A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain.

Abstract

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.

Full text

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A novel slow-inactivation-specific ion channel modulator attenuates
neuropathic pain
Michael E. Hildebranda, Paula L. Smitha, Chris Bladenb, Cyrus Eduljeea, Jennifer Y. Xiec, Lina Chenb,
Molly Fee-Makia, Clint J. Doeringb, Janette Mezeyovaa, Yongbao Zhua, Francesco Belardettia,
Hassan Pajouhesha, David Parkera, Stephen P. Arnerica, Manjeet Parmara, Frank Porrecac,
Elizabeth Tringhama, Gerald W. Zamponib, Terrance P. Snutcha,d,⇑
aZalicus Pharmaceuticals, 301-2389 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3
bDepartment of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada T2N 4N1
cDepartment of Pharmacology and Anesthesiology, University of Arizona, Tucson, AZ 85724, USA
dMichael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada V6T 1Z4
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
a r t i c l e i n f o
Article history:
Received 30 July 2010
Received in revised form 8 December 2010
Accepted 20 December 2010
Available online xxxx
Keywords:
Sodium channel
Calcium channel
Slow inactivation
Neuropathic pain
Dorsal root ganglia
Dorsal horn
a b s t r a c t
Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms
within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments
have shown that specific channel isoforms, including NaV1.7 and NaV1.8 sodium channels and CaV3.2
T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized
a novel small organic compound (Z123212) that modulates both recombinant and native sodium and cal-
cium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactiva-
tion of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability,
and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal
cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration
of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve
ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At ther-
apeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse
effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the
peripheral and central pain signaling pathways may provide a synergistic mechanism toward the devel-
opment of a novel class of pain therapeutics.
? 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
1. Introduction
Voltage-gated sodium (NaV) and calcium (CaV) channels are cru-
cially involved in nociceptive signaling pathways, in part by medi-
ating ionic currents that contribute to the excitability of peripheral
nociceptors in the dorsal root ganglia (DRG). A specific subtype of
T-type CaVchannel (CaV3.2) is highly expressed in DRG neurons
and is involved in the initiation of action potential (AP) firing and
the generation of burst firing [6,19,31,42]. Both tetrodotoxin
(TTX)-sensitive NaV1.7 and TTX-resistant NaV1.8 channels are also
robustly expressed in DRGs and are important for setting the
threshold and upstroke of AP firing, respectively, and further act
to influence the frequency and sustainability of firing [10].
Within the spinal cord dorsal horn, second-order neurons in
superficial layers (lamina I/II) relay nociceptive-specific signals
from peripheral nociceptors to pain-processing regions of the
brain. Evidence suggests that a variety of NaVand CaVchannel iso-
forms are expressed within lamina I/II neurons [14,42,45] and that
both NaVand CaVchannels may increase the excitability of dorsal
horn neurons linked to neuropathic and inflammatory pain signal-
ing [11,14,20]. Specific NaVand CaVisoforms have been shown to
play pronociceptive roles; knockout of either NaV1.7 or NaV1.8
channels or knockdown of CaV3.2 T-type channels reduces hyper-
algesia and allodynia in animal models of acute and neuropathic
pain [6,25,30]. In humans, loss-of-function mutations in the
NaV1.7 channel lead to complete abolition of pain sensation, while
gain-of-function NaV1.7 mutations cause severe chronic pain syn-
dromes [10].
0304-3959/$36.00 ? 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.pain.2010.12.035
⇑Corresponding author at: Michael Smith Laboratories, University of British
Columbia, 2185 East Mall, Vancouver, BC, Canada V6T 1Z4. Tel.: +1 604 822 6968;
fax: +1 604 822 6470.
E-mail addresses: snutch@msl.ubc.ca, tsnutch@zalicus.com (T.P. Snutch).
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Neuropathic pain results from damage to the peripheral or cen-
tral nervous system and persists long after the nerve injury has re-
solved [46]. Pharmaceutical approaches to the management of
neuropathic pain are limited, and the continued use of some ther-
apeutics can lead to a variety of adverse events and/or desensitiza-
tion of drug effects. It has been hypothesized that the increased AP
firing and sustained depolarization of neurons associated with
neuropathic pain may drive a greater subset of NaVand CaVchan-
nels into a protective slow-inactivated state in order to dampen
neuronal excitability [4,5,17,44]. In this regard, blockers selectively
targeting the slow-inactivated channel state would be predicted to
mitigate off-target effects by preferentially attenuating aberrantly
hyperexcitable neurons while largely sparing normally firing neu-
rons and other nonhyperexcited targets.
In the current study, we have designed and characterized a low-
molecular-weight, orally available organic compound (Z123212)
that stabilizes the slow-inactivated state of NaVand CaVchannels,
including TTX-resistant NaVand T-type CaVchannels in DRG neu-
rons and TTX-sensitive NaVchannels in lamina I/II spinal cord neu-
rons. Z123212 potently reduces the excitability of DRGs and
lamina I/II neurons and is found to reverse thermal and mechanical
hypersensitivity in animal models of acute and neuropathic pain.
The identification of compounds such as Z123212 that have poten-
tially synergistic effects by targeting multiple ion channels in com-
ponents of the peripheral and central nociceptive signaling
pathways through a state-dependent mechanism may lead to the
development ofnovel classes
therapeutics.
ofsafe andeffective pain
2. Methods
2.1. Chemistry
The synthesis of Z123212 is illustrated in Suppl. Fig. 1. Briefly,
ethylenediamine-N,N-diacetic acid was cyclized under acidic con-
ditions followed by N-tert-butoxycarbonyl (Boc) protection of pip-
erazinone nitrogen. Subsequent coupling with bis-CF3 aniline
mediated by
O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HATU) in N,N-dimethylformamide (DMF)
provided the desired intermediate and was followed by deprotec-
tion of the Boc group to generate Z123212.
2.2. HEK 293 cell culture, transfection, and electrophysiology
Human embryonic kidney cells (HEK 293) were cultured and
either stably or transiently transfected with recombinant NaV
and CaVchannel genes as previously described [18]. For NaVchan-
nel recordings, the external recording solution contained (in mM):
137 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, adjusted
to pH 7.4 with NaOH, while for CaV1.2, CaV2.1, and CaV2.2, the
external solution contained (in mM): 139 CsCl, 5 BaCl2, 1 MgCl2,
10 HEPES, 10 glucose, adjusted to pH 7.4 with CsOH. For CaV3.1,
CaV3.2, and CaV3.3 channel recordings, the external recording solu-
tion contained (in mM): 142 CsCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10
glucose, adjusted to pH 7.4 with CsOH. For all recordings, the inter-
nal patch pipette solution contained (in mM): 126.5 CsMeSO4, 2
MgCl2, 11 EGTA, 10 HEPES, 2 Na2-ATP, adjusted to pH 7.3 with
CsOH. For all voltage-clamp protocols liquid junction potentials
were left uncorrected. Recordings were digitized at 5 kHz and
low-pass filtered at 1 kHz.
2.3. Animals
All electrophysiological experiments involving animals and
their care were performed in accordance with the recommenda-
tions of the Canadian Council on Animal Care and were according
to the animal care regulations and policies of the University of
British Columbia. For in vivo pain testing experiments, male
Sprague–Dawley rats (225–300 g; Harlan; Indianapolis, IN) were
maintained on a 12/12 h light/dark cycle and provided food and
water ad libitum. All pain testing experiments were performed
nder protocols approved by the Institutional Animal Care and
Use Committee in compliance with policies set forth by the
