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NEUROPHARMACOLOGY

Pharmacology of 2-[4-(4-Chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide: A Potent, Broad-Spectrum State-Dependent Sodium Channel Blocker for Treating Pain States

Discovery Research, Purdue Pharma LP, Cranbury, New Jersey (V.I.I., P.I.T., S.O.); Algos Therapeutics, St. Paul, Minnesota (J.D.P., S.G.); Wyeth Research, Princeton, New Jersey (G.T.W., J.E.H.); Merck and Company, West Point, Pennsylvania (M.S.P.); Schering Plough Research Institute, Kenilworth, New Jersey (L.M.); Novartis Pharmaceutical Corporation, East Hanover, New Jersey (R.B.C.); Allergan, Inc., Irvine, California (P.N.); Department of Pharmacology, University of California, Irvine, California (D.J.H.); Amicus Therapeutics, Cranbury, New Jersey (E.B.); and Adolor Corporation, Exton, Pennsylvania (R.M.W.)

Received March 16, 2006; accepted May 23, 2006.

	Abstract

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Voltage-gated Na⁺ channels may play important roles in establishing pathological neuronal hyperexcitability associated with chronic pain in humans. Na⁺ channel blockers, such as carbamazepine (CBZ) and lamotrigine (LTG), are efficacious in treating neuropathic pain; however, their therapeutic utility is compromised by central nervous system side effects. We reasoned that it may be possible to gain superior control over pain states and, in particular, a better therapeutic index, by designing broad-spectrum Na⁺ channel blockers with higher potency, faster onset kinetics, and greater levels of state dependence than existing drugs. 2-[4-(4-Chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide (PPPA) is a novel structural analog of the state-dependent Na⁺ channel blocker V102862 [4-(4-fluorophenoxy)benzaldehyde semicarbazone]. Tested on recombinant rat Na_v1.2 channels and native Na⁺ currents in cultured rat dorsal root ganglion neurons, PPPA was approximately 1000 times more potent, had 2000-fold faster binding kinetics, and 10-fold higher levels of state dependence than CBZ and LTG. Tested in rat pain models against mechanical endpoints, PPPA had minimal effective doses of 1 to 3 mg/kg p.o. in partial sciatic nerve ligation, Freund's complete adjuvant, and postincisional pain. In all cases, efficacy was similar to clinically relevant comparators. Importantly, PPPA did not produce motor deficits in the accelerating Rotarod assay of ataxia at doses up to 30 mg/kg p.o., indicating a therapeutic index >10, which was superior to CBZ and LTG. Our experiments suggest that high-potency, broad-spectrum, state-dependent Na⁺ channel blockers will have clinical utility for treating neuropathic, inflammatory, and postsurgical pain. Optimizing the biophysical parameters of broad-spectrum voltage-gated Na⁺ channel blockers may lead to improved pain therapeutics.

Multiple lines of evidence now implicate voltage-gated Na⁺ channels in establishing and maintaining patterns of pathological neuronal excitability that underlie chronic pain. For example, nerve injury and inflammation cause changes in the levels of expression and distribution of Na⁺ channel subunits and subunits in primary afferent neurons, leading to changes in action potential parameters and firing patterns (e.g., Coward et al., 2000; Waxman et al., 2000; Craner et al., 2002; Takahashi et al., 2003; Coggeshall et al., 2004; Hong et al., 2004; Lai et al., 2004). Neuronal hyperexcitability is associated with functional modulation of Na⁺ channels by signal transduction cascades (Lai et al., 2004; Chahine et al., 2005). Blocking and reversing changes in Na⁺ channel expression in dorsal root ganglion (DRG) in vivo reduces hypersensitivity in animal pain models (Lai et al., 2002; Gold et al., 2003; Hains et al., 2004). Mice with selective Na⁺ channel subunits genetically knocked out show reduced sensitivity in pain models. In particular, Na_v1.8-null mice demonstrate altered thresholds to mechanical and thermal stimuli compared with wild-type and show deficits in visceral pain (Akopian et al., 1999; Laird et al., 2002), whereas Na_v1.7 knockout mice have substantially reduced sensitivity to mechanical and thermal stimuli in models of inflammatory pain (Nassar et al., 2004, 2005).

Therefore, it is not surprising that drugs with Na⁺ channel-blocking activity, such as carbamazepine (CBZ), lamotrigine (LTG), mexiletine, and lidocaine, show efficacy in animal pain models (Hunter et al., 1997; De Vry et al., 2004; Kiguchi et al., 2004) and, moreover, have utility in treating pain states in humans (e.g., Mao and Chen, 2000; Jensen, 2002; Petersen et al., 2003). However, these drugs were developed as anticonvulsants, antiarrhythmics, and anesthetics, with no attempt to optimize the Na⁺ channel-blocking activity for treating chronic pain. In general, the therapeutic utility of these agents is compromised by dose-limiting side effects, particularly sedation, headache, dizziness, and motor disturbances (Mao and Chen, 2000; Jensen, 2002; Petersen et al., 2003).

2-[4-(4-Chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide (PPPA) (Fig. 1, insert) is a novel structural analog of the state-dependent Na⁺ channel blocker V102862 (Co 102862) (Dimmock et al., 1996; Ilyin et al., 2005). PPPA was discovered as part of a systematic exploration of V102862 analogs where the phenoxyphenyl portion of the molecule was kept constant and the semicarbazone moiety was substituted by various heterocycles. The goal of the research was to design Na⁺ channel blockers with higher in vitro potency, faster binding kinetics, and more pronounced levels of state dependence compared with existing drugs. The Na⁺ channel subtype selectivity of these compounds was consistently <10-fold (V. I. Ilyin, unpublished data). Selected compounds were profiled in rat pain models to see whether altering the biophysics of inhibition at the molecular level translated into improved potency, efficacy, and side effects compared with clinically relevant drugs. In the current paper, we report the data for PPPA.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Voltage-dependent block of INa and retardation of recovery from inactivation in rNav1.2/HEK-293 cells. The currents shown in this and other figures were measured after 1 min in control or drug-containing solutions, i.e., under steady-state drug binding conditions. INa were elicited by pulsing to 0 mV (maximal INa), either from holding potentials of –120 mV (A) or –70 mV (B), first under control conditions and then in the presence of 1 µM PPPA (lower and upper traces in each pair, respectively). Dashed line, zero current level. C, retardation of recovery from inactivation. The rate of recovery from inactivation was assessed using a double-pulse protocol where the depolarizing prepulse was followed by a step back to the initial negative holding voltage for varying durations (recovery gap) and then by a test pulse to examine the extent of channel recovery from inactivation (repriming). The holding (and gap) voltage was set at –120 mV to remove residual steady-state inactivation, the conditioning prepulse was set to –20 mV to drive all channels into inactivated states, and the test pulse voltage was 0 mV. Duration of the conditioning prepulse was chosen to be sufficiently long (1 s) to permit a steady-state level of binding of the drug to inactivated channels. Hyperpolarizing gap was varied in t = 10-ms increments. As presented in the figure, the recovery was measured first in control, then in the presence of 1 µM PPPA in three different HEK-293 cells, and normalized to the size of current taken without the depolarizing prepulse. The double-exponential fit (solid line) was done for the averaged control data set: fast component has 1 5 ms; slow component has 2 >500 ms. In the presence of 1 µM PPPA, the averaged time course of recovery followed a single exponential with 61 ms in this experimental series. Insert, chemical structure of PPPA.

