A novel slow‐inactivation‐specific ion channel modulator attenuates neuropathic pain

&NA; 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 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. A novel organic compound stabilizes slow‐inactivated sodium and calcium channels to reduce the excitability of nociceptors and dorsal horn neurons and attenuate neuropathic pain signaling.


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 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.
Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Introduction
Voltage-gated sodium (Na V ) and calcium (Ca V ) channels are crucially involved in nociceptive signaling pathways, in part by mediating ionic currents that contribute to the excitability of peripheral nociceptors in the dorsal root ganglia (DRG). A specific subtype of T-type Ca V channel (Ca V 3.2) 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 Na V 1.7 and TTX-resistant Na V 1.8 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 Na V and Ca V channel isoforms are expressed within lamina I/II neurons [14,42,45] and that both Na V and Ca V channels may increase the excitability of dorsal horn neurons linked to neuropathic and inflammatory pain signaling [11,14,20]. Specific Na V and Ca V isoforms have been shown to play pronociceptive roles; knockout of either Na V 1.7 or Na V 1.8 channels or knockdown of Ca V 3.2 T-type channels reduces hyperalgesia and allodynia in animal models of acute and neuropathic pain [6,25,30]. In humans, loss-of-function mutations in the Na V 1.7 channel lead to complete abolition of pain sensation, while gain-of-function Na V 1.7 mutations cause severe chronic pain syndromes [10]. 0304 Neuropathic pain results from damage to the peripheral or central nervous system and persists long after the nerve injury has resolved [46]. Pharmaceutical approaches to the management of neuropathic pain are limited, and the continued use of some therapeutics can lead to a variety of adverse events and/or desensitization 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 Na V and Ca V channels 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 neurons and other nonhyperexcited targets.
In the current study, we have designed and characterized a lowmolecular-weight, orally available organic compound (Z123212) that stabilizes the slow-inactivated state of Na V and Ca V channels, including TTX-resistant Na V and T-type Ca V channels in DRG neurons and TTX-sensitive Na V channels in lamina I/II spinal cord neurons. 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 potentially synergistic effects by targeting multiple ion channels in components of the peripheral and central nociceptive signaling pathways through a state-dependent mechanism may lead to the development of novel classes of safe and effective pain therapeutics.

Chemistry
The synthesis of Z123212 is illustrated in Suppl. Fig. 1. Briefly, ethylenediamine-N,N-diacetic acid was cyclized under acidic conditions followed by N-tert-butoxycarbonyl (Boc) protection of piperazinone nitrogen. Subsequent coupling with bis-CF3 aniline mediated by O-benzotriazol-1-yl-N,N,N 0 ,N 0 -tetramethyluronium hexafluorophosphate (HATU) in N,N-dimethylformamide (DMF) provided the desired intermediate and was followed by deprotection of the Boc group to generate Z123212.

HEK 293 cell culture, transfection, and electrophysiology
Human embryonic kidney cells (HEK 293) were cultured and either stably or transiently transfected with recombinant Na V and Ca V channel genes as previously described [18]. For Na V channel recordings, the external recording solution contained (in mM): 137 NaCl, 4 KCl, 1. 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 NaHCO 3 , 5 KCl, 1.25 NaH 2 PO 4 , 0.5 CaCl 2 , 7 MgSO 4 , and 1 kynurenic acid, and bubbled with 5% CO 2 /95% O 2 . 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 lamina I neurons, while 350-to 400-lm transverse slices were prepared for voltage-clamp Na V 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 NaHCO 3 , 3 KCl, 1.25 NaH 2 PO 4 , 2 CaCl 2 , 1 MgCl 2 , 1 kynurenic acid, and 0.1 picrotoxin, bubbled with 5% CO 2 /95% O 2 . 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 outer layer of lamina II were selected on the basis of their location relative 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 Na V currents in lamina I/II neurons were performed after slowly (2-5 min) pulling 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 MgCl 2 , 4 MgATP, 0.5 Na 2 GTP, adjusted to pH 7.2 with 5 M KOH and to 290 mOsm with D-mannitol (if necessary). Only tonic firing neurons were selected for current-clamp experiments, while phasic, delayed-onset, and single-spike neurons were discarded [34]. For voltage-clamp recordings of pharmacologically isolated Na V currents [38] in ESI lamina I/II neurons, the external recording solution was a modified TEA-Ringer solution containing (in mM): 95 NaCl, 20 TEACl, 11 D-glucose, 25 NaHCO 3 , 5.6 KCl, 1 NaH 2 PO 4 , 0.1 CaCl 2 , 5 MgCl 2 , 1 kynurenic acid, 0.1 picrotoxin, while the internal patch pipette solution contained (in mM): 140 CsCl, 5.8 NaCl, 1 MgCl 2 , 3 EGTA, 10 HEPES, 4 MgATP, 0.5 Na 2 GTP, 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 À100 mV) for voltage-clamp and with resting membrane potentials (V rest ) more negative than À50 mV for current-clamp were used for subsequent 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.