National Institutes of Health of the United States.
2.4. Voltage-clamp recordings on DRG neurons
Male Wistar rats (P25 to P30) were anesthetized with isoflurane
and decapitated. DRG were removed, cut into pieces, and placed in
Ca2+and Mg2+-free Hank balanced salt solution containing (in
mM): 138 NaCl, 5.3 KCl, 0.4 KH2PO4, 0.3 Na2HPO4, 6 D-glucose,
10 HEPES, and 2 mg/mL collagenase (Type I, Worthington, Lake-
wood, New Jersey), and 200 units of DNaseI (Worthington, Lake-
wood, NJ). Ganglia were incubated for 45 min at 37 ?C and
subsequently placed in L-15 media supplemented with 10% fetal
bovine serum, 100 units of penicillin, 100 lg streptomycin, 5 mM
HEPES, and 250 ng/mL nerve growth factor (all from Invitrogen,
Carlsbad, CA). Cells were dispersed with fire-polished Pasteur
pipettes and plated on glass coverslips coated with 1 mg/mL
poly-L-lysine and 5lg/mL laminin (Sigma, St. Louis, MO). Coverslips
were incubated at 37 ?C for 1–2 h and transferred to 4 ?C for
storage. Within 72 h, neurons were subjected to voltage-clamp
analyses with borosilicate glass patch pipettes with resistances of
2.5–5 MX. The external recording solution contained (in mM):
137 NaCl, 5 TEACl, 10 D-glucose, 1.8 CaCl2, 1 MgCl2, 10 HEPES,
0.0005 TTX, 0.001 LaCl3 adjusted to pH 7.4 with NaOH and
300 mOsm with sucrose. The internal patch pipette solution
contained (in mM): 120 CsCl, 2 MgCl2, 10 EGTA, 10 HEPES, 3
MgATP, 6 Tris2-phosphocreatine, and 0.4 Tris-GTP adjusted to pH
7.2 with CsOH and 290 mOsm. Only neurons with stable leak
currents less than 100 pA at ?70 mV were used. Recordings were
digitized at 20 kHz and low-pass filtered at 1 kHz.
2.5. Current-clamp recordings on DRG neurons
Male Sprague–Dawley rats (P1 to P4) were anesthetized with
CO2and decapitated. DRG were removed and placed in Ham F-12
supplemented with 10% horse serum, 50 units of penicillin, and
50 lg streptomycin (all media components from Invitrogen).
Ganglia were incubated for 15 min at 37 ?C in F-12 media supple-
mented with 0.05% collagenase (Type XI, Sigma), rinsed, and
incubated for 10 min at 37 ?C in phosphate-buffered saline supple-
mented with 0.12% trypsin (Invitrogen). Cells were dispersed with
fire-polished Pasteur pipettes and plated on glass coverslips coated
with 15 lg/mL poly-L-ornithine (Sigma) in F-12 media supple-
mented with 40 ng/mL nerve growth factor (Invitrogen). Within
72 h of plating, neurons were current-clamped with borosilicate
glass patch pipettes with resistances of 2.5–5 MX. The external
recording solution contained (in mM): 137 NaCl, 5 KCl, 10 D-glucose,
2 CaCl2, 2 MgCl2, 10 HEPES adjusted to pH 7.4 with NaOH, and
300 mOsm with sucrose. The internal patch pipette solution
contained (in mM): 130 KCl, 5 MgCl2, 1 EGTA, 40 HEPES, 2 MgATP,
and 0.5 Mg-GTP adjusted to pH 7.2 with KOH and 290 mOsm with
sucrose. Only neurons with stable leak currents less than 100 pA at
?70 mV were used. A calculated liquid junction potential of
12.2 mV was corrected in all DRG current-clamp recordings.
Recordings were digitized at 5 kHz and low-pass filtered at 1 kHz.
2.6. Recordings on lamina I/II spinal cord neurons
Male Wistar rats (P6 to P9 for voltage-clamp and P15 to P18
current-clamprecordings)forwereanesthetizedthrough
2
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intraperitoneal injection of Inactin (Sigma). The spinal cord was
then rapidly dissected out and placed in an ice-cold protective
sucrose solution containing (in mM): 50 sucrose, 92 NaCl, 15
D-glucose, 26 NaHCO3, 5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgSO4, and
1 kynurenic acid, and bubbled with 5% CO2/95% O2. The meninges,
dura, and dorsal and ventral roots were then removed from the
lumbar region of the spinal cord under a dissecting microscope.
The ‘‘cleaned’’ lumbar region of the spinal cord was glued to the
vibratome stage and immediately immersed in ice-cold bubbled
sucrose solution. For current-clamp recordings, 300- to 350-lm
parasagittal slices were cut to preserve the dendritic arbor of lam-
ina I neurons, while 350- to 400-lm transverse slices were pre-
pared for voltage-clamp NaV channel recordings. Slices were
allowed to recover for 1 h at 35 ?C in Ringer solution containing
(in mM): 125 NaCl, 20 D-glucose, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4,
2 CaCl2, 1 MgCl2, 1 kynurenic acid, and 0.1 picrotoxin, bubbled with
5% CO2/95% O2. The slice recovery chamber was then returned to
room temperature (20–22 ?C), and all recordings were performed
at this temperature.
Neurons were visualized with IR-DIC optics (Zeiss Axioskop 2 FS
plus, Gottingen, Germany), and neurons from lamina I and the out-
er layer of lamina II were selected on the basis of their location rel-
ative to the substantia gelatinosa layer. Neurons were subjected to
patch-clamp analyses with borosilicate glass patch pipettes with
resistances of 3–6 MX. Voltage-clamp recordings of NaVcurrents
in lamina I/II neurons were performed after slowly (2–5 min) pull-
ing the neurons off the slice to enable adequate space clamp (entire
soma isolation [ESI], technique as in Safronov et al. [38]; see Suppl.
Fig. 4). For current-clamp recordings of lamina I/II neurons in the
intact slice, the external recording solution was the above Ringer
solution, while the internal patch pipette solution contained (in
mM): 140 KGluconate, 4 NaCl, 10 HEPES, 1 EGTA, 0.5 MgCl2, 4
MgATP, 0.5 Na2GTP, adjusted to pH 7.2 with 5 M KOH and to
290 mOsm with D-mannitol (if necessary). Only tonic firing neu-
rons were selected for current-clamp experiments, while phasic,
delayed-onset, and single-spike neurons were discarded [34]. For
voltage-clamp recordings of pharmacologically isolated NaVcur-
rents [38] in ESI lamina I/II neurons, the external recording solu-
tion was a modified TEA–Ringer solution containing (in mM): 95
NaCl, 20 TEACl, 11 D-glucose, 25 NaHCO3, 5.6 KCl, 1 NaH2PO4, 0.1
CaCl2, 5 MgCl2, 1 kynurenic acid, 0.1 picrotoxin, while the internal
patch pipette solution contained (in mM): 140 CsCl, 5.8 NaCl, 1
MgCl2, 3 EGTA, 10 HEPES, 4 MgATP, 0.5 Na2GTP, adjusted to pH
7.3 with NaOH and 290 mOsm with D-mannitol (if necessary). Only
neurons with stable leak currents less than 100 pA (at ?100 mV)
for voltage-clamp and with resting membrane potentials (Vrest)
more negative than ?50 mV for current-clamp were used for sub-
sequent experiments. A calculated liquid junction potential of
14.6 mV was corrected for current-clamp recordings. Recordings
were digitized at 50 KHz and low-pass filtered at 2.4 or 10 kHz
for voltage-clamp and current-clamp recordings, respectively.
2.7. In vivo pain testing
Spinal nerve ligation (SNL) injury was performed by tight liga-
tion of the L5 and L6 spinal nerves according to the procedure of
Kim and Chung [24] in Harlan Sprague–Dawley rats. Rats that
exhibited motor deficiency (such as paw dragging or dropping)
or showed no tactile or thermal hypersensitivity were excluded
from further testing. The experimenter was blinded to the drug
pretreatment. Fourteen days after SNL injury, tactile paw with-
drawal threshold and thermal paw withdrawal latency were mea-
sured. Response thresholds to innocuous mechanical stimuli were
evaluated by determining paw withdrawal threshold after probing
the paw with a series of calibrated Von Frey filaments [7]. The
withdrawal threshold was determined by sequentially increasing
and decreasing the stimulus strength (up-and-down method) and
analyzed by a Dixon nonparametric test. Data are expressed as
the mean withdrawal threshold. Response thresholds to noxious
thermal stimuli were determined by measuring the latency of
paw withdrawal from a focused beam of radiant heat on the sur-
face of the hind paw using a plantar analgesia meter (Ugo Basile,
Italy) by the Hargreaves method [16]. A maximum cutoff of 33 s
was used to prevent tissue damage. For the hot-plate test, naive
rats were placed on a 52 ?C metal hot plate to measure the latency
of paw flinching or licking before or 1 h after drug administration.
A cutoff of 30 s was used.
2.8. Cardiovascular liability studies
Isolated New Zealand White rabbit (2.5–3.5 kg) hearts were AV
ablated, perfused in a retrograde manner, and paced at a stimula-
tion rate of 1 Hz (basic cycle length = 1 s). The stabilization period
was at least 15 min long before obtaining control responses. Exper-