	Materials and Methods

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Materials and reagents were purchased from Sigma-Aldrich Company (St. Louis, MO) unless otherwise noted.

Molecular Biology
A cell line expressing rNa_v1.2 channels was constructed using conventional methods. In brief, the plasmid pCMV-RIIA, which encodes the rat type IIa Na⁺ channel under control of the cytomegalovirus promoter, was obtained from Dr. Alan Goldin (University of California, Irvine, CA). Bacterial growth, DNA preparations, and restriction enzyme characterizations were performed using standard procedures. HEK-293 cells were plated at a density of 3 x 10⁵ cells/well in a six-well plate and transfected the following day with pCMV-RIIA DNA using Transfast reagent (Promega, Madison, WI). After 3 weeks of selection in 400 µg/ml G418, resistant colonies were isolated, expanded, and characterized by electrophysiological analysis. Several clones expressing tetrodotoxin-sensitive (TTX-S) inward currents were identified. Clone B2 (NaIIA-B2), with mean current densities of 250 pA/pF, was expanded and further electrophysiological analyses performed. The currents were completely blocked by 400 nM TTX and reversed at the predicted equilibrium potential for Na⁺, 65 ± 2 mV (n = 15). The data indicate that the currents were due to activation of voltage-gated Na⁺ channels. Untransfected cells displayed small (10 pA/pF) and variable inward currents that were only partially blocked by 1 µM TTX.

Electrophysiology
Cell Preparation. HEK-293 cells. HEK-293 cells were cultured using standard techniques (Ilyin et al., 2005). For electrophysiology, cells were plated onto 35-mm Petri dishes precoated with poly-D-lysine at a density of 2 to 4 x 10⁴ cells/dish on the day of reseeding from confluent cultures. HEK-293 cells were suitable for recordings for 3 to 4 days after plating.

Isolation and acute culture of rat DRG neurons. Acutely dissociated DRG neurons were prepared from newborn (3–8 days postnatal) Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) using a modification of published procedures (Rush et al., 1998). In brief, pups were anesthetized on ice and sacrificed by rapid decapitation. DRG were dissected out in cold calcium- and magnesium-free Hanks' buffer (CMF; Invitrogen, Carlsbad, CA), trimmed, and incubated for 90 min at 35°C in 0.3 mg/ml collagenase in Krebs buffer (120 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, and 25 mM D-glucose, pH 7.4). Ganglia were rinsed in CMF at room temperature and incubated for 30 min at 35°C in 2.5 mg/ml trypsin in CMF and then rinsed with Krebs buffer at room temperature and triturated using fire-polished Pasteur pipettes. Liberated cells were plated on poly-D-lysine-precoated 35-mm plastic Petri dishes in Krebs buffer. Cells were suitable for electrophysiological recordings for up to 2 weeks.

Whole-Cell Patch-Clamp Recordings. Whole-cell voltage-clamp recordings were made using standard patch-clamp technique at room temperature (22°C). Currents were recorded using an Axopatch 200A amplifier (Axon Instruments, Union City, CA) and were leak-subtracted (P/4 or by the built-in analog circuitry), low-pass filtered (3 kHz, eight-pole Bessel), digitized (20–50-µs intervals), and stored using Digidata 1200 B interface and pClamp8/Clampex software (Axon Instruments). The patch-clamp pipettes were pulled from thick-walled borosilicate glass (WPI, Sarasota, FL). The pipette resistances ranged from 1.5 to 3 M. Residual series access resistance was in the range of 0.5 to 1.2 M after partial (75–80%) cancellation using built-in amplifier circuitry. In the recording chamber, cells were continuously superfused at a speed of 1 ml/min with physiological saline solution.

Solutions and Drug Application. HEK-293 cells. The external solution for NaIIA-B2 (HEK-293) cells contained 150 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 10 mM D-glucose, and 5 mM HEPES, pH 7.4 (NaOH). The internal solution contained 130 mM CsF, 20 mM NaCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES, pH 7.4 (CsOH); osmolality was set at 10 mOsmol/kg, lower than that for the external solution.

Rat DRG neurons. Small (<25 µm) and medium (25–40 µm) neurons were used for assaying compounds on fast TTX-S and slow tetrodotoxin-resistant (TTX-R) Na⁺ currents. External solution was 65 mM NaCl, 5 mM KCl, 50 mM choline chloride, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM D-glucose, 5 mM HEPES, 5 mM HEPES-Na⁺ salt, and 20 mM TEA chloride, pH 7.4 (NaOH). The internal solution had the same ionic content as that used for HEK-293 cell recordings. Osmolality was set by 5 mOsmol/kg, lower than that for the bathing solution.

Drug solutions and intervening intervals of wash were applied through a linear array of flow pipes (Drummond Microcaps, 2 µl, 64-mm length; Drummond Scientific, Broomall, PA) producing full solution exchange within a few hundred milliseconds. PPPA, LTG, and CBZ were dissolved in dimethylsulfoxide (DMSO) and then diluted in DMSO to make 0.003 to 200 mM stock solutions. The stock solutions were diluted into external solution on each day of experimentation to generate final concentrations of 0.003 to 200 µM. At the highest concentration (200 µM for LTG and CBZ), the concentration of DMSO was 0.2%. In these experiments, data were corrected for a minor inhibition caused by the vehicle. CBZ was from RBI (Natick, MA), and LTG was synthesized in-house. PPPA was synthesized as described in an issued United States patent (Hogenkamp et al., 2005).

Protocols and Data Analysis. The voltage-pulse protocols and procedures used to measure the state-dependent block of voltagegated Na channels were described in detail in a previous article (Ilyin et al., 2005). Some adjustments were made to allow for kinetic differences. Data are presented as mean ± S.E. (n = number of experiments) with statistical significance assessed with paired t-tests. The level of significance was set at P < 0.05.

