channels. Next, we examined the ability of

lacosamide, lidocaine, and carbamazepine to interact with inactivated channels by

holding cells expressing Nav1.7 currents at –50 mV for 10 seconds (to allow all channels

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to move to inactivated states), then stepping the cells to –120 mV for 100ms (to allow

channels without drug bound to recover from fast-inactivation; Cummins and Waxman

1997; Cummins et al., 2001) and finally eliciting currents with a 20 ms step

depolarization to 0 mV to estimate channel availability (Figure 3C). Peak sodium current

amplitude was measured before and after application of lacosamide (1 to 3000 µM; n=3-

7), lidocaine (1 to 3000 µM; n=3-4) and carbamazepine (1 to 3000 µM; n=3-4). The

concentration response curve for inhibition of inactivated Nav1.7 channels is shown in

Figure 3A. The concentration response curve for inactivated Nav1.3 channels (Figure

3B) was similarly obtained, with the exception that we used a 10 second step to –40 mV

(due to differences in steady-state inactivation; see Figure 4) with the cells expressing

Nav1.3 currents. The different inactivation prepulse potentials were chosen to achieve

comparable extents of inactivation (a 500 ms prepulse to -40 mV inactivates 97.5% of

Nav1.3 channels and a 500 ms prepulse to -50 mV inactivates 96.7% of Nav1.7 channels).

Fits to the concentration response curves estimate that the IC50 for inhibition of

inactivated Nav1.7 channels is 44 µM for lidocaine, 406 µM for carbamazepine, and 182

µM for lacosamide. Fits to the concentration response curves estimate that the IC50 for

inhibition of inactivated Nav1.3 channels is 284 µM for lidocaine, 900 µM for

carbamazepine, and 415 µM for lacosamide. Table 1 shows the parameters for the Hill

equation fits. These data indicate that 1) lacosamide has a much higher affinity for

inactivated Nav1.7 and Nav1.3 channels than for resting Nav1.7 and Nav1.3 channels and

2) inactivated Nav1.3 channels are less sensitive to all three compounds than inactivated

Nav1.7 channels.

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Effects on voltage-dependence of steady-state fast and slow inactivation

Our data indicated that lacosamide, like lidocaine and carbamazepine, exhibits

preferential binding to inactivated channels. We asked whether lacosamide would

enhance fast inactivation of Nav1.7 and Nav1.3 channels in the same manner as lidocaine

and carbamazepine. Therefore we measured the effect of lacosamide, lidocaine, and

carbamazepine on the voltage-dependence of steady-state fast and slow inactivation. We

used 1 mM for each of the compounds in order to help maximize the binding of the

compound to the channels. Higher concentrations were not used for this as solubility

becomes a problem, especially with carbamazepine. Cells expressing Nav1.7 or Nav1.3

channels were held at -120 mV, stepped to inactivating prepulse potentials ranging from –

150 to –30 mV (in 10 mV increments) for 500 ms before stepping the cells to 0 mV for

20 ms to measure the available current (Figure 4C). In order to eliminate the bias of time

dependent shifts in the voltage-dependence of fast inactivation we compared data

recorded 4 minutes after establishing the whole-cell recording configuration from control

cells (in the absence of test compound; n=5) to data recorded 4 minutes after establishing

the whole-cell recording configuration from cells exposed to one of the three test

compounds (n=5-8). Both lidocaine and carbamazepine (1 mM) shifted the voltage-

dependence of steady-state fast inactivation by roughly 20 mV in the negative direction

for both Nav1.7 (Figure 4A) and Nav1.3 (Figure 4B) currents. The magnitude of the shift

in the presence of both lidocaine and carbamazepine is significant for both Nav1.3 and

Nav1.7 compared to control cells (P < 0.005). However, lacosamide (1 mM) did not

affect the voltage-dependence of steady-state fast inactivation for either Nav1.7 or Nav1.3

currents.