In vivo pain testing
Spinal nerve ligation (SNL) injury was performed by tight ligation 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 withdrawal threshold and thermal paw withdrawal latency were measured. 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 surface 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.

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 stimulation rate of 1 Hz (basic cycle length = 1 s). The stabilization period was at least 15 min long before obtaining control responses. Experiments 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 allow for equilibration within the heart tissue. The QT interval and QRS duration were calculated by ECG Auto software (EMKA Technologies, Falls Church, VA).

Pharmacokinetic studies
Z123212 was provided as the HCl salt for pharmacokinetic analysis. All dosing was based on the free base weight of the compound. 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 À70°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 analysis of the plasma data were performed by WinNonlin software, version 5.0.1 (Pharsight, Mountain View, CA).

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 À80°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 detectably 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 cellulose at a concentration of 6 mg/mL.

Data analysis
where V m is the test potential, V 0.5a is the half-activation potential, E rev is the extrapolated reversal potential, G max is the maximum slope conductance, and k a reflects the slope of the activation curve. Data from concentration dependence studies were fitted with the equa- where A 1 is initial amplitude (=0) and A 2 is final block value, x o is IC 50 (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 values were set as indicated in the text and figure legends. All data are given as means ± standard errors.

Design and synthesis of Z123212
Given the well-documented roles of Ca V 3.2 T-type channels and Na V channels 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-type and T-type Ca V blockers, the small organic compound Z121912 (molecular weight = 521; Fig. 1) was initially designed on the basis of previous backbones identified as exhibiting promising Ca V blocking and preclinical characteristics [32,47]. In particular, the bis-CF 3 aryl amide group of Z121912 was a preferred structural feature from the aspect of T-type Ca V blocking potency (IC 50 = 64 nM). However, from a drug discovery perspective Z121912 possessed potential cardiovascular liability in that it also potently blocked the hERG potassium channel (IC 50 $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 significantly improved profiles against the hERG potassium channel (IC 50 s >10 lM) with Z123212 being selected for further preclinical assessment on the basis of its favorable pharmacokinetic properties.

Z123212 stabilizes the slow-inactivated state of Ca V 3.2 T-type channels
The blocking activity of Z123212 was initially tested against recombinant Ca V 3.2 channels expressed in HEK cells by using standard depolarizing test pulses from a hyperpolarized holding potential (V hold ; À110 mV) that would place the channels largely in the closed state. Somewhat surprisingly, even at relatively high concentrations, Z123212 caused minimal inhibition of recombinant Ca V 3.2 channels when activated from the closed state (IC 50 )10 lM, Fig. 2A and B). We assessed the effects of Z123212 on other Ca V 3.2 channel properties and found that 10 lM Z123212 also had no significant (P > .05) effect on the voltage dependence of fast channel inactivation (control, V 1/2FastInact = À61 ± 1 mV, n = 5; 10 lM; Z123212, V 1/2FastInact = À66 ± 3 mV, n = 4; data not shown). As previously observed for Ca V 3.1 channels [17], we next examined whether Ca V 3.2 channels undergo a slow inactivation process (Fig. 2C). Z123212 (10 lM) significantly (P < .05) increased the extent of Ca V 3.2 channel slow inactivation at specific membrane potentials and also caused a significant 6 mV hyperpolarizing shift in the voltage dependence of Ca V 3.2 channel slow inactivation ( Fig. 2C; P < .05). The recovery from inactivation  Ca V 3.2 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 Ca V 3.2 channel slow inactivation (control, V 1/2SlowInact = À74 ± 1 mV, n = 4; 10 lM Z123212, V 1/2SlowInact = À80 ± 2 mV, n = 5). Z123212 also caused a significant ( ⁄ P < .05) enhancement of the extent of Na V channel slow inactivation at potentials of À80, À70, À20, and À10 mV. (D) Z123212 (10 lM) significantly (P < .005, n = 4-5) slowed the recovery from Ca V 3.2 channel slow inactivation. Recordings in both (C) and (D) were unpaired and time-matched between control and Z123212 treatment groups to eliminate potential timedependent 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. parameter for Ca V 3.2 channels has been linked to changes in neuronal membrane excitability [33]; thus, we tested the effect of Z123212 on Ca V 3.2 recovery from slow inactivation. Application of Z123212 significantly (P < .01) slowed the recovery from Ca V 3.2 channel slow inactivation at concentrations of 3 lM and greater (control, s recov = 510 ± 30 ms, n = 4; 3 lM; Z123212, s recov = 650 ± 20 ms, n = 4; 10 lM; Z123212, s recov = 680 ± 20 ms, n = 5; Fig. 2D). The Ca V 3.2 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 recombinant Ca V 3.2 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).