Fluorescence Plate Reader Measurements
Fluorescence Plate Reader Calcium Mobilization Assay for IMR-32 Cells. PPPA was profiled in human neuroblastoma IMR-32 cells expressing significant proportion of N-type Ca²⁺ channels. The channels were opened by addition of KCl, and Ca²⁺ mobilization was measured by fluo-4 fluorescence on a fluorescence plate reader, FLIPR⁹⁶ (Molecular Devices, Inc., Sunnyvale, CA).

The assay is described in detail elsewhere (Benjamin et al., 2006). In brief, 1 day before performing this assay, IMR-32 cells were seeded on poly-D-lysine-coated 96-well clear-bottom black plates (Becton Dickinson, Franklin Lakes, NJ) at 75,000 cells/well. On the day of the assay, the cell plates were washed with assay buffer (127 mM NaCl, 1 mM KCl, 2 mM MgCl₂, 700 µM NaH₂PO₄, 5 mM CaCl₂, 5 mM NaHCO₃, 8 mM HEPES, 10 mM glucose, pH 7.4). Cells were loaded with 0.1 ml of assay buffer containing 20 µM nifedipine and 6 µM fluo-4-AM for 1 h at 37°C. Cells were then washed once with 0.1 ml of assay buffer containing 20 µM nifedipine, then replaced with 0.05 ml of the same. Plates were transferred to the FLIPR⁹⁶. Basal fluo-4 fluorescence was measured for 15 s, then 0.05 ml of each compound diluted at a 4x concentration in assay buffer was added, and fluorescence was read for another 5 min. Then, 0.1 ml of 180 mM KCl dissolved in assay buffer was added, and fluorescence was measured for another 45 s. Final test compound concentrations on the cells after FLIPR read ranged from 1 nM to 20 µM [10 pM to 20 µM for -conotoxin (-ctx) MVIIA, positive control], final nifedipine concentration was 5 µM, and final KCl concentration was 90 mM. Final DMSO concentration was held constant at 0.5%. Data were collected over the entire time course of the FLIPR read and then analyzed using Prism (version 3.02; GraphPad, San Diego, CA) software.

Data Analysis. Experiments were expressed as percentage of control. This refers to the average of multiple determinations normalized to the maximal average counts in the presence of 90 mM KCl and to the minimal average counts in the presence of 50 nM -ctx MVIIA block of 90 mM KCl. Normalization calculations were conducted using Prism software or Microsoft Excel (Microsoft, Redmond, WA). Theoretical curves were generated using nonlinear regression curve-fitting analysis in Prism software.

In Vivo Pharmacology
Compounds, Animals, and General Procedures. For all in vivo pharmacological studies, PPPA was administered p.o. as a suspension in 0.5% methylcellulose dissolved in distilled water. The µ-opioid receptor agonist morphine was dissolved in 0.9% saline solution and administered s.c. The anticonvulsant gabapentin (Kemprotec, Middlesborough, UK) was dissolved in 0.9% saline and administered by i.p. injection. The cyclooxygenase-2 inhibitor celecoxib (Toronto Research Chemicals, Toronto, ON, Canada), the nonsteroidal anti-inflammatory drug indomethacin, and the anticonvulsant CBZ were suspended in 0.5% methylcellulose and administered p.o. The anticonvulsant LTG was dissolved in 25% hydroxypropyl--cyclodextrin and administered via i.p. injection. The Purdue Institutional Animal Care and Use Committee approved all animal procedures according to the guidelines of the Office of Laboratory Animal Welfare. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 90 to 110 g at the start of nerve ligation experiments or 180 to 200 g at the start of acute, inflammatory, incisional, and Rotarod experiments were used. Animals were group-housed and had free access to food and water at all times, except before oral administration of drugs when food was removed 12 h before dosing. For comparison with compound-treated groups, animals treated with appropriate drug vehicle were included in each experiment. The volume of administration and all other experimental procedures and conditions for vehicle and compound-treated rats were identical. All experiments were blinded to behavioral testers through use of randomization codes.

Postsurgical Hyperalgesia. The effect of PPPA on postsurgical pain was assessed using an incisional pain model, as described previously by Brennan et al. (1996). Hind paw withdrawal thresholds (PWTs) to a noxious mechanical stimulus were determined using a model 7200 analgesimeter (Ugo Basile, Varese, Italy). Cut-off was set at 250 g, and the endpoint was taken as complete paw withdrawal. PWT was determined once for each rat at each time point. Baseline PWTs were determined, and a 1-cm longitudinal incision was made through skin and fascia of the plantar aspect of the paw. Unoperated rats served as controls. Twenty-four hours following plantar incision, predrug PWTs were measured, and the rats (n = 9–20/group) received a single dose of 3, 10, or 30 mg/kg p.o. PPPA, 30 mg/kg p.o. celecoxib, or vehicle. PWTs were determined again 1, 3, 5, and 24 h postdrug administration. Percent reversal of hyperalgesia for each animal was defined by eq. 1:

(1)

Hyperalgesia Due to Nerve Injury. The partial sciatic nerve ligation model (PSN) was used as a model of nerve injury-related pain in rats, as described previously by Seltzer et al. (1990). PWTs to a noxious mechanical stimulus were determined as described above. Baseline PWTs were determined and partial ligation of the left sciatic nerve was performed under isoflurane (2% in oxygen) inhalation anesthesia. Three weeks following nerve ligation, predrug PWTs were measured, and the rats received a single dose of 3, 10, or 30 mg/kg p.o. PPPA, 100 mg/kg i.p. gabapentin, or vehicle. PWT was again determined 1, 3, 5, and 24 h postdrug administration. In separate experiments, the potency and efficacy of CBZ and LTG were examined using doses of 30, 100, and 300 mg/kg p.o. for CBZ and 10, 30, and 100 mg/kg i.p. for LTG. In both cases, 100 mg/kg i.p. gabapentin served as the positive control.

Inflammatory Hyperalgesia. The efficacy of PPPA against hyperalgesia associated with inflammation was investigated using the Freund's complete adjuvant (FCA) model in rats. PWTs to a noxious mechanical stimulus were determined as described above. Baseline PWT was determined, the rats (n = 8–28/group) were anesthetized with isoflurane (2% in oxygen) and received an intraplantar injection of 50% FCA (50 µl diluted in 0.9% saline) to the left hind paw. Twenty-four hours following FCA injection, predrug PWTs were measured, and the rats received a single dose of 1, 3, or 10 mg/kg p.o. PPPA, 30 mg/kg p.o. celecoxib (the positive control), or vehicle (i.p. volume = 2 ml/kg). PWT was again determined 1, 3, 5, and 24 h postdrug administration.