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Our data demonstrates that although lacosamide seems to exhibit preferential

binding to inactivated channels, this was not seen with 500 ms prepulses. Therefore we

next measured the effect of lacosamide, lidocaine, and carbamazepine (all at 1 mM) on

the voltage-dependence of steady-state slow inactivation. Cells expressing Nav1.7 or

Nav1.3 channels were held at –120 mV, stepped to inactivating prepulse potentials

ranging from –140 to 0 mV (in 10 mV increments) for 10 s, hyperpolarized to –160 mV

for 100 ms, then stepped to 0 mV for 20 ms to measure the available current (Figure 4F).

The brief hyperpolarization was used to allow channels (both with and without drug

bound) to recover from fast inactivation while limiting the recovery from slow

inactivation. In order to eliminate the bias of time dependent shifts in the voltage-

dependence of fast inactivation we compared data recorded 4 minutes after establishing

the whole-cell recording configuration from control cells (in the absence of test

compound; n=5) to data recorded 4 minutes after establishing the whole-cell recording

configuration from cells exposed to one of the three test compounds (n=5-8).

Carbamazepine did not alter the voltage-dependence of steady-state slow inactivation for

either Nav1.7 (Figure 4D) or Nav1.3 (Figure 4E) currents. However, both lacosamide and

lidocaine shifted the apparent voltage-dependence of steady-state slow inactivation for

both Nav1.7 and Nav1.3 currents.

Rate of development of inhibition

In order to better understand the interaction between lacosamide and Nav1.7 and

Nav1.3 sodium channels, we investigated the time course for development of block by

1 mM lacosamide, lidocaine and carbamazepine. Development of block was examined

by holding the cells (n=5-7 for each of the four conditions) expressing Nav1.7 (Figure

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5A) or Nav1.3 (Figure 5B) at –120 mV in the presence of one of the three compounds,

prepulsing the cells to 0 mV for varying amounts of time to allow block to develop,

hyperpolarizing the cells to –120 mV for 20 ms to allow unbound channels to recover

from fast inactivation, then stepping the cells to 0 mV for 20 ms to determine the fraction

of channels available for activation (Figure 5C). In control cells the reduction in the

fraction of available current represents the time course for the development of slow

inactivation. The time course for reduction of channel availability in the presence of

lidocaine and carbamazepine was clearly biphasic, with a fast component that likely

represents development of block of fast-inactivated channels and a slow component that

was consistent with the time course for development of slow inactivation in control cells.

The time course for reduction in channel availability in the presence of lacosamide

showed a different pattern. The rate of development of block was 4-7 fold faster than the

time course for development of slow inactivation, but the shape of the curve was very

similar to that for slow inactivation.

Recovery from inactivation/block

We also examined the rate at which Nav1.3 and Nav1.7 channels recovered from

inactivation and/or block by lidocaine, carbamazepine, and lacosamide (all at 1 mM;

n = 5-6 for each condition). In the first set of experiments cells were held at –120 mV,

depolarized to 0 mV for 10 ms, then hyperpolarized to –120 mV for variable increasing

durations before testing the available current with a step depolarization to 0 mV (Figure

6C). The time course for recovery from the 10 ms depolarization was not different from

control for currents recorded in the presence of 1 mM lacosamide, but the time course

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was more complex and slower in the presence of lidocaine and carbamazepine, indicating

that these two compounds can rapidly alter Nav1.7 (Figure 6A) and Nav1.3 (Figure 6B)

channel activity. In the second set of experiments cells (n=5-9) were held at –120 mV,

depolarized to 0 mV for 5 s, then hyperpolarized to –120 mV for a variable duration

before testing the available current with a step depolarization to 0 mV. The 5 s

depolarization provides time for the channels to undergo slow inactivation. The time

course for recovery from the 5 s depolarization was not different from control for currents

recorded in the presence of 1 mM lidocaine and carbamazepine. However, the time

constant for recovery from the 5 s prepulse was significantly greater in the presence of

1 mM lacosamide (P < 0.01 at 500 ms; Figure 6D,E).