iments were performed at 37 ± 3 ?C. Each heart acted as its own
vehicle control before application of Z123212. Concentrations of
3, 10, and 30 lM Z123212 were applied sequentially, in ascending
order, for exposure periods of at least 15 min/concentration to al-
low for equilibration within the heart tissue. The QT interval and
QRS duration were calculated by ECG Auto software (EMKA Tech-
nologies, Falls Church, VA).
2.9. Pharmacokinetic studies
Z123212 was provided as the HCl salt for pharmacokinetic anal-
ysis. All dosing was based on the free base weight of the com-
pound. Harlan male Sprague–Dawley rats were fasted overnight
before dose administration of Z123212 in 0.5% carboxy methyl
cellulose. Plasma samples were collected via jugular cannulae from
3 animals per time point at 0.25, 0.5, 0.75, 1, 2, and 4 h. Brains were
collected from 3 animals per time point at 1 and 4 h. Plasma and
brain samples were stored below ?70 ?C until analysis could be
performed by a research-grade liquid chromatography/tandem
mass spectrometry assay. Mean Z123212 concentrations in the
plasma and brain and noncompartmental pharmacokinetic analy-
sis of the plasma data were performed by WinNonlin software, ver-
sion 5.0.1 (Pharsight, Mountain View, CA).
2.10. Compounds and perfusion
Unless otherwise indicated, all compounds were obtained from
Sigma. For in vitro studies, Z123212 was prepared as 30 or 100 mM
stock solutions in dimethyl sulfoxide and stored at ?80 ?C. Stock
aliquots were thawed and used for a maximum of 2 weeks. The
highest concentration of dimethyl sulfoxide in the extracellular
solutions did not exceed 0.1%, a concentration that did not detecta-
bly affect current-clamp or voltage-clamp recording properties. A
closed perfusion system (10 mL) was used for spinal cord slice
recordings, with a flow rate of between 2 and 4 mL/min. For
in vivo studies, Z123212 was dissolved in 0.5% carboxy methyl cel-
lulose at a concentration of 6 mg/mL.
2.11. Data analysis
Figures and fittings utilized Microcal Origin 7.5 (Northampton,
MA). Current–voltage relationships were fitted with the modified
Boltzmann equation: I = [Gmax? (Vm? Erev)]/[1 + exp(/ka)], where
Vmis the test potential, V0.5ais the half-activation potential, Erev
is the extrapolated reversal potential, Gmaxis the maximum slope
conductance, and kareflects the slope of the activation curve. Data
from concentration dependence studies were fitted with the equa-
tion y = [(A1? A2)/{1 + (x/xo)P} + A2], where A1is initial amplitude
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(=0) and A2is final block value, xois IC50(concentration causing
50% inhibition of currents), and P gives a measure of the steepness
of the curve. Statistical significance was determined by paired or
unpaired Student’s t tests and 1-way or repeated measures ANOVA
followed by Tukey’s multiple comparison test, and significant val-
ues were set as indicated in the text and figure legends. All data are
given as means ± standard errors.
3. Results
3.1. Design and synthesis of Z123212
Given the well-documented roles of CaV3.2 T-type channels and
NaVchannels in modulating the excitability of DRG neurons, we
set out to rationally design a mixed ion channel blocker that may
have synergistic effects in attenuating pain signaling. As part of a
rationale scaffold-based design and screening program to develop
subtype-selective N-typeand T-type CaVblockers, the smallorganic
compound Z121912 (molecular weight = 521; Fig. 1) was initially
designedon the basis of previousbackbones identifiedas exhibiting
promising CaVblocking and preclinical characteristics [32,47]. In
particular, the bis-CF3aryl amide group of Z121912 was a preferred
structural feature from the aspect of T-type CaVblocking potency
(IC50= 64 nM). However, from a drug discovery perspective
Z121912 possessed potential cardiovascular liability in that it also
potently blocked the hERG potassium channel (IC50?100 nM). In
order to reduce the hERG liability, the benzhydrol group of
Z121912 was either removed (Z123875; molecular weight = 355)
or replaced with an oxygenated piperazine (Z123212; molecular
weight = 369; Fig. 1; Suppl. Fig. 1). Both derivatives exhibited sig-
nificantly improved profiles against the hERG potassium channel
(IC50s >10 lM) with Z123212 being selected for further preclinical
assessmentonthebasis of
properties.
itsfavorable pharmacokinetic
3.2. Z123212 stabilizes the slow-inactivated state of CaV3.2 T-type
channels
The blocking activity of Z123212 was initially tested against re-
combinant CaV3.2 channels expressed in HEK cells by using stan-
dard depolarizing test pulses from a hyperpolarized holding
potential (Vhold; ?110 mV) that would place the channels largely
in the closed state. Somewhat surprisingly, even at relatively high
concentrations, Z123212 caused minimal inhibition of recombi-
nant CaV3.2 channels when activated from the closed state (IC50
?10 lM, Fig. 2A and B). We assessed the effects of Z123212 on
other CaV3.2 channel properties and found that 10 lM Z123212
also had no significant (P > .05) effect on the voltage dependence
of fast channel inactivation (control, V1/2FastInact= ?61 ± 1 mV,
n = 5; 10 lM; Z123212, V1/2FastInact= ?66 ± 3 mV, n = 4; data not
shown). As previously observed for CaV3.1 channels [17], we next
examined whether CaV3.2 channels undergo a slow inactivation
process (Fig. 2C). Z123212 (10 lM) significantly (P < .05) increased
the extent of CaV3.2 channel slow inactivation at specific mem-
brane potentials and also caused a significant 6 mV hyperpolariz-
ing shift in the voltage dependence of CaV3.2 channel slow
inactivation (Fig. 2C; P < .05). The recovery from inactivation
Fig. 1. Structural features of Z123212. Z123875 and Z123212 are derivatives of the
high-affinity piperazine T-type antagonist, Z121912. The dipeptide backbone of
Z123212 is highlighted in red.
0 8
0.6
1.0
0.8
1.2
10 μM Z123212
Current
ormalized C
No
-110 mV
-30 mV
500 pA
10 μM
Z123212
0 2
0.0
0.4
0.2
-30 mV
t
zed Current
Normaliz
20 ms
Control
Time (min)
-110 mV
0.6
0.4
0.8
1.0
0 4
0.2
0.6
0.4
0.8
1.0
Normalized
P2/P1 N
**
*
-10 mV
0.0
0.2
R I tl ()
-120-100 -80 -60 -40 -20
Voltage(mV)
0
0.0
**
P2 P1
-110 mV
-30 mV
-110 mV, 1 min
-110 mV
-30 mV, 1 min
Δ Δt = 40 to 5120 ms
4000
P1
-30 mV
ecovery In erva ms
AB
CD
EF
1.0
0.8
ed
1 Normalize
P2/P1
10 μM Z123212
μ
-90 mV
-120 mV
-80 mV, 10 s
P1 P2
-30 mV
0.4
0.2
0.6
Control
10 μ μM Z123212
P1P2
012345
020006000
02468 10
0.0
Time (min)
Fig. 2. Z123212 selectively alters the slow-inactivated state of CaV3.2 channels. (A)
Representative traces of recombinant CaV3.2 channels during depolarizing steps
from ?110 mV to ?30 mV demonstrating that perfusion of 10 lM Z123212 caused
minimal inhibition of CaV3.2 channels when activated from hyperpolarized
potentials. Scale bar x = 20 ms, y = 500 pA. (B) Average time course of normalized
CaV3.2 channel peak current values before and during perfusion of 10 lM Z123212
(n = 11). (C) Z123212 (10 lM) caused a significant (P < .05) hyperpolarizing shift in
the voltage dependence of CaV3.2 channel slow inactivation (control, V1/2SlowInact=
?74 ± 1 mV, n = 4; 10 lM Z123212, V1/2SlowInact= ?80 ± 2 mV, n = 5). Z123212 also
caused a significant (⁄P < .05) enhancement of the extent of NaV channel slow
inactivation at potentials of ?80, ?70, ?20, and ?10 mV. (D) Z123212 (10 lM)
significantly (P < .005, n = 4–5) slowed the recovery from CaV3.2 channel slow
inactivation. Recordings in both (C) and (D) were unpaired and time-matched
between control and Z123212 treatment groups to eliminate potential time-
dependent changes in parameters. (E) Representative traces demonstrating that
Z123212 selectively inhibited slow-inactivated T-type currents (P2 traces) in
dissociated dorsal root ganglia (DRG) neurons. Scale bar: x = 5 ms, y = 1000 pA. (F)
Average time course of the ratio of P2 peak current P1 peak current demonstrating
that Z123212 increases the extent of T-type channel slow inactivation in DRG
neurons (n = 4). Insets illustrate voltage step waveforms.
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M.E. Hildebrand et al./PAIN
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Please cite this article in press as: Hildebrand ME et al. A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain. PAIN
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?