The Rotarod Assay of Ataxia/Motor Coordination. To assess effects of PPPA, CBZ, and LTG on motor performance, rats were tested using the accelerating Rotarod (Accuscan, Columbus, OH). The Rotarod was set to accelerate from 4 to 40 rpm over 300 s with the maximal time spent on the Rotarod set at 300 s. Rats (n = 10/group) received two training trials on the first day and then received a single dose of 3, 10, or 30 mg/kg p.o. PPPA, 100 mg/kg i.p. gabapentin (positive control), or vehicle. Animals were re-tested on the Rotarod 1, 3, 5, and 24 h following drug administration. In separate experiments, rats received 30, 100, or 300 mg/kg p.o. CBZ, 10, 30, or 100 mg/kg i.p. LTG, 100 mg/kg i.p. gabapentin (positive control), or vehicle, and were tested according to the same schedule.

Acute Analgesia. The effect of PPPA on acute analgesia was investigated using the tail-flick assay. Rats (n = 9–12/group) were placed on the apparatus (Ugo Basile), and an infrared beam was focused onto the tail, 5 cm from the tip. The latency to tail flick was assessed. Cut-off was set at 20 s, and the intensity was set to 35%. Latency was determined once for each rat at each time point. Baseline latency was determined and approximately 1 h later the rats received a single dose of 1, 3, or 10 mg/kg PPPA, 10 mg/kg morphine, or vehicle. Latencies were determined again 1, 3, and 5 h postdrug administration.

Statistical Analysis. Data are presented as the mean ± S.E.M. Untransformed data (thresholds/latencies) were analyzed using a one-way analysis of variance. In instances where a main effect was detected, planned comparisons were made using Fisher's protected least significant difference post hoc analysis. The level of significance was set at P < 0.05. Minimal effective dose (MED) and minimal ataxic dose (MAD) represent the lowest tested dose that resulted in a statistically different result as compared with vehicle-treated rats at any time point tested.

	Results

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State-Dependent Inhibition of rNa_v1.2 Channels by PPPA
Voltage Dependence of Inhibition. rNa_v1.2 currents in HEK-293 cells are half-maximally activated by depolarization from –120 to –18.2 ± 0.3 mV (n = 13) with a steepness of 5.7 ± 0.3 mV and reach maximum at 0 mV (Ilyin et al., 2005). With 100-ms depolarizing conditioning prepulses, half-maximal inactivation occurs at –53.9 ± 1.7 mV with the slope of 6.4 ± 0.1 mV per e-fold change in membrane potential (n = 17). The currents were >95% inactivated at membrane voltages above –20 mV.

Exploratory studies showed that inhibition of recombinant Na⁺ currents by PPPA was clearly voltage-dependent. For example, when the holding voltage was set at –120 mV, 1 µM PPPA blocked only 5% of peak current (Fig. 1A). However, when the holding voltage was –70 mV, inhibition rose to 85% (Fig. 1B). At 1 µM, PPPA did not obviously affect the time course of either activation or inactivation of I_Na or the voltage dependence of activation (data not shown). The inhibition was fully reversible upon removal of the drug, although at depolarized voltages the recovery required a longer interval of wash; for example, the time constant of the washout of PPPA, 3 µM was 35 ± 4 s (n = 4) and 96 ± 9 s (n = 4) at –120 mV and –80 mV, respectively (P < 0.006). The profound voltage dependence of inhibition is qualitatively consistent with PPPA preferentially binding to Na⁺ channels in their inactivated states rather than to the resting states. Thus, the overall affinity of the drug toward the channel at any membrane voltage is a weighted average of the affinity toward resting and inactivated states and should increase with depolarization as the proportion of inactivated channels increases.

Inhibition of Resting Channels. The dissociation constant K_r toward resting state was estimated by measuring the degree of inhibition of the peak I_Na elicited by depolarizing pulses from a rather negative membrane voltage, i.e., –120 mV (Fig. 1A), assuring nearly zero steady-state inactivation. At this voltage, block of the peak I_Na is weak since the affinity of the drug toward resting channels is low. Assuming 1:1 binding ratio, K_r, calculated/extrapolated from the equation K_r = {fractional response/(1 – fractional response)} x [antagonist] was 22 ± 5 µM (n = 10) (Table 1).

Retardation of Repriming Kinetics. In the absence of drug, recovery of rNa_v1.2 channels from inactivation caused by 1-s-long depolarizing prepulse from –120 to –20 mV was biphasic. Approximately 90% of channels recovered with a time constant of 5.0 ms and the remaining 10% with a time constant of >500 ms (Fig. 1C; also see Ilyin et al., 2005). In 1 µM PPPA, the fast component of recovery was retarded to 53 ± 5 ms (n = 8) (Table 1), whereas the slower component was essentially unchanged. Retardation of recovery did not depend on PPPA concentration; the repriming constant was 62 ± 3(n = 3) and 60 ± 4(n = 3) ms for 0.1 and 1.0 µM PPPA, respectively, when measured at –120 mV in one and the same cell (P > 0.2). The lack of concentration dependence of repriming time constant is easy to understand given that the transition from the state "PPPA bound to inactivated channel" to the state "PPPA bound to resting channel" is a monomolecular reaction (e.g., see the kinetic schema in Ilyin et al., 2005); thus, it should not depend on the drug concentration. However, this transition is strongly affected by the membrane voltage. Hyperpolarization promotes faster recovery from inactivation both in control and in the presence of drug (data not shown).