Effects on TTX-R current

We wanted to examine the effects of lacosamide, carbamazepine and lidocaine on

Nav1.8 currents and determine how the effects compared with those on Nav1.3 and

Nav1.7 currents. Nav1.8 currents are very difficult to record from HEK293 cells,

therefore we recorded Nav1.8-type currents from rat L4/L5 DRG neurons 12-18 hours

after being cultured. Two distinct types of TTX-R currents (Nav1.8-type and Nav1.9-

type) have been identified in DRG neurons (Akopian et al., 1996; Dib-Hajj et al., 1999;

Cummins et al., 1999). We focused on Nav1.8 currents as its role in pain is much better

understood than that of Nav1.9 and because it is difficult to obtain stable recordings of

Nav1.9 currents, even in DRG sensory neurons (Leffler et al., 2005). In our recordings

we isolated Nav1.8-type currents by using 500 nM TTX to eliminate TTX-S currents and

a holding potential (-80 mV) which is efficient at eliminating the persistent TTX-R

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current carried by Nav1.9 (Cummins et al., 1999, Priest et al. 2005, Amaya et al. 2006).

The effects of lacosamide, carbamazepine, and lidocaine were tested on resting and

inactivated channels. Peak sodium current amplitude was measured before and after

application of drug (300 µM), and projected IC50 values from our results using the

equation: (% remaining current)/((1-% remaining current) x [drug]). As before, resting

channels were tested by holding the neurons at -80 mV and pulsing to –120 mV for 10

seconds (to allow all channels to move to the resting (closed state) and then eliciting

currents with a 20 ms step depolarization to 0 mV. Using this protocol we observed

decreases of 7.3 ± 4.3% (n = 6), 26.4 ± 3.2% (n = 4), 48.6 ± 7.1% (n = 5) for lacosamide,

carbamazepine, and lidocaine, respectively (Figure 7A). Based on these values, the

estimated IC50 values of lacosamide, carbamazepine, and lidocaine for resting Nav1.8-

type TTX-R channels are 4 mM, 840 µM, and 320 µM, respectively.

To test the ability of lacosamide, lidocaine, and carbamazepine to inhibit

inactivated Nav1.8-type TTX-R channels, we pulsed the neurons to –20 mV (which

produces ~95% inactivation) for 10 seconds (to allow all channels to move to the

inactivated state and to allow for drug binding to reach equilibrium), then stepped the

cells to –120 mV for 100ms (to allow channels without drug bound to recover from

inactivation; Cummins and Waxman, 1997) and finally elicited currents with a 20 ms step

depolarization to 0 mV to estimate channel availability. Peak sodium current amplitudes

were measured before and after application of lacosamide (n = 6), carbamazepine (n = 8),

or lidocaine (n = 9), all at a concentration of 300 µM. Using this protocol, we observed

decreases of 95.1 ± 1.3%, 68.5 ± 5.1%, 89.1 ± 2.6% for lacosamide, carbamazepine, and

lidocaine, respectively. Based on these values, we estimate that the IC50 for inhibition of

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inactivated Nav1.8-type TTX-R channels is 16 µM for lacosamide, 138 µM for

carbamazepine, and 37 µM for lidocaine. Figure 7B shows the comparison of current

inhibition on inactivated Nav1.7, Nav1.3, and Nav1.8-type channels by all three drugs at

concentrations of 300 µM. In this case each channel subtype was held at different pre-

pulse potentials, due to differences in their voltage dependence of steady-state

inactivation, to ensure >95% inactivation. Figure 7C shows the comparison of current

inhibition of Nav1.7, Nav1.3, and Nav1.8-type channels held at -50 mV, which would

simulate a slightly depolarized potential for a sensory neuron. Lacosamide inhibition at –

50 mV was similar for Nav1.8-type, Nav1.7 and Nav1.3 currents. Lidocaine inhibition at

–50 mV was also similar for all three types of currents. However, differences were still

observed between the current subtypes for carbamazepine inhibition at -50 mV.