Z123212 inhibits recombinant Na V and Ca V channels by modulating slow inactivation
Both TTX-sensitive Na V 1.7 and TTX-resistant Na V 1.8 channels are highly expressed in DRG neurons and exhibit overlapping distributions and functional roles with Ca V 3.2 T-type channels [10,19]. Hypothesizing that Z123212 may also alter Na V channel activity, we tested whether Z123212 could inhibit slowinactivated recombinant Na V channels. In order to induce slow inactivation, sweeps were elicited every 30 s that included a 10-s conditioning pulse to À20 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 Na V 1.7 and Na V 1.8 P2 currents that was even greater than that observed for Ca V 3.2 channels (Fig. 3A and B). Concentration-dependent response studies revealed that Z123212 inhibited Na V 1.7 channels with an IC 50 = 17 lM and Na V 1.8 channels with an IC 50 = 9.2 lM (Fig. 3C).
Because Z123212 stabilized the slow-inactivated state of Ca V 3.2 T-type and Na V 1.7/Na V 1.8 channels, it was of interest to test whether it also acted on other Na V and Ca V channel classes. Similar to that for Ca V 3.2 T-type and Na V 1.7/Na V 1.8 channels, application of 10 lM Z123212 did not cause significant tonic block of other Ca V and Na V isoforms tested (Suppl. Fig. 2). In contrast, 10 lM Z123212 was shown to selectively stabilize the putative slowinactivated states of exogenously expressed Ca V 1.2 (L-type), Ca V 3.1 (T-type), Ca V 3.3 (T-type), and Na V 1.5 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 inhibition) on the Ca V 2.1 (P/Q-type) and Ca V 2.2 (N-type) isoforms (Suppl. Fig. 2). Taken together, Z123212 selectively stabilizes the slowinactivated 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 relevance to the putative hyperexcitable processes associated with various pain states compared to ion channel functioning during normal physiological processes. For example, cardiac Na V 1.5 channels 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 Na V 1.5 channels under certain experimental conditions (involving step depolarization to À20 mV for 10 s; Suppl. Fig. 3A), its effects on Na V 1.5 channels activated by simulated cardiac AP waveforms is greatly reduced (Suppl. Fig. 3B).

Z123212 selectively stabilizes the slow-inactivated state of TTXsensitive Na V channels in lamina I/II spinal cord neurons
We next set out to determine whether Z123212 could alter native Na V channels 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 performed to examine the effects of Z123212 on TTX-sensitive Na V currents in nociceptive spinal cord neurons. In order to ensure adequate voltage-clamp of Na V currents, the ESI technique pioneered by Safronov et al. [38] was used to remove healthy lamina I/II neurons from the slice surface (see Section 2.6 and Suppl. Fig. 4). In this recording configuration, Na V currents in lamina I/II neurons were completely blocked by TTX (data not shown; see [38]). Similar to that for recombinant Na V and Ca V channels, application of 10 lM Z123212 did not result in tonic block of native TTX-sensitive Na V currents during depolarizations from a hyperpolarized state (V hold = À100 mV; Fig. 4A and B). The perfusion of 10 lM Z123212 did result in a small (À2.4 ± 0.5 mV, n = 5) but significant (P < .01) hyperpolarizing shift in the voltage dependence of Na V 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 hyperpolarizing shift was likely not mediated by Z123212.
Application of 10 lM Z123212 also had no effect on the voltage dependence of Na V channel fast inactivation (100-ms conditioning pulses, Fig. 4C). The voltage dependence of Na V channel slow inactivation could not be directly assayed by using the native recording system because the neurons would not tolerate the highly hyperpolarized holding potential (V hold = À120 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 Na V currents during P2 pulses by 45 ± 7% (n = 5; P < .02; Fig. 4D). Analysis of Na V current amplitudes in P1 control pulses versus P2 test pulses further demonstrated that Z123212 selectively stabilized the slow-inactivated state of native TTX-sensitive Na V  Fig. 5). The inhibition by Z123212 of Na V channels reaching the slow-inactivated state (P2) was significant (P < .05) at concentrations of 3 lM and higher, with an IC 50 = 13 lM (Fig. 4E).