Binding Kinetics. Significant retardation of repriming from inactivation by PPPA provided a way to measure the rate of binding and affinity of the drug to inactivated channels using a double-pulse protocol. The duration of the conditioning prepulse, from –120 to –20 mV, was increased in increments of 10 to 40 ms, whereas the duration of the hyperpolarizing gap was set at 5 ms. The 5-ms gap produced only a negligible contribution to the recovery of liganded channels. Thus, the decrease in the amplitude of I_Na, compared with the current in control, was a satisfactory measure of drug-bound (and inhibited) inactivated channels. Subsequent episodes of stimulation were repeated every 15 s to allow all the recovery processes to occur. Figure 2, A and B, show examples of I_Na traces obtained from this type of experiment. Under control conditions, the peak I_Na showed only a slight trend to drop with the duration of the depolarizing prepulse, whereas in the presence of 0.03 µM PPPA, the current decreased in subsequent stimulation episodes due to progressive binding of the drug to inactivated channels. Binding reached its apparent steady state at the prepulse duration of 480 ms (the 12th stimulation cycle). The drug was potent causing approximately 50% inhibition at 0.03 µM. The amplitude of the peak I_Na was plotted against the duration of the inactivating prepulse to give the binding curve. Contamination with nonrecovered fast inactivating and slow inactivating channels was corrected by calculating the difference between I_Na in control and in the presence of PPPA. The corrected time course of inhibition was well fit by a monoexponential (Fig. 2C). Figure 2D shows that the microscopic binding rates increase linearly with drug concentration consistent with Langmuir binding theory. This supports the idea that PPPA, and other inhibitors of this class, bind to Na⁺ channels in a simple 1:1 mode. From the linear fit, the microscopic rate constant k₊ (the pseudo-first order rate constant) for PPPA binding to the inactivated channels was 97 µM^–1s^–1 (Table 1). This value indicates that the binding of PPPA to inactivated channels is fast but is still about an order of magnitude slower than the rate of diffusion-limited reactions.

View larger version (28K): [in this window] [in a new window]   Fig. 2. The binding rate of PPPA to inactivated rNav1.2 channels. A, control: from a holding voltage of –120 mV, the cell was pulsed in duration increments of 40 ms to –20 mV every 15 s. Gap voltage (–120 mV, 5 ms) was applied after the depolarizing prepulse to recover inactivated and unliganded channels. The 10-ms test pulse to 0 mV gave a measure of channels available for activation. Thirteen successive traces (episodes) were superimposed; downward deflections represent inward Na currents evoked by the test pulses in successive episodes. In control, currents show a small reduction in amplitude with increasing duration of conditioning prepulse. B, same protocol in the presence of 0.03 µM PPPA. Progressive drop in the size of current over time reflects the increase in the proportion of Na+ channels bound and inhibited by PPPA. C, peak current amplitudes from A and B were plotted against duration of inactivating prepulse to get the binding curves. The time course of current decay in control (filled circles) was subtracted from the time course of the decay in the presence of the drug (open circles). The resulting curve (crossed circles) tracks the binding of PPPA to inactivated channels. All three curves were fitted by single exponentials. Two parameters were determined for the binding curve at each particular drug concentration: the time constant, , of binding to inactivated channels and fractional response FR in steady state. The latter was measured as a ratio of peak currents in control and in the presence of the drug. D, similar analysis was done for various PPPA concentrations, 0.03, 0.1, 0.3, and 1.0 µM. The microscopic binding rates for PPPA (the inverses of the time constants as measured in C) taken from six cells and plotted against concentration of the drug. The line is linear regression fit to mean values. The slope is 97 µM–1s–1 and numerically equal to the pseudo-first order constant k+ of PPPA binding to inactivated channels.

Affinity for the Inactivated State. To estimate the apparent dissociation constant (K_i) of PPPA for inactivated rNa_v1.2 channels, we constructed partial, steady-state concentration-inhibition curves and fit the data with the Hill equation: 1/(1 + ([PPPA]/K_i)^p), where p is the slope (Fig. 3A). The K_i was 0.041 ± 0.008 µM (n = 5) (Table 1). The slope of the individual fits varied between 0.7 and 1.0.

View larger version (25K): [in this window] [in a new window]   Fig. 3. A, set of semilogarithmic concentration-inhibition curves for inhibition of rNav1.2 channels by PPPA taken for conditioning (inactivating) prepulses to –20 mV in five different cells. Fractional responses were measured in steady state as illustrated in Fig. 2, A and B. The curves are the best sigmoidal fits according to the eq. 1/(1 + ([PPPA]/Ki)p). B, frequency dependence (use dependence) of inhibition of 10 Hz trains of maximal rNav1.2 currents measured from a holding voltage of –70 mV with 5-ms depolarizing pulses to 0 mV. The peak currents in each train were normalized to the size of the first current in a series and plotted against the pulse number (20 pulses in total). The curves are monoexponential fits. The size of the current to the 20th depolarizing pulse is 0.94, 0.69, and 0.40 for control and 0.1 and 3 µM PPPA, respectively.

Use-Dependent Block. In addition to the voltage-dependent inhibition observed with low excitation frequencies (<0.5 Hz), state-dependent blockers can also produce the phenomenon of use dependence, where inhibition is enhanced during sustained or higher frequency depolarizations. Mimicking the repetitive discharge seen in hyperexcitable neurons, we assessed the ability of PPPA to "filter" high-frequency electrical activity by measuring use-dependent block of trains of depolarizing voltage pulses of varying frequency. Figure 3B shows the amplitudes of currents evoked by 10 Hz trains of 20 5-ms pulses from –70 (the voltage with <10% of channels in inactivated state) to 0 mV in control conditions and in the presence of 0.1 and 3 µM PPPA. In control, there was a small, steady decline in the current amplitude. In the presence of PPPA, there was an additional decline in the current due to accumulation of use-dependent block. The use-dependent block became more pronounced with increasing concentration of the drug and frequency of the pulse train (data not shown). A stimulus frequency of 10 Hz was sufficient to clearly show use-dependent block with 0.1 µM PPPA, whereas with 3 µM PPPA, the block became very strong. The results of studies on inhibition of rNa_v1.2 channels in HEK-293 cells by PPPA are summarized in Table 1.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Traces and I-V curve for TTX-R current, Vh = –90 mV. A, current traces at different depolarizing pulses. Little inactivation is seen with low-threshold, type R2 (Rush et al., 1998) TTX-R current (up to –20 mV). High levels of inactivation are seen for type R1 current measured at 0 mV. B, I-V-curve for the inward current has two distinct peaks at approximately –20 and 0 mV. Current reverses at approximately + 55 mV, close to the Nernstian potential for Na+ ions. C to E, measuring fractional response (FR) for inhibition of R1 type TTX-R current in a rat DRG neuron by PPPA. C, TTX-R current in response to double-pulse protocol in control. One-second-long depolarizing prepulse to 0 mV was followed up by the 10-ms hyperpolarizing step back to –90 mV and then by a 10-ms-long test pulse to 0 mV. D, current in the presence of 0.03 µM PPPA. Calibration bars are the same for C and D. E, current traces (from C and D) in response to a second, testing voltage pulse shown at a higher gain and speed. The ratio of peak amplitudes was used as the FR to plot inhibition-concentration curves.