We further investigated the effects lacosamide, lidocaine, and carbamazepine on

Nav1.8-type channels by looking at their effects on the voltage-dependence of steady-

state fast and slow inactivation. We used 1 mM for each of the compounds in order to

help maximize the interaction of the channels with the compound. Neurons were held at

-80 mV, stepped to inactivating prepulse potentials ranging from –100 to 30 mV (in

10 mV increments) for 500 ms before stepping the cells to 0 mV for 20 ms to measure the

available current. In order to eliminate the bias of time dependent shifts in the voltage-

dependence of fast inactivation we compared data recorded 3 minutes after establishing

the whole-cell recording configuration from control cells (in the absence of test

compound; n = 9) to data recorded 3 minutes after establishing the whole-cell recording

configuration from cells exposed to one of the three test compounds (n = 5-7).

Surprisingly, none of the drugs altered the voltage-dependence of steady-state fast

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inactivation (Figure 8A). The estimated V1/2 of steady-state fast inactivation (obtained

with Boltzmann fits) of Nav1.8-type current in the presence of lacosamide (-33.4 ±

2.0 mV; slope: 3.5 ± 0.1; n = 5), lidocaine (-39.5 ± 2.5 mV; slope: 4.5 ± 0.2, n = 7), and

carbamazepine (-37.4 ± 2.5 mV; slope: 4.1 ± 0.2, n = 6) was not statistically different

from that of control Nav1.8-type current (-34.7 ± 1.3 mV; slope: 4.0 ± 0.1; One-way

ANOVA analysis).

To study the effects of these drugs on Nav1.8-type current slow inactivation, we

stepped to inactivating prepulse potentials ranging from –100 to 30 mV (in 10 mV

increments) for 10 s, hyperpolarized to –140 mV for 100 ms, then stepped to 0 mV for

20 ms to measure the available current. Effects were observed with all of the drugs on

the steady-state slow inactivation curve of Nav1.8-type currents (Figure 8B). The

estimated V1/2 of steady-state slow inactivation of Nav1.8-type current in the presence of

lacosamide (-54.8 ± 3.2 mV; slope: 5.2 ± 0.2, n = 6), lidocaine (-57.9 ± 2.6 mV; slope:

5.1 ± 0.2, n = 7), and carbamazepine (-53.1 ± 3.2 mV; slope: 4.4 ± 0.1, n = 6) were

significantly (p < 0.001) hyperpolarized from that of control Nav1.8-type current (-37.1 ±

1.2 mV; slope: 10.4 ± 0.1; n = 9; One-way ANOVA analysis). In addition, the slope of

the steady-state slow inactivation curve for Nav1.8-type current was significantly (p <

0.001; One-way ANOVA analysis) different from the slope values observed in the

presence of lacosamide, lidocaine, or carbamazepine. Both lacosamide and lidocaine

markedly increased the maximal extent to which slow inactivation can reduce the

availability of Nav1.8, i.e. at 20 mV reduction of channel availability for control and

carbamazepine reached a maximum of about 50%, whereas it amounted to 80% and 90%

for lidocaine and lacosamide respectively.

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Discussion

This study was aimed at determining the inhibitory effects of a new drug,

lacosamide, on voltage-gated sodium channels that are expressed in peripheral

nociceptive neurons and that have been identified as playing important roles in pain. We

focused our efforts on examining the effects of lacosamide on Nav1.7 and Nav1.3

channels and comparing them to clinically used traditional analgesics carbamazepine and

lidocaine. We found clinically relevant concentrations of lacosamide substantially reduce

Nav1.7 current amplitudes at a holding potential of –80 mV, while Nav1.3 currents were

inhibited to a slightly lesser extent, indicating that they are less sensitive. We found that

lacosamide has a significantly higher IC50 for resting Nav1.3 and Nav1.7 channels than

either carbamazepine or lidocaine. Lacosamide exhibited substantially greater inhibition

of inactivated Nav1.7 and Nav1.3 channels than resting Nav1.7 and Nav1.3 channels.

Lacosamide is more potent at inhibiting inactivated channels than carbamazepine but

slightly less potent than lidocaine. These data show lacosamide has a much greater

ability to discriminate between resting and inactivated Nav1.7 and Nav1.3 channels than

either lidocaine or carbamazepine.

The effects of lacosamide on Nav1.8-type currents in DRG neurons were similar

to that seen with Nav1.7 and Nav1.3 as lacosamide inhibited inactivated Nav1.8-type

channels to a significantly greater extent then resting channels and suggest lacosamide is

the most potent of the three drugs at inhibiting inactivated Nav1.8-type channels.