Page 5

parameter for CaV3.2 channels has been linked to changes in neu-
ronal membrane excitability [33]; thus, we tested the effect of
Z123212 on CaV3.2 recovery from slow inactivation. Application
of Z123212 significantly (P < .01) slowed the recovery from
CaV3.2 channel slow inactivation at concentrations of 3 lM and
greater(control, srecov= 510 ± 30 ms,
srecov= 650 ± 20 ms, n = 4; 10 lM; Z123212, srecov= 680 ± 20 ms,
n = 5; Fig. 2D). The CaV3.2 T-type channel isoform mediates the
majority of whole cell T-type current within DRG neurons
[1,6,8,42]; thus, we also tested the effects of Z123212 on T-type
currents within isolated DRG neurons. Consistent with the recom-
binant CaV3.2 results, Z123212 (10 lM) was found to selectively
stabilize the slow-inactivated state of T-type currents in isolated
DRG neurons (Fig. 2E and F).
n = 4;3 lM;Z123212,
3.3. Z123212 inhibits recombinant NaVand CaVchannels by
modulating slow inactivation
Both TTX-sensitive NaV1.7 and TTX-resistant NaV1.8 channels
are highly expressed in DRG neurons and exhibit overlapping dis-
tributions and functional roles with CaV3.2 T-type channels
[10,19]. Hypothesizing that Z123212 may also alter NaVchannel
activity, wetestedwhether
inactivated recombinant NaV channels. In order to induce slow
inactivation, sweeps were elicited every 30 s that included a 10-s
conditioning pulse to ?20 mV, followed by a short hyperpolarizing
step to remove fast inactivation and then a depolarizing test pulse
(P2; as described in [40]). We found that perfusion of Z123212
caused a robust inhibition of both recombinant NaV1.7 and
NaV1.8 P2 currents that was even greater than that observed for
CaV3.2 channels (Fig. 3A and B). Concentration–dependent re-
sponse studies revealed that Z123212 inhibited NaV1.7 channels
with an IC50= 17 lM and NaV1.8 channels with an IC50= 9.2 lM
(Fig. 3C).
Z123212couldinhibitslow-
Because Z123212 stabilized the slow-inactivated state of CaV3.2
T-type and NaV1.7/NaV1.8 channels, it was of interest to test
whether it also acted on other NaVand CaVchannel classes. Similar
to that for CaV3.2 T-type and NaV1.7/NaV1.8 channels, application
of 10 lM Z123212 did not cause significant tonic block of other
CaVand NaVisoforms tested (Suppl. Fig. 2). In contrast, 10 lM
Z123212 was shown to selectively stabilize the putative slow-
inactivated states of exogenously expressed CaV1.2 (L-type), CaV3.1
(T-type), CaV3.3 (T-type), and NaV1.5 channels, with an inhibition
of P2 currents (after 10-s depolarizing conditioning pulses) ranging
from ?30% to 60%. However, Z123212 did not uniformly alter slow
inactivation states as the compound had no effect (<15% P2 inhibi-
tion) on the CaV2.1 (P/Q-type) and CaV2.2 (N-type) isoforms (Suppl.
Fig. 2). Taken together, Z123212 selectively stabilizes the slow-
inactivated states of a subset of ion channel types and does not
seem to act as a tonic channel blocker. The selective action of
Z123212 on channel slow inactivation may be of particular rele-
vance to the putative hyperexcitable processes associated with
various pain states compared to ion channel functioning during
normal physiological processes. For example, cardiac NaV1.5 chan-
nels are adapted to have reduced slow inactivation during the
repetitive (>1 Hz) and prolonged (?200 ms) depolarizations that
occur during AP firing of cardiac myocytes [36]. In support,
although Z123212 is able to stabilize the slow-inactivated state
of NaV1.5 channels under certain experimental conditions (involv-
ing step depolarization to ?20 mV for 10 s; Suppl. Fig. 3A), its ef-
fects on NaV1.5 channels activated by simulated cardiac AP
waveforms is greatly reduced (Suppl. Fig. 3B).
3.4. Z123212 selectively stabilizes the slow-inactivated state of TTX-
sensitive NaVchannels in lamina I/II spinal cord neurons
We next set out to determine whether Z123212 could alter na-
tive NaVchannels implicated in the nociceptive signaling pathway
selectively through its effect on slow inactivation. Voltage-clamp
recordings on lamina I/II neurons from spinal cord slices were per-
formed to examine the effects of Z123212 on TTX-sensitive NaV
currents in nociceptive spinal cord neurons. In order to ensure ade-
quate voltage-clamp of NaVcurrents, the ESI technique pioneered
by Safronov et al. [38] was used to remove healthy lamina I/II neu-
rons from the slice surface (see Section 2.6 and Suppl. Fig. 4). In this
recording configuration, NaVcurrents in lamina I/II neurons were
completely blocked by TTX (data not shown; see [38]). Similar to
that for recombinant NaVand CaVchannels, application of 10 lM
Z123212 did not result in tonic block of native TTX-sensitive NaV
currents during depolarizations from a hyperpolarized state
(Vhold= ?100 mV; Fig. 4A and B). The perfusion of 10 lM
Z123212 did result in a small (?2.4 ± 0.5 mV, n = 5) but significant
(P < .01) hyperpolarizing shift in the voltage dependence of NaV
channel activation (Fig. 4A); however, time-dependent negative
shifts in the voltage dependence of activation were also observed
during control recordings (data not shown). Thus, this small hyper-
polarizing shift was likely not mediated by Z123212.
Application of 10 lM Z123212 also had no effect on the voltage
dependence of NaVchannel fast inactivation (100-ms conditioning
pulses, Fig. 4C). The voltage dependence of NaVchannel slow inac-
tivation could not be directly assayed by using the native recording
system because the neurons would not tolerate the highly hyper-
polarized holding potential (Vhold= ?120 mV) required to allow
recovery from slow inactivation between pulses. However, during
slow inactivation-inducing sweeps, perfusion of 10 lM Z123212
caused a robust reduction in the amplitude of lamina I/II neuron
NaVcurrents during P2 pulses by 45 ± 7% (n = 5; P < .02; Fig. 4D).
Analysis of NaVcurrent amplitudes in P1 control pulses versus P2
test pulses further demonstrated that Z123212 selectively stabi-
lized the slow-inactivated state of native TTX-sensitive NaV
AB
1.0
0.8
1.2
zed
P2 NormalizP
10 µM Z123212
1.0
0.8
1.2
zed
P2 Normali
10 µM Z123212
0.2
0.0
0.4
0.6
P2
0 mV
P1
-20 mV, 10 s
- 120 mV
120 mV
- 160 mV
0.2
0.0
0.4
0.6
P2
+20 mV
P1
-20 mV, 10 s
- 80 mV
C
ion in P2
% Reduct
024681012
Time (min)
02468 1012
Time (min)
- 120 mV
60
80
100
NaV1.7
IC50 = 17 µ µM
50
110
[Z123212] (µ µM)
100 1000
0
20
40
NaV1.8
IC50 = 9.2 µ µM
Fig. 3. Z123212 inhibits slow-inactivated NaV1.7 and NaV1.8 channels. The effects
of Z123212 on slow-inactivated recombinant NaV1.7 (A) and NaV1.8 (B) channels
were assessed using a test pulse (P2) that followed a 10-s conditioning prepulse to
?20 mV. (A) Plot of time course of normalized P2 current amplitude showing that
application of 10 lM Z123212 reduced the amplitude of recombinant NaV1.7
currents by 36 ± 7% (n = 4). (Top inset) Representative P2 traces with (gray) and
without (black) 10 lM Z123212. Scale bar: x = 5 ms, y = 1000 pA. (B) Plot of time
course of normalized P2 current amplitude showing that application of 10 lM
Z123212 reduced the amplitude of recombinant NaV1.8 currents by 40 ± 4% (n = 4).
(Top inset) Representative P2 traces with (gray) and without (black) 10 lM
Z123212. Scale bar: x = 5 ms, y = 250 pA. (C) Concentration–dependent response
curves for Z123212 inhibition of P2 currents for recombinant NaV1.7 channels
(IC50= 17 lM) and NaV1.8 channels (IC50= 9.2 lM). n = 3–5 for all concentrations.
M.E. Hildebrand et al./PAIN
?xxx (2011) xxx–xxx
5
Please cite this article in press as: Hildebrand ME et al. A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain. PAIN
(2011), doi:10.1016/j.pain.2010.12.035
?