TTX-R Currents. Of the different TTX-R currents seen in DRG neurons (Fig. 4A), we assessed PPPA effects on the component (R1) that activates close to –30 mV, peaks at around 0 mV, and shows relatively slow but profound inactivation with V_half of –35 mV (complete at 0 mV) (Rush et al., 1998; Lai et al., 2004). There is now compelling evidence to suggest that Na_v1.8-containing channels are the major channels underlying this high-threshold TTX-R current (Lai et al., 2004). A double-pulse protocol was again used to measure K_i. In this case, the holding voltage was –90 mV. One-second-long conditioning prepulse to –10 mV elicited Na⁺ current that inactivated by the end of the depolarization. Then, a 10-ms hyperpolarizing gap (to –90 mV) was applied to recover nonliganded channels from the fast inactivation. The subsequent test pulse to 0 mV was used to assay the fraction of the channels available for activation. The protocol was run before and after drug application (Fig. 4, C–E). Even under control conditions, the size of the current to the second pulse was 20% of the current to the first pulse. This was due to marked slow inactivation of TTX-R1 channels, which needed a few seconds at polarized potentials for recovery. Nevertheless, the size of the test current was still big enough to assay inhibition caused by PPPA (Fig. 4D). Fractional inhibition was collected for various concentrations of the blocker and used to draw the partial concentration-inhibition curve as shown in Fig. 3. The K_i was 0.046 ± 0.014 µM (n = 5) (Table 2).

TTX-Sensitive Currents. For assaying effects on TTX-S currents, the holding voltage was –120 mV, with a 1-s prepulse to –10 mV (causing complete inactivation), followed by a 5-ms hyperpolarizing gap (–120 mV) and a test pulse to 0 mV. The protocol was run before and after drug application. The current traces looked similar to those shown in Fig. 1. The overall kinetics of the TTX-S current was faster, and, in the absence of drug, a 5-ms hyperpolarizing gap was sufficient for >85% channels to recover from inactivation. The fractional responses were measured at three different concentrations of PPPA to plot partial concentration-inhibition curves. The K_i was 0.011 ± 0.001 µM (n = 3) (Table 2).

Inhibition of rNa_v1.2 Channels and Native Rat DRG Neuron Currents by CBZ and LTG. For comparison purposes, we characterized inhibition of Na⁺ channels by the clinically relevant drugs carbamazepine and lamotrigine.

rNa_v1.2 Channels. Biophysical parameters for CBZ and LTG inhibition of recombinant rNa_v1.2 channels are given in Table 1 (Ilyin et al., 2005). In addition, PPPA was compared with the anticonvulsants in its capacity to filter electrical discharge as assessed by inhibition of 20-Hz trains of 40 depolarizing pulses from –70 to 0 mV. When administered at concentrations giving comparable steady-state inhibition (0.1 µM PPPA, 30 µM LTG, and 50 µM CBZ), the drugs inhibited the 40th pulse by 13.5 ± 3.2% (n = 4), 15.5 ± 2.0% (n = 3), and 17 ± 1.6% (n = 3), respectively. However, steady-state inhibition was achieved appreciably faster for PPPA ( = 4.5 ± 0.6 s, n = 4) than for CBZ ( = 8.7 ± 3.2 s, n = 3) and LTG ( = 25 ± 10.6 s, n = 3). The difference was not significant for comparison of PPPA and CBZ (P < 0.08) but was significant compared with LTG (P < 0.02).

Neuronal Recordings. Protocols for isolated DRG neurons recordings using CBZ and LTG were identical to those described for PPPA. K_i values for CBZ and LTG inhibition of TTX-R and TTX-S Na⁺ currents are given in Table 2. As seen with the recombinant channels, PPPA was significantly more potent than classic anticonvulsants (P < 0.001).

PPPA Does Not Block Voltage-Gated N-Type Ca²⁺ Channels in Human Neuroblastoma IMR-32 Cells
In the concentration range tested, PPPA showed no inhibition of Ca²⁺ signal in FLIPR⁹⁶ [IC₅₀ > 20 µM (n = 4), data not shown]. In the same experimental series, -ctx MVIIA, a selective blocker of N-type Ca²⁺ channels, inhibited the fluorescent signal with an IC₅₀ = 46.6 ± 10.8 nM (n = 4) (data not shown).

PPPA Reduces Hyperalgesia following Nerve Injury
Partial ligation of the sciatic nerve resulted in mechanical hyperalgesia present 21 days after surgery (Fig. 5A). Administration of PPPA (3–30 mg/kg p.o.) produced a dose-dependent inhibition of mechanical hyperalgesia 1 (F_5,40 = 10.39, P < 0.0001), 3 (F_5,40 = 5.56, P = 0.0006), and 5 (F_5,40 = 7.81, P < 0.0001) h after administration. PPPA (30 mg/kg) significantly increased PWTs 1, 3, and 5 h after administration, whereas administration of 3 or 10 mg/kg PPPA produced a significant effect only at the 3-h time point. The antihyperalgesic effects of PPPA were no longer evident 24 h after dosing. The efficacy of 30 mg/kg PPPA was similar to and the duration of action was greater than that of the positive control, 100 mg/kg i.p. gabapentin. The antihyperalgesic effects of PPPA were limited to the injured paw; there was no effect of PPPA on PWTs in the contralateral paw (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 5. Reversal of mechanical hyperalgesia by PPPA in a rat partial spinal nerve ligation model (A), in a rat FCA model of chronic inflammatory pain (B), and in a rat model of incisional pain (C). Hind PWTs to a noxious mechanical stimulus were determined using a model 7200 analgesimeter (Ugo Basile). Cut-off was set at 250 g, and the endpoint was taken as complete paw withdrawal. PWT was determined once for each rat at each time point. Baseline PWTs were taken, and either the sciatic nerve was exposed and one-third to one-half ligated, or 50 µl of a 50% FCA solution in saline was injected into the paw, or a 1-cm longitudinal incision was made through skin and fascia of the plantar aspect of the paw. Twenty-four hours (FCA and incision) or 21 days (PSN) later, predrug PWTs were measured, and the rats (n = 9–20/group) received a single dose of PPPA, and PWT was again determined 1, 3, 5, and 24 h postdrug administration. x axis, base, baseline; pre, predrug reading.

PPPA Reduces Mechanical Hyperalgesia Induced by Paw Incision
Incision of the plantar surface of the hind paw resulted in the development of mechanical hyperalgesia 24 h after surgery (Table 3). Administration of PPPA (0.3–30 mg/kg, p.o.) resulted in a dose-dependent reduction in mechanical hyperalgesia 1 (F_7,112 = 25.416, P < 0.0001), 3 (F_7,112 = 20.051, P < 0.0001), and 5 (F_7,112 = 21.495, P < 0.0001) h after dosing (Fig. 5C). Administration of 30 mg/kg PPPA significantly reduced incision-induced hyperalgesia 1 and 3 h after dosing, and administration of 3 or 10 mg/kg PPPA resulted in a significant reduction in mechanical hyperalgesia at the 3-h time point only. There was no effect of PPPA administration on mechanical hyperalgesia 24 h after dosing. The maximal reversal of mechanical hyperalgesia by PPPA was comparable with that produced by the positive control indomethacin (30 mg/kg p.o.).