Although the ratio of lacosamide resting IC50 to the –50 mV IC50 was smaller for Nav1.8-

type channels than for Nav1.3 or Nav1.7 channels, this ratio was still greater for

lacosamide than for either lidocaine or carbamazepine (Table 2). These data, showing

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that lacosamide is much more effective at distinguishing between resting and inactivated

Nav1.7, Nav1.3, and Nav1.8-type channels than lidocaine and carbamazepine, suggest

lacosamide should be selective at inhibiting the activity of neurons with depolarized

membrane potentials compared to neurons with normal resting membrane potentials than

either lidocaine or carbamazepine. This could be an important part of the analgesic

mechanism of action of lacosamide as nociceptor hyperexcitability and increased pain

sensations are likely to be associated with depolarized membrane potentials in neurons.

The differential effects of lacosamide, carbamazepine, and lidocaine on the

voltage-dependence of steady-state fast inactivation and steady-state slow inactivation are

intriguing. These data showed that the inhibition induced by the three drugs was

different depending on the length of the depolarizing pulse used to inactivate the

channels. When using 500 ms prepulses, we observed a significant hyperpolarizing shift

in the voltage-dependence of steady-state fast inactivation for Nav1.7 and Nav1.3 treated

with carbamazepine and lidocaine but not when treated with lacosamide. In contrast

while carbamazepine had no effect on the voltage-dependence of slow inactivation for

Nav1.7 and Nav1.3, lacosamide and lidocaine both produced a significant hyperpolarizing

shift in the voltage-dependence of slow inactivation of these channels. These results

suggest while carbamazepine and lidocaine interact with fast-inactivated Nav1.7 and

Nav1.3 channels, lacosamide does not. Our data are consistent with the proposal that

lacosamide interacts predominantly with slow inactivated sodium channels (Errington et

al., 2008) and that this effect could be a mechanistic rather than a kinetic difference

compared to lidocaine and carbamazepine. However, based on these data we cannot rule

out the alternative possibility that lacosamide may bind and unbind very slowly from

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fast-inactivated channels. Mutagenesis experiments that selectively alter fast- or slow-

inactivation of the sodium channels should help definitively determine the mechanism of

action for lacosamide. Interestingly, none of the 3 drugs had any effect on the voltage-

dependence of Nav1.8-type channel fast inactivation. This result is not surprising for

lacosamide due to its ineffectiveness on fast-inactivation of Nav1.7 and Nav1.3 channels,

but is somewhat surprising for lidocaine and carbamazepine. However, previous studies

have indicated that lidocaine does not alter fast-inactivation of Nav1.8 channels in the

same manner that it does Nav1.7 channels (Chevrier et al., 2004) and that carbamazepine

might even preferentially target slow inactivated Nav1.8 channels (Cardenas et al., 2006).

This raises the possibility that access to the drug-binding site in fast-inactivated state of

Nav1.8-type channels might be substantially different from the site in Nav1.7 and Nav1.3

channels.

An interesting finding was that carbamazepine and lidocaine show significant

inhibition of Nav1.7 and Nav1.3 channels prepulsed to 0 mV for durations ranging from 3

to100 ms (Figure 5A and 5B). In that same range of prepulse durations lacosamide

induces minimal inhibition of Nav1.7 and Nav1.3 channels. At prepulse durations ranging

100ms to 11s, lacosamide induces inhibition of Nav1.7 and Nav1.3 while inhibition by

carbamazepine and lidocaine levels off. All three drugs eventually reached maximal

inhibition around 9 to 11s for both channels. Interestingly, the time point where

development of slow inactivation starts to occur for control channels is near the time

point at which lacosamide begins to cause inhibition and also where carbamazepine and

lidocaine inhibition plateaus. This suggests lacosamide interaction with Nav1.7 and

Nav1.3 channels is dependent on the channels transitioning to slow inactivated states and,

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in contrast, that transition to the slow inactivated state might impede further Nav1.7 and

Nav1.3 channel inhibition by carbamazepine and lidocaine. While recovery from slow

inactivation appeared to be slower in the presence of lacosamide, due to an increase in the

steady state fraction of slow inactivated channels by lacosamide, it was not altered by

lidocaine or carbamazepine, providing additional evidence that the mechanism of block

by lacosamide is possibly distinct from that of lidocaine or carbamazepine.

