| | Carisbamate, a novel neuromodulator, inhibits voltage-gated sodium channels and action potential firing of rat hippocampal neuronsReceived 28 April 2008; received in revised form 18 September 2008; accepted 23 September 2008. Summary Carisbamate (RWJ-333369; (S)-2-O-carbamoyl-1-o-chlorophenyl-ethanol) is a novel investigational antiepileptic drug that exhibits a broad-spectrum of activity in a number of animal models of seizure and drug refractory epilepsy. In an effort to understand the molecular mechanism by which carisbamate produces its antiepileptic actions, we studied its effects on the function of voltage-gated, rat brain sodium and potassium channels and on the repetitive firing of action potentials in cultured rat hippocampal neurons. In whole-cell patch clamp recording, carisbamate resulted in a concentration-, voltage- and use-dependent inhibition of rat Nav1.2, with an IC50 value of 68 μM at −67mV. In rat hippocampal neurons, carisbamate similarly blocked voltage-gated sodium channels, with an IC50 value of 89μM at −67mV, and inhibited repetitive firing of action potentials in a concentration-dependent manner (by 46% at 30μM and 87% at 100μM, respectively). Carisbamate had no effect on the steady-state membrane potential or voltage-gated potassium channels (Kv) in these neurons. These inhibitory effects of carisbamate occurred at therapeutically relevant concentrations in vivo, raising the possibility that block of voltage-gated sodium channels by carisbamate contributes to its antiepileptic activity. 1. Introduction  Epilepsy, a group of neurological conditions with diverse etiology and symptomology, is characterized by recurrent, unprovoked seizures and is among the most prevalent chronic neurological disorders. Although currently available antiepileptic drugs (AEDs) and other treatments can effectively treat seizures in most of the epilepsy population, seizures are still not fully controlled in nearly 30% of the patients. Carisbamate (RWJ-333369; (S)-2-O-carbamoyl-1-o-chlorophenyl-ethanol; Fig. 1) is a structurally novel antiepileptic drug currently undergoing clinical evaluation. Preclinically, carisbamate is distinguished from many other AEDs by an ability to suppress seizures in almost all animal seizure models tested to date (Grabenstatter and Dudek, in press, Nehlig et al., 2005, Novak et al., 2007, Rogawski, 2006, White et al., 2006). Because of the critical role various ion channels play in modulating neuronal excitability and the fact that sodium channel inhibition is a common mechanism of several AEDs (Rogawski, 2006), we studied the effects of carisbamate on the function of voltage-gated, rat brain sodium and potassium channels, as part of an extensive effort to identify the molecular mechanism(s) by which carisbamate exerts its antiepileptic activity. We found that carisbamate inhibited repetitive firing of action potentials and voltage-gated sodium channels (Nav), but had no effect on voltage-gated potassium (Kv) channels. Our results suggest that the inhibition of voltage-gated sodium channels may contribute to the antiepileptic activity of carisbamate. 2. Materials and methods CHL1610 cells (derived from a Chinese hamster lung fibroblast cell line) stably expressing rat Nav1.2 (rNav1.2) were plated on glass coverslips and incubated in 5% CO2 at 37°C for 1–2 days before patch clamp recording. The culture media contained DMEM supplemented with 10% fetal bovine serum, 100units/ml penicillin, 100μg/ml streptomycin and 200μg/ml G418. Hippocampal neuronal cultures were prepared from embryonic day 18 Sprague–Dawley rat pups. Briefly, embryos were removed from anesthetized, timed-pregnant mothers by cesarean section and then decapitated. The hippocampi were dissected and incubated with 20units/ml papain (Worthington Biochemicals, Lakewood, NJ) at 37°C for 1h. Enriched neuronal cultures were prepared by suspending the papain-treated cell pellet in Neurobasal/B27 media (Invitrogen, Carlsbad, CA). The cells were then plated onto poly-d-lysine-coated glass coverslips at a density of 1×104 to 1×105cells/coverslip and incubated in 5% CO2 at 37°C. Isolated neurons that were in culture for 8–9 days were used for patch clamp studies. That adequate space clamp was achieved in our experiments is also supported by the good agreement of our data (e.g., the time constant of fast inactivation=0.60±0.04ms at −7mV (n=13), the maximum peak current occurred between −7 and −17mV (n=5), and V1/2 for the fast steady-state inactivation=−50mV (Fig. 4A inset)) with published reports using acutely isolated hippocampal neurons (Costa, 1996, Ribeiro and Costa, 2003). All studies were carried out using a protocol approved by the Institutional Animal Care and Use Committee of Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Spring House, PA, and in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Electrophysiological recordings were performed using the whole-cell patch clamp technique. For recording of sodium channel currents, the holding potential was −107mV. Unless otherwise stated, a 45-s preconditioning pulse (at either −107 or −67mV) was followed by 3s of brief (5ms) depolarizations to −7mV at 10Hz. The membrane potential during the period between brief depolarizations was the same as the preconditioning voltage. The time interval between preconditioning pulses was 15s, during which time the cell was held at −107mV. Both fast and slow inactivation was thus allowed to develop in this protocol at the preconditioning voltage of −67mV. For measurement of voltage-dependent fast inactivation, a 100-ms preconditioning pulse from −97 to +3mV (in 10mV increments) was given, followed by a brief (5ms) depolarizing pulse to −7mV. For recording of potassium currents, a 20-ms depolarization pulse from −107 to +63mV (in 10mV increments) was given, followed by a voltage step to −87mV to measure the tail current. Leak current was subtracted using a P/4 protocol. The time interval between consecutive 20-ms pulses was 2s, during which time the cell was held at −107mV. For action potential recording, a hyperpolarizing holding current was injected to maintain the steady-state membrane potential at −79±2mV (n=9) before drug application. A depolarizing current was then injected for 1.5s to induce repetitive firing of action potentials, followed by a return to the same holding current prior to the depolarization. The time interval between consecutive depolarizing current injections was 10s. The extracellular solution was applied at 0.5ml/min via a gravity-fed perfusion system. For recording of sodium currents in both CHL1610 cells and hippocampal neurons, the extracellular solution contained (mM): NaCl (132), KCl (5.4), CaCl2 (1.8), MgCl2 (0.8), HEPES (10) and glucose (10) at pH 7.4. The pipette solution contained (mM): CsCl (45), CsF (100), EGTA (5), HEPES (10) and glucose (5) at pH 7.4. For recording of potassium currents in hippocampal neurons, the extracellular solution contained (mM): choline chloride (132), KCl (5.4), CaCl2 (1.8), MgCl2 (0.8), HEPES (10) and glucose (10) at pH 7.4. The pipette solution contained (mM): KCl (140), EGTA (5), HEPES (10) and glucose (5) at pH 7.4. For action potential recording in hippocampal neurons, the extracellular solution contained (mM): NaCl (132), KCl (5.4), CaCl2 (1.8), MgCl2 (0.8), HEPES (10) and glucose (10) at pH 7.4. The pipette solution contained (mM): KCl (140), EGTA (5), HEPES (10) and glucose (5) at pH 7.4. The junction potential was corrected using pClamp 9 (Molecular Devices, Sunnyvale, CA). Pipette resistance ranged from 1 to 2MΩ. Capacitance transients were cancelled, and series resistance was 85% compensated. Data were amplified, filtered (2KHz; Axopatch 200B), sampled (20kHz, except for action potential measurements, which were sampled at 10kHz; Digidata 1322A) and acquired using pClamp 9 (all from Molecular Devices). All experiments were conducted at 22°C. Phenytoin was obtained from Sigma (St. Louis, MO) and stored as a 100mM stock in 100% dimethyl sulfoxide at −20°C. Carisbamate was synthesized in-house and stored as a 1M stock in 100% dimethyl sulfoxide at −20°C. The stock solutions were freshly diluted before each use. For sodium channels, compound effects were calculated by taking the ratio of the peak current amplitude during a brief (5ms) depolarization to −7mV in the presence of a compound over the corresponding peak current amplitude in the control solution. Concentration–response relationships were fitted to a logistic function to determine the IC50 value. To calculate the voltage dependence of drug effects on potassium channels, both the peak current amplitude during the 20-ms depolarizing pulse and the tail current amplitude at −87mV were used. The voltage dependence of activation, calculated from the tail currents, was fitted to a Boltzmann function to determine V1/2, the voltage at which 50% of the