PPPA Has No Effect on Acute Nociception
Administration of PPPA (1–10 mg/kg p.o.) had no effect on acute nociception as measured by the tail-flick assay (Table 3). Tail-flick latencies were measured 1, 3, and 5 h after drug administration. The positive control, morphine (10 mg/kg, s.c.), produced significant antinociception 1, 3, and 5 h after drug administration (P < 0.0001, Fisher's protected least significant difference test at each time point).

PPPA Has No Effect on Motor Performance
Administration of PPPA (3–30 mg/kg p.o.) did not induce ataxia, nor did it have other effects on motor performance as measured by the accelerating Rotarod assay (Table 4). Latencies to fall off of the Rotarod apparatus were measured 1, 3, 5, and 24 h after drug administration. Although there was no effect of PPPA on motor performance, the positive control, gabapentin (100 mg/kg i.p.), resulted in significant motor deficits 1, 3, and 5 h after administration.

Efficacy and Side Effect Profiles of Carbamazepine and Lamotrigine
As reported previously (Hunter et al., 1997), CBZ and LTG each produced significant reversal of nerve injury-induced mechanical hyperalgesia as well as significant motor impairment as measured by the Rotarod assay. Administration of CBZ (1–100 mg/kg p.o.) 3 weeks after partial ligation of the sciatic nerve resulted in a significant reversal of mechanical hyperalgesia with an MED of 100 mg/kg 1 and 3 h after administration. When the effect of CBZ on motor performance was measured, the MAD was 30 mg/kg 1 h after administration, rising to 300 mg/kg 3 h after drug administration. Therefore, the therapeutic index (TI) for CBZ was 0.3 at 1 h and 3 at 3 h after drug administration (Table 4).

The efficacy and side effect profiling of LTG was similar to that of CBZ. Administration of LTG (3–100 mg/kg i.p.) produced a dose-dependent reversal of nerve injury-induced mechanical hyperalgesia 1, 3, and 5 h after administration. At each of these time points, the MED of LTG was 30 mg/kg. LTG also induced profound motor deficits after administration, with an MAD of 30 mg/kg 1 and 5 h after administration and 10 mg/kg 3 h after drug administration. As such, the TI for LTG was 1 at 1, 3, and 5 h after drug administration (Table 4).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
PPPA is a potent, state-dependent blocker of voltage-gated Na⁺ channels. Assayed on recombinant rNa_V1.2 channels and native Na⁺ currents in rat DRG neurons, PPPA has 1000 times higher potency and >2000-fold faster onset kinetics than the clinical comparators CBZ and LTG. Moreover, PPPA has 10- to 20-fold higher levels of selectivity for inactivated channels versus resting-state channels (i.e., state dependence) than CBZ and LTG. PPPA was inactive against N-type Ca²⁺ channels in IMR-32 cells and across a standard panel of CNS targets (General Side Effects Profile I; NovaScreen, Hanover, MD). In vivo studies show that PPPA is potent and efficacious in reversing mechanical hyperalgesia in rat models of neuropathic, postsurgical, and inflammatory pain. PPPA has no effect on paw withdrawal thresholds in the contralateral paw or in models of acute thermal nociception, indicating that efficacy is not due to a local anestheticlike nerve block. It is noteworthy that PPPA has a TI >10 versus ataxia. PPPA, CBZ, and LTG all have limited (<10-fold) levels of subtype selectivity for different recombinant Na⁺ channel subtypes and native Na⁺ currents (V. I. Ilyin, unpublished data). Thus, it seems that differences in the biophysics of Na⁺ inhibition confer PPPA with an improved efficacy to side effects profile compared with anticonvulsants like CBZ and LTG.

Studies investigating CBZ and LTG in rat neuropathic pain models are limited, and direct comparisons with the current work are difficult due to differences in rat strain, model, routes of administration, and behavioral measurement (e.g., Hunter et al., 1997; Erichsen et al., 2003; Decosterd et al., 2004; Lindia et al., 2005). In most cases, however, doses associated with efficacy in neuropathic pain models are in the same range that would have produced ataxic/sedative effects in our accelerating Rotarod assay. Thus, for CBZ and LTG, it is difficult to distinguish "efficacy" from side effects. This lack of TI in rats seems to translate directly to a poor TI for treating neuropathic pain in humans (Mao and Chen, 2000; Jensen, 2002; Petersen et al., 2003). In contrast, PPPA has a TI >10, suggesting a meaningful separation between doses that are efficacious in reversing mechanical hyperalgesia and those producing ataxia. To confirm this, exploratory pharmacokinetic studies in rats were conducted and showed that, in the range 1 to 30 mg/kg p.o., PPPA has dose-proportional increases in C_max and area under the curve, with a half-life of 6 h and oral bioavailability of 100% (Y. Rotstheyn and P. Bullock, unpublished data). This indicates PPPA has a TI that is 10 both in terms of dose and, more importantly, in terms of drug exposure. Na⁺ channel blockers are widely used clinically to treat neuropathic pain (e.g., Mao and Chen, 2000; Jensen, 2002; Petersen et al., 2003). In contrast, their effectiveness for treating other types of human pain, for example pain associated with osteoarthritis, visceral pain arising from gastrointestinal disorders, and postsurgical pain (beyond their use of local anesthetics), is minimal or unreported. To further explore the therapeutic potential of PPPA as an analgesic, it will be worthwhile to test the compound in more sophisticated animal models of osteoarthritis and in models of visceral pain (Laird et al., 2001; Blackburn-Munro et al., 2002; Su et al., 2002).

The results of our study suggest that the biophysical parameters of Na⁺ channels inhibition contribute to an improved TI for PPPA as compared with CBZ and LTG; however, the exact mechanism(s) remains unclear. Pathological nerve hyperexcitability is characterized by sustained depolarizations with superimposed high-frequency bursts of action potentials. Under these conditions, Na⁺ channels accumulate in inactivated states, and their availability for repetitive excitation is lowered. An additional reduction in the channel availability by drug-induced use-dependent block is thought to lead to selective attenuation of spike bursts in the depolarized neurons. PPPA exerts highly state-dependent block of Na⁺ channels. For rNa_v1.2, PPPA discriminates inactivated versus resting channels with a factor of K_r/K_i > 500-fold (Table 1). Such a high level of state dependence minimizes the risk of interfering pharmacologically with signaling in nonpathological neuronal circuits. Levels of state dependence for CBZ (30-fold) and LTG (60-fold) are substantially lower.