Lacosamide has been shown in acute, inflammatory and neuropathic animal pain

models (Beyreuther et al. 2006, 2007a; Hao et al., 2006), as well as in phase II clinical

trials for painful diabetic neuropathy (Rauck et al., 2007), to effectively inhibit pain with

few adverse side-effects. Based on the data reported here, it is likely lacosamide’s effects

on sodium channel activity contribute to its analgesic activity. Tissue injury and

inflammatory modulators can cause prolonged depolarization of peripheral nociceptors

and this likely contributes to inflammatory and neuropathic pain. A recent study suggests

that the effectiveness of drugs at inhibiting peripheral nerve activity following nerve

injury does not depend so much on the frequency at which action potentials are

generated, but rather on the nature of the injury and the pattern of action potential activity

(Ritter et al., 2007). Since a substantial degree of slow inactivation can be observed for

TTX-R sodium channels at small, subthreshold membrane depolarizations without any

spiking activity (Blair and Bean, 2003), a compound like lacosamide which targets the

slow inactivated channel might show enhanced therapeutic benefit.

CNS side-effects can limit the usefulness of sodium channel blockers in treating

pain and therefore drugs that are better able to differentiate between normal and abnormal

activity are desired. The enhanced ability of lacosamide to discriminate between resting

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channels and channels that have been subjected to prolonged depolarizations should

contribute to a better safety profile and increased tolerability.

Overall, our data show lacosamide is effective at blocking inactivated voltage-

gated Nav1.3, Nav1.7 and Nav1.8-type channels, voltage-gated sodium channels that have

been implicated as playing important roles in pain mechanisms. Furthermore our data

show that the kinetics of lacosamide inhibition is much slower than that of two other

sodium channel blockers, carbamazepine and lidocaine, at least for Nav1.3 and Nav1.7

channels. Our data are consistent with the proposal that lacosamide inhibition of voltage-

gated sodium channels involves selective interactions with slow-inactivated channels

(Errington et al. 2008). Ratios of resting IC50 to inactivated IC50 suggest lacosamide

should be more effective than carbamazepine and lidocaine at selectively blocking the

electrical activity of neurons that are chronically depolarized while having little or no

effect on neurons with normal resting potentials and we propose that this could contribute

to the therapeutic action of lacosamide.

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Footnotes

This work was supported by Schwarz BioSciences. 1Current address: Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany

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Legends for Figures Figure 1: A, Current-voltage (IV) family trace of an HEK293 cell stably expressing the

human Nav1.7 channel before (left) and after (middle) application of 30 µM lacosamide

(LCM). The voltage protocol is shown on the right. B and C, IV relationship of Nav1.7

current in the absence and presence of 30 µM (B) and 100 µM (C) lacosamide. D and E,

IV relationship of Nav1.3 current in the absence and presence of 30 µM (D) and 100 µM

(E) lacosamide.

Figure 2: A, Concentration-response curve for the inhibitory effects of lacosamide

(LCM), lidocaine, and carbamazepine (CBZ) on resting Nav1.7 channels. B,

Concentration-response curve for the inhibitory effects of lacosamide (LCM), lidocaine,

and carbamazepine (CBZ) on resting Nav1.3 channels. C, The voltage protocol for testing

current amplitude is shown.

Figure 3: A, Concentration-response curve for the inhibitory effects of lacosamide

(LCM), lidocaine, and carbamazepine (CBZ) on inactivated Nav1.7 channels. B,

Concentration-response curve for the inhibitory effects of lacosamide (LCM), lidocaine,

and carbamazepine (CBZ) on inactivated Nav1.3 channels. C, The voltage protocol for

testing current amplitude is shown.

Figure 4: Effects of lacosamide, lidocaine, and carbamazepine on the voltage-

dependence of steady-state fast and slow inactivation for Nav1.7 and Nav1.3 channels.