maximal tail currents are produced. Data fitting was performed using Origin 7.0 (OriginLab, Northampton, MA). A two-tailed t-test was performed as appropriate to determine statistical significance. Data are expressed as the mean±S.E.M. 3. Results We first studied the effects of carisbamate on rNav1.2 channels stably expressed in CHL1610 cells, using the voltage protocol depicted in Fig. 2A. Carisbamate (100μM) had little effect on rNav1.2 currents preconditioned at −107mV, regardless of the pulse number; in contrast, it significantly blocked the currents preconditioned at −67mV (Fig. 2B), a voltage at which significant channel inactivation occurred (Fig. 3B inset). This block was concentration-dependent and, to some degree, use-dependent (Fig. 2C). Fig. 3A plots the concentration dependence of the carisbamate inhibition at these two preconditioning voltages for the 1st and 30th pulses. At −67mV, carisbamate blocked rNav1.2 with IC50 values of 106μM and 68μM for the 1st and 30th pulses, respectively, inhibiting 16% and 36% of the current at 10 and 30μM, respectively (30th pulse). Phenytoin, a marketed AED, also blocked rNav1.2 channels with a similar voltage and use dependence profile, albeit with somewhat higher potency (IC50 values of 32 and 22μM at −67mV for the 1st and 30th pulses, respectively; Fig. 3B). Next, we studied the effects of carisbamate in cultured rat hippocampal neurons. As shown in Fig. 4A, carisbamate blocked voltage-gated sodium channels expressed in these neurons in a fashion similar to its block of rNav1.2 channels in CHL1610 cells. Specifically, the block was concentration- and voltage-dependent, with comparable IC50 values at −67mV (117μM and 90μM for the 1st and 30th pulses, respectively). As with rNav1.2, significant inhibition of voltage-gated, hippocampal sodium channels occurred at carisbamate concentrations as low as 10 and 30μM (11% and 30%, respectively, at −67mV for the 30th pulse). Furthermore, carisbamate reversibly (within 2–3min; Fig. 4B) and concentration dependently (Fig. 4C) inhibited the repetitive firing of action potentials in these neurons over a concentration range within which carisbamate blocked rNav1.2 in CHL1610 cells and voltage-gated sodium channels in these neurons. This inhibition occurred without any change in the steady-state membrane potential in these cells (Fig. 4D). Finally, we examined the ability of carisbamate to affect Kv channels in cultured rat hippocampal neurons. A series of brief (20ms) depolarizing steps (in 10mV increments) elicited voltage-dependent and fast-activating outward potassium currents at voltages above −57mV (Fig. 5A). Carisbamate, at a relatively high concentration of 300μM, produced no significant effect on the peak current over a wide range of membrane potentials (from −47 to +63mV; Fig. 5B). In addition, it did not affect the voltage-dependent activation of these channels (Fig. 5C). 4. Discussion Given the abundant (molecular, genetic and physiological) evidence that links voltage-gated sodium channels to epilepsy (Köhling, 2002), it is perhaps not surprising that many AEDs have been shown to modulate these channels. Among them, phenytoin, lamotrigine and carbamazepine are thought to exert their antiepileptic effects primarily via sodium channel blockade (Köhling, 2002). In addition, a number of other AEDs (e.g., felbamate and topiramate) that are thought to possess multiple mechanisms of action often block sodium channels as well (Rogawski and Löscher, 2004). Carisbamate is a novel, investigational AED that exhibits broad-spectrum antiepileptic activities in a battery of rodent seizure models (White et al., 2006) as well as in clinical trials. In a phase IIb adjunctive therapy clinical trial in patients with refractory partial seizures, carisbamate significantly reduced seizure frequency at a dose of 300mg/day (Faught et al., 2007). This dose corresponds to a plasma level (Cmax at steady state) of approximately 5μg/ml (∼23μM; Yao et al., 2006). A study in photosensitive epilepsy patients showed a clinically significant suppression of photosensitivity in 2 of 4 patients following a single 500mg dose of carisbamate (corresponding to Cmax of 10.2μg/ml or approximately 48μM; Kasteleijn-Nolst Trenite et al., 2007). Furthermore, a 50% reduction in spontaneous recurrent seizure frequency was observed in rats with kainate-induced epilepsy at approximately 5μg/ml (∼20μM; Grabenstatter and Dudek, in