Z123212 selectively stabilizes the slow-inactivated state of TTXresistant Na V channels in peripheral nociceptors
The majority of TTX-resistant Na V current in nociceptive DRG neurons is composed of the Na V 1.8 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 Na V currents, perfusion of 10 lM Z123212 did not result in tonic block of native TTX-resistant Na V currents when depolarized from a relatively hyperpolarized potential (V hold = À70 mV; Fig. 5A and B) and also had no significant (P > .05) effect on the voltage dependence of Na V channel activation (Fig. 5A). Further, while application of 10 lM Z123212 did not affect the voltage dependence of fast channel inactivation (Fig. 5C), it significantly (P < .05) enhanced the fraction of Na V channels that reached the slow-inactivated state during conditioning prepulses of À20 mV (as well as more depolarized potentials; Fig. 5D). The concentration dependence for the reduction in P2 current amplitude by Z123212 is shown in Fig. 5E. Using 10-s conditioning prepulses to À20 mV, we calculated an IC 50 = 12 lM.

Z123212 reduces the excitability of peripheral nociceptors and second order spinal cord neurons
Because Z123212 can modulate the activity of several Na V and Ca V channel isoforms linked to neuronal excitability, we used current-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 (À220 ± 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).  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 repeated every 2 min with 1200 ms hyperpolarizing/depolarizing current injection steps ranging from À50 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 depolarizing current injection step (40 ± 6 pA, n = 7) that caused only a minor 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 recordings 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 properties including input resistance (R N ) and V rest remained unchanged (control, R N = 490 ± 60 MX, n = 5, V rest = À63 ± 2 mV, n = 7; 10 lM; Z123212, R N = 460 ± 50 MX, n = 5, V rest = À63 ± 2 mV, n = 7). The inhibition of AP firing by Z123212 was concentration dependent with an IC 50 = 480 nM (Fig. 7C).
Lacosamide is an anticonvulsant that shares some structural features with Z123212 and has been reported to specifically stabilize the slow inactivation of Na V channels [4,12,40]. When tested in the spinal cord slice preparation, lacosamide inhibited lamina I/II neuron AP firing approximately 300 times less potently (IC 50 = 150 lM) than Z123212 (Fig. 7C). Lacosamide did reach a higher level of AP reduction than Z123212, but only at concentrations (>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 conditions where V rest remained unaltered by tonic current injection and the depolarizing current injection steps under analysis followed depolarizing current injection steps using an IV protocol. Thus, Z123212 may exert its inhibitory effects on AP firing by stabilizing accumulated slow inactivation of native Na V and Ca V channels. In support, repeating the above experiments during tonic hyperpolarizing current injection to elicit a more hyperpolarized V rest (À86 ± 3 mV, n = 3) completely eliminated the inhibitory effect of Z123212 (Fig. 7B).

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 SNLinduced model of neuropathic pain (Fig. 8). More specifically, Z123212 significantly (P < .05) reversed both tactile allodynia   Pharmacokinetic analysis revealed that Z123212 was well adsorbed 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 Na V and Ca V channel slow inactivation and to reduce the excitability of nociceptive DRG and lamina I/II neurons (Figs. 2-7).

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 Na V and Ca V channels. The data suggest that by enhancing the slow inactivation of a combination of TTX-sensitive and TTX-resistant Na V and T-type Ca V currents, 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 administered Z123212 to significantly attenuate thermal and mechanical hypersensitivity in rodent models of both chronic neuropathic pain and acute pain.

Mechanism underlying pain-attenuating effects of Z123212
Z123212 inhibits certain Na V and Ca V channels by selectively stabilizing the slow-inactivated state over the fast inactivated state. As channel slow inactivation is significantly enhanced during prolonged depolarizations (eg, À50 mV for 10 s; Fig. 4), Z123212 is predicted to cause pronounced inhibition of Na V and Ca V channels 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 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. of fast depolarized firing due to changes in inhibitory and excitatory 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 hyperexcited neurons within the peripheral and central pain pathways. Future in vivo experiments could verify this by examining the effects of Z123212 on peripheral and spinal cord neurons from animals with enhanced sensitivity for pain [3].