Another biophysical parameter that could contribute to PPPA's improved TI is onset-binding kinetics. The on-rate of binding of PPPA to inactivated channels is comparatively fast. At equimolar concentrations, PPPA binds to the inactivated Na_V1.2 channels >2000 times faster than CBZ and LTG. If the drugs are available at their respective K_i values, a more reasonable assumption for the situation in vivo at efficacious doses, room temperature microscopic rates of binding (the product of K_i and k₊) are 4, 1.8, and 0.7 s^–1 for PPPA, CBZ, and LTG, respectively. This means PPPA exerts a 2- to 6-fold faster, and more pronounced, block of hyperexcitability episodes of comparable durations, which may be important in pain states where hyperexcitability hinges on the short-lived inactivated states (Rush et al., 1998; Waxman et al., 2000).

Retardation of recovery from inactivation is another feature of state-dependent block of voltage-gated Na⁺ channels and underlies use (or frequency)-dependent inhibition (Ilyin et al., 2005). Interestingly, although highly potent, PPPA shows only moderate levels of retardation, i.e., 70% of channels recovered from inactivation with _repr 53 ms, which are comparable with LTG and CBZ (Table 1). In this respect, PPPA and the two anticonvulsants are very different from anesthetics such as bupivacaine, which retards repriming for many seconds (V.I. Ilyin, unpublished observations). Modest effects on retardation may be important to minimize the impact on nonpathological neuronal activity, particularly in the CNS.

Compared with PPPA, the prototype compound V102862 (Ilyin et al., 2005) was 20- and 6-fold less potent on native TTX-S and TTX-R currents in rat DRG neurons. On the recombinant rNa_v1.2 channels, V102862 was 10-less potent than PPPA in binding to inactivated channels and had a lower state dependence ratio, K_r/K_i 70. At equal concentrations, V102862 was 50-fold slower in binding to inactivated channels; at the same time, it caused more significant retardation of repriming from inactivation (_repr 700 ms at V_h = –120 mV as compared with 50 ms for PPPA). From a mechanistic point of view, higher potency combined with higher rate of binding to inactivated channels may provide PPPA an advantage in exerting higher levels of inhibition of sodium channels within shorter intervals of hyperexcitability. The experiments with frequency pulse trains similar to that shown in Fig. 3B support this hypothesis: at 3 µM, PPPA caused steady-state inhibition of 60% at the second pulse in the train as compared with 34% inhibition seen at the 20th pulse for V102862 (V. I. Ilyin, unpublished data). This feature may play especially important role for effective blocking of rapidly repriming TTX-R channels in DRG neurons (Rush et al., 1998) during short episodes of hyperexcitability.

There is now little question, at least in rat pain models, that Na⁺ channel regulation contributes to hyperexcitability of primary afferent neurons after nerve injury and inflammation (for review, see Waxman et al., 2000; Lai et al., 2004; Priestley, 2004). However, recent data also calls into question whether selective inhibition of a single subtype of Na⁺ channel, e.g., Na_V1.8 or Na_V1.7, will be sufficient to produce robust analgesic effects across diverse pain states (Lindia et al., 2005; Nassar et al., 2005). The improved TI of PPPA, in the absence of clear subtype selectivity, suggests that researchers reporting on novel subtype-selective blockers (e.g., Silos-Santiago et al., 2005) should be cautious in ascribing improvements in TI to subtype selectivity when it may be due to more optimal biophysical parameters (Brochu et al., 2006).

Whether PPPA acts both centrally and peripherally, and which is more important in various pain states, is unknown. Separate experiments showed that PPPA is active in a rat model of maximal electroshock-induced seizures over the same dose range (ED₅₀ = 2.5 mg/kg p.o.) and time course that produced efficacy in the pain models (K. Vanover and R. Carter, unpublished data). This clearly implies that PPPA has access to the CNS. Drug levels of CBZ and LTG in the cerebrospinal fluid at clinical therapeutic doses are 13 and 20 µM, respectively (e.g., Kuo and Lu, 1997). If approximately the same free drug levels are found in the periphery, the anticonvulsants reach sufficient levels to block central and peripheral TTX-S currents but will have little effect on TTX-R currents in primary afferents, where the K_i values are >100 µM (Table 2). PPPA is 2000 times more potent at blocking TTX-R currents. Therefore, PPPA blockade of TTX-S currents, centrally and peripherally, combined with blockade of peripheral TTX-R currents, may be contributing to the improved potency, efficacy, and TI.

In conclusion, alterations in the excitability of injured neurons appear to play a role in spontaneous pain, allodynia, and hyperalgesia. Numerous different Na⁺ channel subtypes seem to play roles in establishing and maintaining ectopic discharge in primary afferent neurons and hyperexcitability in the periphery and CNS. PPPA's broad activity in pain models and improved TI suggest that potent, broad-spectrum state-dependent, Na⁺ channel blockers will have utility for treating pain in humans.

	Acknowledgements

 
We acknowledge the work of Dianne D. Hodges in establishing the rNaIIA-B2 (rNa_v1.2)-HEK-293 cell line; Daniela Leumer, Silvia Robilos, and Michael Suruki for technical support; and Kenneth Valenzano for critical reading of the manuscript.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.104737.

ABBREVIATIONS: DRG, dorsal root ganglion; CBZ, carbamazepine; LTG, lamotrigine; PPPA, 2-[4-(4-chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide; V102862, 4-(4-fluorophenoxy)benzaldehyde semicarbazone; rNa_v1.2, rat brain type IIa Na⁺ channel; HEK, human embryonic kidney; TTX-S, tetrodotoxin-sensitive; CMF, calcium- and magnesium-free Hanks' buffer; TTX-R, tetrodotoxin-resistant; DMSO, dimethyl sulfoxide; FLIPR, fluorescence plate reader; -ctx, -conotoxin; PWT, paw withdrawal threshold; PSN, partial spinal nerve ligation; FCA, Freund's complete adjuvant; MED, minimal effective dose; MAD, minimal ataxic dose; TI, therapeutic index; CNS, central nervous system.

Address correspondence to: Victor I. Ilyin, Discovery Research, Purdue Pharma LP, 6 Cedar Brook Drive, Cranbury, NJ 08512. E-mail: victor.ilyin{at}pharma.com

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