Page 6

channels (Suppl. Fig. 5). The inhibition by Z123212 of NaVchannels
reaching the slow-inactivated state (P2) was significant (P < .05) at
concentrations of 3 lM and higher, with an IC50= 13 lM (Fig. 4E).
3.5. Z123212 selectively stabilizes the slow-inactivated state of TTX-
resistant NaVchannels in peripheral nociceptors
The majority of TTX-resistant NaVcurrent in nociceptive DRG
neurons is composed of the NaV1.8 channel isoform [10]. We next
evaluated the effects of Z123212 on pharmacologically isolated
TTX-resistant currents in dissociated small diameter DRG neurons.
Similar to that for TTX-sensitive NaVcurrents, perfusion of 10 lM
Z123212 did not result in tonic block of native TTX-resistant NaV
currents when depolarized from a relatively hyperpolarized poten-
tial (Vhold= ?70 mV; Fig. 5A and B) and also had no significant
(P > .05) effect on the voltage dependence of NaVchannel activation
(Fig.5A).Further,whileapplicationof 10 lM Z123212did notaffect
the voltage dependence of fast channel inactivation (Fig. 5C), it sig-
nificantly (P < .05) enhanced the fraction of NaV channels that
reached the slow-inactivated state during conditioning prepulses
of ?20 mV (as well as more depolarized potentials; Fig. 5D). The
Please cite this article in press as: Hildebrand ME et al. A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain. PAIN
(2011), doi:10.1016/j.pain.2010.12.035
concentration dependence for the reduction in P2 current ampli-
tude by Z123212 is shown in Fig. 5E. Using 10-s conditioning pre-
pulses to ?20 mV, we calculated an IC50= 12 lM.
3.6. Z123212 reduces the excitability of peripheral nociceptors and
second order spinal cord neurons
Because Z123212 can modulate the activity of several NaVand
CaVchannel isoforms linked to neuronal excitability, we used cur-
rent-clamp recordings to test for possible direct effects of Z123212
on the overall excitability of both DRG and spinal cord lamina I/II
neurons. Current-clamp recordings were initially performed on
dissociated small diameter (25 ± 3 pF, n = 6) DRG neurons from
neonatal rats. A 350-ms depolarizing current injection step
(?220 ± 120 pA, n = 6) was used to elicit 4–6 APs (Fig. 6A), and this
sweep was repeated every 30 s to ensure that a stable baseline
number of APs was reached before applying compound (Fig. 6B).
Subsequent perfusion of 10 lM Z123212 was found to significantly
(P < .01) reduce the number of APs elicited during the depolarizing
pulse by 60 ± 9% (n = 6; Fig. 6C).
0 8
0.6
1.0
0.8
1.2
Current
ormalized C
N
10 µM Z123212
-0.2
-0.4
0.0
urrentA
action of Cu
Fra
B
0.2
0.0
0.4
-1.0
-1.2
-0.8
1.0
-0.6
- 20 mV
P1
012345
Time (min)
1 2
1.0
10 M Z12321210 µM Z123212
t
zed Current
Normaliz
-60-40-200
Voltage (mV)
C
D
- 100 mV
- 120 mV, 100 ms
0.6
0.4
0.8
1.2
ormalized
P2 N
0 4
0.2
0.6
0.4
0.8
1.0
n=4
012345678
0.0
0.2
-120 -100 -80 -60 -40
V ltVoltage (mV)
-20
0.0
( V)
P2
- 20 mVP1
-50 mV, 10 s
- 80 mV
- 120 mV
-30 mV
-20 mV
P1
-120 mV
100 ms
-100 mV
100
P2
eduction in
% Re
Time (min)
E
40
60
80
TTX-Sensitive NaV(Lamina I/II)
IC50 = 13 µ µM
110
[Z123212] (µ µM)
1001000
0
20
V()
Fig. 4. Z123212 selectively alters the slow inactivation of TTX-sensitive NaV
channels in lamina I/II neurons. (A) Voltage-clamp recordings of TTX-sensitive NaV
currents in lamina I/II spinal cord neurons. Current values were normalized to the
peak current of the control IV relationship, demonstrating no tonic block by
Z123212 at Vhold= ?100 mV. (Inset) Representative current traces during depolar-
ization steps from ?60 mV to ?10 mV in 10 mV increments. Scale bar: x = 5 ms,
y = 500 pA. (B) Average tonic block time course of normalized NaVpeak current
values before and during perfusion of 10 lM Z123212. (C) Z123212 (10 lM) caused
no significant (P > .05, n = 4) shift in the voltage dependence of NaVchannel fast
inactivation. (D) Plot of time course of normalized P2 current showing that
application of 10 lM Z123212 significantly reduced the amplitude of slow-
inactivated NaV currents. (Top inset) Representative P2 traces with (gray) and
without (black) 10 lM Z123212. Scale bar: x = 5 ms, y = 100 pA. (E) Concentration–
response curve for Z123212 inhibition of P2 currents for TTX-sensitive lamina I/II
NaVchannels (IC50= 13 lM). n = 3–5 for all concentrations.
0 8
0.6
1.0
0.8
1.2
10 μM Z123212
Current
ormalized C
No
-0.2
-0.4
0.0
rrent
ction of Cu
Frac
AB
0 2
0.0
0.4
0.2
1 0
-1.2
-0.8
-1.0
-0.6
P1, 50 ms, 20 mV
012345
Time (min)
-40-20
Voltage (mV)
02040
C
ormalized
P2/P1 No
D
ormalized
P2/P1 No
- 70 mV
0.6
0.4
0.8
1.0
0.6
0.4
0.8
1.0
-80 -60 -40 -20
Voltage (mV)
020
0.0
0.2
-80 -60 -40 -20
Voltage (mV)
E
0 20
0.0
0.2
*
*
*
*
- 70 mV
-100 mV
0.5 s
20 mV
P2
P1
- 70 mV
-80 mV
10 s
P2
10 mV
20 mV
P1
100
n P2
eduction in
% Re
40
60
80
TTX-Resistant NaV (DRG)
110
[Z123212] ( (μ μM)
100 1000
0
20
IC50 = 12 μ μM
Fig. 5. Z123212 selectively alters the slow inactivation of TTX-resistant NaV
channels in nociceptive dorsal root ganglia (DRG) neurons. Voltage-clamp record-
ings of TTX-resistant NaV currents in small-diameter rat DRG neurons. (A)
Treatment with 10 lM Z123212 had no effect on the voltage dependence of NaV
channel activation. (Inset) Representative current traces during depolarizing steps
from ?40 to +10 mV in 10-mV increments. Scale bar: x = 50 ms, y = 2000 pA. (B)
Average time course of normalized NaVchannel peak current values before and
during perfusion of 10 lM Z123212 (n = 5). (C) Application of 10 lM Z123212 had
no significant (P > .05, n = 4) effect on the voltage dependence of fast inactivation.
(D) Z123212 (10 lM) caused a significant (P < .05, n = 5) enhancement of the extent
of NaVchannel slow inactivation at conditioning prepulses of ?20 mV and more
depolarized potentials. (E) Concentration–response curve for Z123212 inhibition of
P2 currents for TTX-resistant DRG NaV channels (IC50= 12 lM). n = 3–5 for all
concentrations.
6
M.E. Hildebrand et al./PAIN
?xxx (2011) xxx–xxx
?