Molecular targets of Z123212 action
Relevant to nociceptive signaling, Z123212 was found to stabilize the slow inactivation of recombinant TTX-sensitive Na V 1.7 channels as well as recombinant TTX-resistant Na V 1.8 channels. Both Na V isoforms are highly expressed and involved in modulating excitability within peripheral nociceptors [10], and we directly demonstrated that Z123212 inhibits endogenous TTX-resistant Na V current within DRG neurons. Slow inactivation is induced at potentials near neuronal resting membrane potentials (Figs. 3 and 5; [40]) for both Na V 1.7 and Na V 1.8 isoforms. In this regard, Z123212 likely reduces DRG excitability by acting on multiple Na V channel isoforms, which may also include other prominently expressed DRG Na V isoforms such as Na V 1.1, Na V 1.6, and Na V 1.9 [10].
To determine how Z123212 influences neurons downstream of nociceptors in the pain pathway, we evaluated the effects of Z123212 on uncontaminated TTX-sensitive Na V currents from spinal cord lamina I/II neurons using the ESI recording technique [38]. Z123212 inhibited lamina I/II Na V currents via their slowinactivated state with a similar potency compared to both recombinant Na V 1.7/Na V 1.8 currents and DRG TTX-resistant Na V currents (IC 50 values between 9 and 17 lM). Lamina I/II neurons have been shown to contain several functional components of TTX-sensitive Na V currents [38], although the exact Na V channel isoforms involved have not been thoroughly explored. As Z123212 stabilizes the slow-inactivated state of functionally distinct Na V isoforms, the observed reduction of lamina I/II neuronal excitability may be mediated by one or multiple Na V isoforms. It is known that the expression of Na V 1.3 channel protein is upregulated within dorsal horn neurons in both peripheral and central neuropathic pain models [14,15] and that this isoform (along with Na V 1.2) is localized within lamina I/II of the spinal cord [13] (our unpublished observations). In this regard, Z123212 may in part reduce neuropathic pain signaling by enhancing the natural brakes (slow inactivation) of aberrantly expressed Na V 1.3 channels in dorsal horn neurons. This could be tested in future studies by examining the effects of Z123212 on recombinant Na V 1.3 channels as well as endogenous Na V currents from rats with enhanced sensitivity to pain.
T-type Ca V channels are functionally expressed and implicated in modulating the excitability of both DRG and lamina I spinal cord neurons [20,31,37,39]. More specifically, Ca V 3.2 T-type channels 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 Ca V 3.2 channels for the first time and found properties consistent with those previously described for Ca V 3.1 channels (Fig. 2C; [17]). Z123212 significantly altered the voltage dependence and extent of Ca V 3.2 slow inactivation as well as the recovery from slow inactivation (Fig. 2). Given the contributions of Ca V 3.2 T-type channels toward nociceptive signaling, the effects of Z123212 observed on AP firing, hyperalgesia and allodynia may be partly mediated by interactions with Ca V 3.2 channels. A definitive role for functionally expressed Ca V 3.3 channels in peripheral and central nociception pathways remains to be elucidated. In contrast, attenuation of central Ca V 3.1 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 Na V and Ca V currents act synergistically 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 individual effects on specific Na V and Ca V 3.2 channel isoforms. Taken together, we predict that Z123212 exerts its effects on neuronal excitability and nociceptive signaling by enhancing the combined slow inactivation of multiple pronociceptive Na V and Ca V channel 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 Na V 1.3, Na V 1.7, and TTX-resistant DRG Na V currents [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 spinal cord neurons at high micromolar concentrations (IC 50 = 150 lM; Fig. 7). In the lamina I/II neuron preparation, Z123212 inhibits AP firing approximately 300 times more potently than lacosamide (IC 50 = 0.48 lM; Fig. 7). The concentration of lacosamide shown to alter neuronal excitability and affect Na V channel 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 Na V channel slow inactivation are directly linked to its antinociceptive properties [4]. We find that Z123212 reduces lamina I/II neuronal excitability and enhances Na V channel slow inactivation at concentrations (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 slowinactivated state of both Na V and Ca V channels might create an additive effect not previously demonstrated for lacosamide.

Potential development of novel mixed Na V /Ca V channel therapeutics
We have identified Z123212 as the first dual modulator of Na V / Ca V channel 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 nonspecifically inhibit T-type Ca V channel isoforms (eg, phenytoin and ethosuximide), and mechanistically, blockade occurs through the channel resting state [2,43]. These compounds also nonselectively block Na V channels and act on other molecular targets [27,28]. Z123212 represents a novel class of small organic blocker that acts across the Na V and Ca V ion 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 Na V 1.5 and Ca V 1.2 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.