A and B, Lidocaine and carbamazepine (CBZ) caused a significant hyperpolarizing shift

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in the voltage-dependence of steady-state fast inactivation for Nav1.7 (A) and Nav1.3 (B)

channels. Lacosamide (LCM) had no effect on the voltage-dependence of steady-state

fast inactivation for either channel. Midpoints of Nav1.7 steady-state inactivation in (A)

were –76.1±1.9 (n=6), -99.3±1.5 (n=7), -97.0±3.3 (n=5), -78.7±1.2 (n=6) for control,

+lidocaine, +carbamazepine, and +lacosamide, respectively. Midpoints of Nav1.3

steady-state inactivation in (A) were –62.1±2.7 (n=9), -82.5±1.6 (n=9), -81.0±2.6 (n=8),

-65.4±1.8 (n=11) for control, +lidocaine, +carbamazepine, and +lacosamide, respectively.

C, Voltage protocol for steady-state fast inactivation is shown. Data sweeps were

acquired at 0.2 Hz. D and E, Lidocaine and lacosamide (LCM) caused a significant

hyperpolarizing shift in the voltage-dependence of slow inactivation for Nav1.7 (D) and

Nav1.3 (E) channels. Carbamazepine (CBZ) had no effect on the voltage-dependence of

slow inactivation for either channel. F, Voltage protocol for steady-state slow

inactivation is shown. The 100 ms pulse at –160 mV is used to allow for recovery of

fast-inactivated and drug-bound fast-inactivated channels, but not slow-inactivated or

drug-bound slow-inactivated channels. Data sweeps were acquired at 0.09 Hz.

Figure 5: Effects of lacosamide, lidocaine, and carbamazepine on the development of

slow inactivation/inhibition for Nav1.7 and Nav1.3 channels. For the control channels, a

brief (20 ms) hyperpolarizing pulse to -120 mV was given after the inactivating pulse to

0 mV at increasing durations. A and B, Lidocaine and carbamazepine (CBZ) caused an

increase in the rate of development of inactivation/inhibition at pre-pulse durations

ranging from 1 to 100 ms before slowing at pre-pulse durations greater than 100 ms for

both Nav1.7 (A) and Nav1.3 (B) channels. An increase in the rate of development of

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inactivation/inhibition for both Nav1.7 (A) and Nav1.3 (B) channels was observed in the

presence of lacosamide (LCM) but at pre-pulse durations longer than those seen with

lidocaine and carbamazepine. C, The voltage protocol is shown. Following a variable

conditioning pulse to 0 mV, a 20 ms pulse to –120 mV allows recovery from fast

inactivation (but not block) before the fraction of current available with the 20 ms pulse

to 0 mV. Data sweeps were acquired at 0.5 Hz for short pre-pulse durations and at

slower rates for the longer pre-pulse durations.

Figure 6: Effects of lacosamide, lidocaine, and carbamazepine on the recovery of Nav1.7

and Nav1.3 channels from inactivation. Cells were held at –120 mV, depolarized to 0 mV

for 10 ms or 5s, then hyperpolarized to –120 mV for variable increasing durations before

testing the available current with a step depolarization to 0 mV. A and B, Lidocaine and

carbamazepine (CBZ) caused an decrease in the rate of recovery from

inactivation/inhibition (10 ms) for both Nav1.7 (A) and Nav1.3 (B) channels while

lacosamide (LCM) had no effect. C, The voltage protocol used for (A) and (B) is shown.

Data sweeps were acquired at 0.5 Hz. D and E, Lidocaine and carbamazepine (CBZ) did

not alter recovery kinetics after induction of slow inactivation compared to control. In the

presence of lacosamide (LCM) the fraction of available channels after 5 s depolarization

was lower compared to control, lidocaine and carbamazepine and the duration of

recovery longer (significant at 500 ms) compared to the other conditions. F, The voltage

protocol used for (D) and (E) is shown. Data sweeps were acquired at 0.5 Hz for short

recovery durations and at slower rates for the longer recovery durations.