Page 7

Current-clamp recordings were also performed on intact tonic
firing lamina I/II neurons in parasagittal lumbar spinal cord slices
from juvenile rats [34]. A current–voltage relationship was re-
peated every 2 min with 1200 ms hyperpolarizing/depolarizing
current injection steps ranging from ?50 pA to +80 pA in +10 pA
increments. From this IV relationship, the effect of Z123212 on
the number of elicited APs was analyzed for a moderate depolariz-
ing current injection step (40 ± 6 pA, n = 7) that caused only a min-
or decay in AP amplitude (15 ± 2%, n = 5) over the entire train but
still elicited a robust number of APs (16 ± 2, n = 7). Control record-
ings demonstrated no time-dependent changes in the number of
APs during the moderate depolarizing steps, whereas the number
of APs decreased with time for depolarizing current injection steps
of greater magnitude (data not shown). Application of 10 lM
Z123212 resulted in a 47 ± 12% (n = 7) decrease in AP firing during
the moderate depolarizing steps (Fig. 7A), while membrane prop-
erties including input resistance (RN) and Vrest remained un-
changed(control,
RN= 490 ± 60 MX,
n = 7; 10 lM; Z123212, RN= 460 ± 50 MX, n = 5, Vrest= ?63 ± 2 mV,
n = 7). The inhibition of AP firing by Z123212 was concentration
dependent with an IC50= 480 nM (Fig. 7C).
Lacosamide is an anticonvulsant that shares some structural
features with Z123212 and has been reported to specifically stabi-
lize the slow inactivation of NaVchannels [4,12,40]. When tested in
the spinal cord slice preparation, lacosamide inhibited lamina I/II
neuronAPfiringapproximately
(IC50= 150 lM) than Z123212 (Fig. 7C). Lacosamide did reach a
higher level of AP reduction than Z123212, but only at concentra-
tions (>100 lM) well above therapeutic plasma levels for this
agent (see Fig. 7C and [4]).
Z123212 reduced AP firing in lamina I/II neurons under condi-
tions where Vrestremained unaltered by tonic current injection
n = 5,
Vrest= ?63 ± 2 mV,
300timeslesspotently
and the depolarizing current injection steps under analysis fol-
lowed depolarizing current injection steps using an IV protocol.
Thus, Z123212 may exert its inhibitory effects on AP firing by sta-
bilizing accumulated slow inactivation of native NaVand CaVchan-
nels. In support, repeating the above experiments during tonic
hyperpolarizing current injection to elicit a more hyperpolarized
Vrest(?86 ± 3 mV, n = 3) completely eliminated the inhibitory ef-
fect of Z123212 (Fig. 7B).
3.7. Z123212 effectively reverses multiple pain modalities
We next tested whether the highly state-dependent mechanism
of action of Z123212 on nociceptor/spinal cord neuron inhibition
translated to an ability to reverse behavioral hypersensitivity in
animal models of pain. Oral administration of Z123212 (30 mg/
kg) was shown to attenuate both acute thermal hypersensitivity
by using the hot-plate test and to a greater extent, chronic
mechanical and thermal hypersensitivity assessed using the SNL-
induced model of neuropathic pain (Fig. 8). More specifically,
Z123212 significantly (P < .05) reversed both tactile allodynia
40
20
mV)A
e Potential (
Membran
0 pA
45 pA
40
mV)
e Potential (
Membran
Control
10 μ μM Z123212
-20
-40
0
-20
0
20
0.00.20.40.6
-60
Time (s)
0.00.20.40.6
-60
-40
Time (s)
B
6
ing Step
0 ms Depolarizi
# of APs in 350
C
5
s
mber of AP
Num
10 μ μM Z12321210 μ μM Z123212
4
*
2
3
4
0
ControlZ123212
2
0 1 2 3 4 5 6 7 8
Time(min)
0
1
Fig. 6. Z123212 reduces the excitability of small, nociceptive dorsal root ganglia
(DRG) neurons. Current-clamp recordings were performed on small (25 ± 3 pF,
n = 6) DRG neurons from P1 to P4 Sprague–Dawley rats. Of 3 DRG neurons tested, 2
were found to be capsaicin sensitive. Firing of 4–6 action potentials (APs) was
elicited by 350-ms depolarizing current injections (ranging from 20 to 400 pA,
depending on cell). (A) Representative traces demonstrating that perfusion of
10 lM Z123212 (right, gray traces) reduced the number of elicited APs. (B) Time
course from another representative neuron demonstrating how perfusion of
Z123212 reduced the number of APs during 350-ms depolarizing steps elicited
every 30 s. (C) Bar graph showing that 10 lM Z123212 caused a significant
(P < .002, n = 6) reduction in DRG AP firing.
A
15
20
olarizing Step
APs in 1.2 s Depo
# of
0 pA
70 pA
5
10
*
B
Control Z123212
0
20
tep
2 s Depolarizing St
# of APs in 1.2
-64 mV
10
15
C
AP Firing
duction in A
% Re
Control
0
5
-89 mV
Z123212
40
30
50
40
60
10
10
20