press). In the present study, we found that, in rat hippocampal neurons, carisbamate inhibited the repetitive firing of action potentials and voltage- and use-dependently blocked voltage-gated sodium, but not potassium, channels. Similarly, in CHL1610 cells, it also blocked an isoform of voltage-gated sodium channels, Nav1.2, which is highly expressed in the hippocampus as well as in many other regions of the central nervous system (Gordon et al., 1987). These effects occurred at concentrations that are therapeutically relevant, given that carisbamate is found at comparable concentrations in the brain and plasma (data not shown). Various voltage-gated potassium channels, including fast activating Kv1.4, Kv1.5, Kv2.1, Kv2.2 and Kv4.2, among others, have been shown to express in hippocampal neurons (Maletic-Savatic et al., 1995). Our study demonstrated that carisbamate had virtually no effect on these channels at a concentration as high as 300μM. (It should be pointed out that the protocols we used were not designed to study more slowly activating potassium channels, such as Kv7, Kv10 to Kv12 families.) Carisbamate, at 30 and 100μM, also did not significantly change the membrane input resistance (at least near Vm=−80mV). Taken together, these results suggest that the antiepileptic activities of carisbamate may, at least in part, be mediated by its ability to block voltage-gated sodium channels in vivo. Consistent with our conclusion, a recent study (Deshpande et al., 2008, Deshpande et al., in press) showed that carisbamate inhibited spontaneous recurrent seizure discharges and sustained repetitive firing in cultured hippocampal neurons, with potencies similar to what we report here. Carisbamate blocked rNav1.2 channels only weakly at hyperpolarized voltages (e.g., at −107mV). However, when the membrane was depolarized, such that substantial channel inactivation occurred (e.g., at −67mV), there was a marked increase in the ability of the drug to induce channel block, strongly suggesting that carisbamate preferentially binds to and promotes the inactivated state(s) of the channel. Channel inhibition by carisbamate was further enhanced by repeated, high-frequency channel use as a result of repetitive activation and inactivation (i.e., the block was more potent at the 30th pulse than at the 1st pulse). These characteristics of carisbamate are similar to those of lamotrigine (Liu et al., 2006) and phenytoin (Fig. 3B), and are likely responsible for selective inhibition of high-frequency repetitive firing (such as what is believed to occur during the spread of seizure activity) without affecting normal, ongoing neuronal firing. In addition to its activity in the standard seizure models, carisbamate is also efficacious in two models of refractory epilepsy, the 6Hz seizure model and the lamotrigine-resistant amygdala kindled rat model (White et al., 2006), in which other sodium channel-blocking AEDs are inactive. For example, in the 6-Hz seizure model, carisbamate maintains potent anti-seizure activity even when the stimulus intensity is increased from 22 to 44mA (White et al., 2006), while phenytoin and lamotrigine are not active above the minimal 22mA stimulus (Barton et al., 2001). Unlike phenytoin and carbamazepine, carisbamate was active against spike–wave seizures in the GAERS model of absence seizures and did not aggravate spike-and-wave discharges in the GAERS model (Nehlig et al., 2005). In addition, a recent study showed that carisbamate, but not phenytoin, prevented the development and generation of epileptiform discharges in vitro when administered following status epilepticus-like injury (Deshpande et al., 2008, Deshpande et al., in press). These results clearly differentiate carisbamate from conventional sodium channel blocking AEDs in its breadth of antiepileptic efficacy. Thus, while the present studies provide a reasonable molecular mechanism (i.e., voltage-gated sodium channel blockade) that may partially account for the antiepileptic actions of carisbamate in vivo, it is likely that additional mechanisms/targets are also involved. Future studies should help to identify and characterize these mechanisms. Acknowledgements The authors wish to thank Drs. Brian Klein and Richard Shank for their critical reading of the manuscript and helpful suggestions. References Barton et al., 2001. 1.Barton ME, Klein BD, Wolf HH, White HS. Pharmacological characterization of the 6Hz psychomotor seizure model of partial epilepsy. Epilepsy Res. 2001;47:217–227. Abstract | Full Text |