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Figure 7: Comparison of inhibition on resting and inactivated Nav1.7, Nav1.3, and TTX-

R channels by lacosamide, lidocaine, and carbamazepine (all at 300 µM). A, Lacosamide

(LCM) had the smallest inhibitory effect on resting Nav1.7, Nav1.3, and TTX-R channels

compared to lidocaine and carbamazepine. Lidocaine and carbamazepine (CBZ) had the

greatest inhibitory effect on resting TTX-R channels compared to Nav1.7 and Nav1.3

channels. B, Effects of lacosamide (LCM), lidocaine, and carbamazepine (CBZ) on

completely inactivated Nav1.7, Nav1.3, and TTX-R channels. For complete inactivation,

the voltage of the inactivating pre-pulse was varied due to differences in the voltage-

dependence of inactivation between channels. Nav1.7 channels were pre-pulsed to -50

mV, Nav1.3 channels to -40 mV, and TTX-R channels to -20 mV. Lacosamide showed

considerably more inhibition on TTX-R channels compared to Nav1.7 and Nav1.3

channels. Lidocaine and carbamazepine were effective at inhibiting TTX-R channels

more than Nav1.3 channels but not Nav1.7 channels. C, Effects of lacosamide (LCM),

lidocaine, and carbamazepine (CBZ) on Nav1.7, Nav1.3, and TTX-R channels pre-pulsed

to -50 mV. This pre-pulse voltage was chosen to theoretically represent a slightly

depolarized potential for a sensory neuron. Lacosamide and lidocaine showed

comparable inhibition on TTX-R channels, Nav1.7, and Nav1.3 channels. Carbamazepine

displayed more inhibition on TTX-R channels compared to Nav1.3 channels. Values and

statistical significance was determined using a one-way ANOVA followed with a

Tukey’s means comparison test (** = p < 0.01, *** = p < 0.001).

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Figure 8: Effects of lacosamide, lidocaine, and carbamazepine (all at 1 mM) on the

voltage-dependence of steady-state fast and slow inactivation for TTX-R channels. A,

Representative family of Nav1.8-type currents recorded from a small diameter DRG

neuron. B, Lacosamide (LCM), lidocaine, and carbamazepine (CBZ) had no effect on

the voltage-dependence of steady-state fast inactivation for TTX-R channels. Data were

acquired at 0.2 Hz. C, Lacosamide (LCM), lidocaine, and carbamazepine (CBZ) caused a

significant hyperpolarizing shift in the voltage-dependence of slow inactivation for TTX-

R channels. Data were acquired at 0.09 Hz. Lacosamide caused the greatest decrease in

elicited TTX-R current followed by lidocaine and carbamazepine, respectively. At

20 mV lacosamide and lidocaine reduced available current by 90 and 80%, whereas

maximal reduction by carbamazepine at this voltage was similar to control (about 50%).

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Table 1. Hill equation fit parameters to dose response relationships in Figures 2 and 3. Resting Inhibition (Figure 2) Inactivated Inhibition (Figure 3) fmax IC50 h R2 fmax IC50 h R2 Nav1.3 Lidocaine .997 1462 µM 1.35 .99 .949 284 µM .48 .95 Carbamazepine .996 2464 µM 1.10 .97 .942 900 µM .89 .95 Lacosamide .998 51,200 µM 1.0 .96 .986 415 µM .61 .98 Nav1.7 Lidocaine .994 718 µM 1.02 .99 .992 44 µM .43 .95 Carbamazepine 1.0 1584 µM 1.0 .96 .94 406 µM 1.12 .96 Lacosamide 1.0 40,300 µM .78 .95 .954 182 µM .90 .974

R2 is the square of the correlation between the fit and the actual inhibition values and is an indication of the goodness of the fit.

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Table 2. Ratio of IC50 for closed channels to IC50 at depolarized potentials Nav1.3

at -40 mV Nav1.3 at -50 mV

Nav1.7 at -50 mV

Nav1.8-type at -50 mV

Nav1.8-type at -20 mV

Lacosamide 123 130 221 16 250 Lidocaine 5 5 16 3 9 Carbamazepine 3 3 4 7 6