Z123212
IC50 = 0.48 μ μM
Lacosamide
IC50 = 150 μ μM


0.1110100
0
Concentration ( μ μM)
Fig. 7. Z123212 reduces the excitability of spinal cord lamina I/II neurons in a state-
dependent manner. (A) Current-clamp recordings were performed on dorsal horn
lamina I/II neurons from P15 to P18 Wistar rats. Tonic firing of these neurons was
induced by 1.2-s depolarizing current injections (ranging from 20 to 70 pA,
depending on cell). (Left) Representative traces demonstrating that perfusion of
10 lM Z123212 (gray traces) reduced the number of elicited action potentials (APs).
(Right) Bar graph showing that 10 lM Z123212 caused a significant (P < .05, n = 7)
reduction in lamina I/II neuron AP firing. Vrest= ?63 ± 2 mV, n = 7 for control group.
(B) Same experimental conditions as above, except a constant hyperpolarizing
current was injected (?47 ± 4 pA, n = 3) to elicit a more hyperpolarized resting
membrane potential (Vrest= ?86 ± 3, n = 3). Perfusion of 10 lM Z123212 had no
significant (P > .05) effect on lamina I/II neuron AP firing in this more hyperpolar-
ized state. (C) Concentration–response curve for Z123212 and lacosamide on
lamina I/II neuron AP firing under conditions as in (A). Z123212 inhibited AP firing
with an IC50value of 0.48 lM and a maximal inhibition of 44%, while lacosamide
inhibited AP firing with an IC50 value of 150 lM and a maximal inhibition
approaching 100% (at concentrations greater than 300 lM) (n = 3–7 for all
concentrations).
M.E. Hildebrand et al./PAIN
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7
Please cite this article in press as: Hildebrand ME et al. A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain. PAIN
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?

Page 8

(measured by Von Frey filaments) and thermal hyperalgesia (mea-
sured by the Hargreaves method) in SNL rats with an efficacy sim-
ilar to equivalent doses of gabapentin (Fig. 8A). Z123212 did not
cause observable motor deficits at the oral doses used for pain test-
ing (30 mg/kg, oral administration). More comprehensive Rotarod
experiments revealed that at doses of 30 mg/kg (n = 8) and
100 mg/kg (n = 20), Z123212 delivered through the more direct
intraperitoneal route caused no detectable motor impairment from
0.25 to 4 h after administration (data not shown). As Z123212 was
shown to stabilize NaV1.5 and CaV1.2 L-type channel slow inactiva-
tion under certain experimental conditions, we also tested whether
Z123212 could affect cardiovascular properties directly. Z123212
tested at 3, 10, and 30 lM doses on isolated rabbit hearts did not
cause significant (n = 3; P > .05) alterations in either the QT interval
or QRS duration of electrocardiograms. For example, perfusion of
Z123212 at doses relevant to the present study (10 lM) caused a
?4.7 ± 2.4% change (n = 3; P > .05) in the QT interval and a
?2.3 ± 2.4% (n = 3; P > .05) change in the QRS duration of isolated
rabbit hearts.
Pharmacokinetic analysis revealed that Z123212 was well ad-
sorbed at the oral dose used for testing animal efficacy (30 mg/
kg) with mean plasma levels ranging between approximately 10
and 17 lM for up to 4 h after oral administration (Fig. 8C). Mean
levels of Z123212 in the brain ranged between approximately 3
and 5 lM over the same time period (Fig. 8C). Overall, in both
the blood and brain, the concentrations of Z123212 reached would
be predicted to effectively stabilize NaVand CaVchannel slow inac-
tivation and to reduce the excitability of nociceptive DRG and lam-
ina I/II neurons (Figs. 2–7).
4. Discussion
We report the design, synthesis and functional characterization
of a novel small organic agent (Z123212) that uniquely stabilizes
the slow-inactivated state of a subset of NaVand CaVchannels.
The data suggest that by enhancing the slow inactivation of a
combination of TTX-sensitive and TTX-resistant NaVand T-type
CaVcurrents, Z123212 reduces AP firing in peripheral DRG and
lamina I/II spinal cord neurons. We predict that this highly
state-specific mechanism underlies the ability of orally adminis-
tered Z123212 to significantly attenuate thermal and mechanical
hypersensitivity in rodent models of both chronic neuropathic
pain and acute pain.
4.1. Mechanism underlying pain-attenuating effects of Z123212
Z123212 inhibits certain NaVand CaVchannels by selectively
stabilizing the slow-inactivated state over the fast inactivated
state. As channel slow inactivation is significantly enhanced during
prolonged depolarizations (eg, ?50 mV for 10 s; Fig. 4), Z123212 is
predicted to cause pronounced inhibition of NaVand CaVchannels
in neurons that are either tonically depolarized or firing in bursts
that create sustained depolarizations. In this regard, we observed
a Z123212-mediated reduction in AP firing in lamina I/II neurons
during prolonged depolarizations from rest (in slices where both
excitatory and inhibitory synaptic inputs are blocked) but not
when the neurons were tonically hyperpolarized (Fig. 7). During
chronic neuropathic pain states, dorsal horn spinal cord neurons
can become tonically depolarized and/or exhibit prolonged periods
*
#
*
##
y (s)
drawal Latency
Paw Withd
15
15
*
#
ld (g)A
rawal Thresho
Paw Withdr
20
25
#
#
#*
10
*
*
#
*
*
#
#
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#
#
-14 Days01 Hour 2 Hours 4 Hours
Time
10
15
#

-14 Days01 Hour 2 Hours 4 Hours
Time
0
5
#

25
*#
(s)
plate Latency
Hotp
Baseline
60 Min. Treatment
B
C
16
16
20
(μ μM)
ncentration
Con
30 mg/kg Z123212, mean brain
30 mg/kg Z123212, mean plasma
10
15
20
*#
8
12
VehicleNP123212
Z123212
Morphine
0
5
012
Time (h)
34
0
4
Fig. 8. Orally administered Z123212 is efficacious in reversing both acute and chronic neuropathic pain in rats. (A) Z123212 (30 mg/kg, n = 9) reversed both tactile allodynia
(left) and thermal hyperalgesia (right) 14 days after L5/L6 spinal nerve ligation (SNL) with similar efficacy to the gabapentin (30 mg/kg, n = 9) positive control. Measurements
were made before (time 0) or 1, 2, and 4 h after oral drug administration.⁄P < .05 compared to vehicle control (n = 8);#P < .05 compared to predrug (post-SNL) baseline. (B)
Z123212 (30 mg/kg, exposure for 60 min; n = 10) produced acute antinociception (52 ?C hot-plate assay) to a lesser degree than the morphine (100 mg/kg, n = 9) positive
control.⁄P < .05 compared to 0.5% carboxy methyl cellulose vehicle control (n = 11);#P < .05 compared to baseline. (C) Pharmacokinetic analysis reveals that Z123212 is
present at concentrations of approximately 3–5 lM in rat brain tissue (n = 3) and 10–17 lM in the plasma (n = 3) for the 4 h after oral administration.
8
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of fast depolarized firing due to changes in inhibitory and excit-
atory inputs [41,48], the remodeling of ion channel expression
[26], and alteration of electrical gradients [22]. Z123212 exhibited
somewhat greater antinociceptive effects in models of neuropathic
pain compared to acute pain (Fig. 8), and we predict that this may
be due to the ability of Z123212 to preferentially attenuate hyper-
excited neurons within the peripheral and central pain pathways.
Future in vivo experiments could verify this by examining the ef-
fects of Z123212 on peripheral and spinal cord neurons from ani-
mals with enhanced sensitivity for pain [3].
4.2. Molecular targets of Z123212 action
Relevant to nociceptive signaling, Z123212 was found to stabi-
lize the slow inactivation of recombinant TTX-sensitive NaV1.7
channels as well as recombinant TTX-resistant NaV1.8 channels.
Both NaVisoforms are highly expressed and involved in modulat-
ing excitability within peripheral nociceptors [10], and we directly
demonstrated that Z123212 inhibits endogenous TTX-resistant
NaVcurrent within DRG neurons. Slow inactivation is induced at
potentials near neuronal resting membrane potentials (Figs. 3
and 5; [40]) for both NaV1.7 and NaV1.8 isoforms. In this regard,
Z123212 likely reduces DRG excitability by acting on multiple
NaVchannel isoforms, which may also include other prominently
expressed DRG NaVisoforms such as NaV1.1, NaV1.6, and NaV1.9
[10].
To determine how Z123212 influences neurons downstream of
nociceptors in the pain pathway, we evaluated the effects of
Z123212 on uncontaminated TTX-sensitive NaV currents from
spinal cord lamina I/II neurons using the ESI recording technique
[38]. Z123212 inhibited lamina I/II NaVcurrents via their slow-
inactivated state with a similar potency compared to both recom-
binant NaV1.7/NaV1.8 currents and DRG TTX-resistant NaVcurrents
(IC50values between 9 and 17 lM). Lamina I/II neurons have been
shown to contain several functional components of TTX-sensitive
NaVcurrents [38], although the exact NaVchannel isoforms in-
volved have not been thoroughly explored. As Z123212 stabilizes
the slow-inactivated state of functionally distinct NaVisoforms,
the observed reduction of lamina I/II neuronal excitability may
be mediated by one or multiple NaVisoforms. It is known that
the expression of NaV1.3 channel protein is upregulated within
dorsal horn neurons in both peripheral and central neuropathic
pain models [14,15] and that this isoform (along with NaV1.2) is
localized within lamina I/II of the spinal cord [13] (our unpublished
observations). In this regard, Z123212 may in part reduce neuro-
pathic pain signaling by enhancing the natural brakes (slow inacti-
vation) of aberrantly expressed NaV1.3 channels in dorsal horn
neurons. This could be tested in future studies by examining the ef-
fects of Z123212 on recombinant NaV1.3 channels as well as
endogenous NaVcurrents from rats with enhanced sensitivity to
pain.
T-type CaVchannels are functionally expressed and implicated
in modulating the excitability of both DRG and lamina I spinal
cord neurons [20,31,37,39]. More specifically, CaV3.2 T-type chan-
nels have been directly linked to hyperalgesia and allodynia in
various pain animal models [1,6,9,21,29]. We characterized the
slow inactivation properties of recombinant CaV3.2 channels for
the first time and found properties consistent with those previ-
ously described for CaV3.1 channels (Fig. 2C; [17]). Z123212 sig-
nificantly altered the voltage dependence and extent of CaV3.2
slow inactivation as well as the recovery from slow inactivation
(Fig. 2). Given the contributions of CaV3.2 T-type channels toward
nociceptive signaling, the effects of Z123212 observed on AP fir-
ing, hyperalgesia and allodynia may be partly mediated by inter-
actions with CaV3.2 channels. A definitive role for functionally
expressed CaV3.3 channels in peripheral and central nociception
pathways remains to be elucidated. In contrast, attenuation of
central CaV3.1 channel activity has actually been shown to be
pronociceptive [23]; thus, in the central nervous system at least
Z123212 is unlikely to induce antinociceptive effects through this
T-type isoform.
It has been previously shown that NaVand CaVcurrents act syn-
ergistically to prolong subthreshold depolarizations within lamina
I neurons [35], which may account for the greater effect of
Z123212 on lamina I/II neuron excitability compared to its individ-
ual effects on specific NaVand CaV3.2 channel isoforms. Taken to-
gether, we predict that Z123212 exerts its effects on neuronal
excitability and nociceptive signaling by enhancing the combined
slow inactivation of multiple pronociceptive NaVand CaVchannel
isoforms.
Z123212 shares some structural features with lacosamide (the
dipeptide backbone highlighted in Fig. 1), an antiepileptic drug
shown to attenuate chronic pain and enhance slow inactivation
of NaV1.3, NaV1.7, and TTX-resistant DRG NaVcurrents [40]. To
date, the effects of lacosamide on in situ neuronal firing patterns
have only been characterized for cultured neocortical neurons
[12]. We find that lacosamide reduces the AP firing of lamina I/II
spinalcord neuronsat high
(IC50= 150 lM; Fig. 7). In the lamina I/II neuron preparation,
Z123212 inhibits AP firing approximately 300 times more potently
than lacosamide (IC50= 0.48 lM; Fig. 7). The concentration of laco-
samide shown to alter neuronal excitability and affect NaVchannel
slow inactivation (predominantly 100 lM and above; Suppl. Fig. 6)
are generally beyond therapeutic plasma levels achieved by oral
dosing (10–60 lM) [4,12,40]. Of note, a recent study showed that
direct systemic injection of lacosamide could reduce evoked dorsal
horn neuronal responses in vivo [3]. Lacosamide also binds to the
signaling protein collapsin-response mediator protein 2 (affinity
?5 lM) involved in neuroprotection and axonal remodeling, and
it is currently unclear whether the effects of lacosamide on NaV
channel slow inactivation are directly linked to its antinociceptive
properties [4]. We find that Z123212 reduces lamina I/II neuronal
excitability and enhances NaVchannel slow inactivation at concen-
trations (1–3 lM) that are within both therapeutic plasma and
brain tissue levels (10–17 lM and 3–5 lM, respectively). The
ability of Z123212 to target multiple mechanistic elements that
contribute to neuronal hyperexcitability by stabilizing the slow-
inactivated state of both NaVand CaVchannels might create an
additive effect not previously demonstrated for lacosamide.
micromolarconcentrations
4.3. Potential development of novel mixed NaV/CaVchannel
therapeutics
We have identified Z123212 as the first dual modulator of NaV/
CaVchannel slow inactivation and have shown that it is efficacious
in reversing mechanical and thermal hypersensitivity in animal
models of pain. A number of currently marketed therapeutics non-
specifically inhibit T-type CaVchannel isoforms (eg, phenytoin and
ethosuximide), and mechanistically, blockade occurs through the
channel resting state [2,43]. These compounds also nonselectively
block NaVchannels and act on other molecular targets [27,28].
Z123212 represents a novel class of small organic blocker that acts
across the NaVand CaVion channel families but specifically targets
the slow-inactivated state. We predict that the specificity for
affecting channel slow inactivation could enable the preferential
targeting of channels associated with pathophysiological states
linked to hyperexcitability (eg, epilepsy and neuropathic pain). In
support, although Z123212 also affects NaV1.5 and CaV1.2 slow
inactivation under certain experimental conditions, we did not find
off-target cardiovascular effects in isolated rabbit hearts. Further,
we did not observe any adverse effects of high doses of Z123212
in regards to motor coordination.
M.E. Hildebrand et al./PAIN
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Conflict of interest statement
Zalicus
Cambridge, MA. Paula Smith, Cyrus Eduljee, Janette Mezeyova,
Molly Fee-Maki, Yongbao Zhu, Francesco Belardetti, Hassan
Pajouhesh, David Parker, Manjeet Parmar, Elizabeth Tringham,
Gerald Zamponi and Terrance P. Snutch all hold shares and/or
options in Zalicus Inc.
Pharmaceuticalsisasubsidiary of ZalicusInc,
Acknowledgments
We thank Drs Peter Smith, Yishen Chen, Patrick Whelan, and
Pengcheng Han for training in the spinal cord slice technique.
TPS is supported by an operating grant from the Canadian Insti-
tutes of Health Research and a Tier 1 Canada Research Chair in
Biotechnology and Genomics-Neurobiology. MEH was supported
by an Industrial Research and Development Fellowship from the
Natural Sciences and Engineering Research Council of Canada.
GWZ is a scientist of the Alberta Heritage Foundation for Medical
Research and is also supported by a Canada Research Chair in
Molecular Neurobiology.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.pain.2010.12.035.
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