Full-Text PDF (406 KB)
|
CrossRef
Costa, 1996. 2.Costa PF. The kinetic parameters of sodium currents in maturing acutely isolated rat hippocampal CA1 neurones. Brain Res. Dev. 1996;91:29–40. Deshpande et al., 2008. 3.Deshpande LS, Nagarkatti N, Sombati S, Delorenzo RJ. The novel antiepileptic drug carisbamate (RWJ 333369) is effective in inhibiting spontaneous recurrent seizure discharges and blocking sustained repetitive firing in cultured hippocampal neurons. Epilepsy Res. 2008;79:158–165. Abstract | Full Text |
Full-Text PDF (665 KB)
|
CrossRef
Deshpande et al., in press. 4.Deshpande, L.S., Nagarkatti, N., Ziobro, J.M., Sombati, S., Delorenzo, R.J., in press. Carisbamate prevents the development and expression of spontaneous recurrent epileptiform discharges and is neuroprotective in cultured hippocampal neurons. Epilepsia. Faught et al., 2007. 5.Faught E, Rosenfeld W, Holmes G, Novak G, Greenspan AJ, Haas M, et al. A double-blind, placebo-controlled, dose ranging study to evaluate the efficacy and tolerability of carisbamate (RWJ-333369) as adjunctive therapy in patients with refractory partial seizures. Epilepsia. 2007;48(Suppl. 6):330. MEDLINE |
CrossRef
Gordon et al., 1987. 6.Gordon D, Merrick D, Auld V, Dunn R, Goldin AL, Davidson N, et al. Tissue-specific expression of the RI and RII sodium channel subtypes. Proc. Natl. Acad. Sci. U.S.A. 1987;84:8682–8686. MEDLINE |
CrossRef
Grabenstatter and Dudek, in press. 7.Grabenstatter, H.L., Dudek, F.E., in press. A new potential AED, carisbamate, substantially reduces spontaneous motor seizures in rats with kainate-induced epilepsy. Epilepsia. Kasteleijn-Nolst Trenite et al., 2007. 8.Kasteleijn-Nolst Trenite DGA, French JA, Hirsch E, Macher JP, Meyer BU, Grosse PA, et al. Evaluation of carisbamate, a novel antiepileptic drug, in photosensitive patients: an exploratory, placebo-controlled study. Epilepsy Res. 2007;74:193–200. Abstract | Full Text |
Full-Text PDF (336 KB)
|
CrossRef
Köhling, 2002. 9.Köhling R. Voltage-gated sodium channels in epilepsy. Epilepsia. 2002;43:1278–1295. MEDLINE |
CrossRef
Liu et al., 2006. 10.Liu Y, Qin N, Reitz T, Wang Y, Flores CM. Inhibition of the rat brain sodium channel Nav1, 2 after prolonged exposure to gabapentin. Epilepsy Res. 2006;70:263–268. Abstract | Full Text |
Full-Text PDF (323 KB)
|
CrossRef
Maletic-Savatic et al., 1995. 11.Maletic-Savatic M, Lenn NJ, Trimmer JS. Differential spatiotemporal expression of K+ channel polypeptides in rat hippocampal neurons developing in situ and in vitro. J. Neurosci. 1995;15:3840–3851. MEDLINE Nehlig et al., 2005. 12.Nehlig A, Rigoulot M-A, Boehrer A. A new drug, RWJ-333369 displays potent antiepileptic properties in genetic models of absence and audiogenic epilepsy. Epilepsia. 2005;46(Suppl. 8):215;. Novak et al., 2007. 13.Novak GP, Kelley M, Zannikos P, Klein B. Carisbamate (RWJ-333369). Neurotherapeutics. 2007;4:106–109. Abstract | Full Text |
Full-Text PDF (65 KB)
|
CrossRef
Ribeiro and Costa, 2003. 14.Ribeiro MA, Costa PF. The sensitivity of sodium channels in immature and mature rat CA1 neurones to the local anaesthetics procaine and lidocaine. Brain Res. Dev. 2003;146:59–70. Rogawski, 2006. 15.Rogawski MA. Diverse mechanisms of antiepileptic drugs in the development pipeline. Epilepsy Res. 2006;69:273–294. Abstract | Full Text |
Full-Text PDF (408 KB)
|
CrossRef
Rogawski and Löscher, 2004. 16.Rogawski MA, Löscher W. The neurobiology of antiepileptic drugs. Nat. Rev. Neurosci. 2004;5:553–564. White et al., 2006. 17.White HS, Srivastava A, Klein B, Zhao B, Choi YM, Gordon R, et al. The novel investigational neuromodulator RWJ 333369 displays a broad-spectrum anticonvulsant profile in rodent seizure and epilepsy models. Epilepsia. 2006;47(Suppl. 4):200. Yao et al., 2006. 18.Yao C, Doose DR, Novak G, Bialer M. Pharmacokinetics of the new antiepileptic and CNS drug RWJ-333369 following single and multiple dosing to humans. Epilepsia. 2006;47:1822–1829. MEDLINE |
CrossRef
a Analgesics Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Welsh & McKean Roads, P.O. Box 776, Spring House, PA 19477-0776, United States b CNS Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Welsh & McKean Roads, P.O. Box 776, Spring House, PA 19477-0776, United States  Corresponding author. Tel.: +1 215 628 5427; fax: +1 215 628 3297.
PII: S0920-1211(08)00277-5 doi:10.1016/j.eplepsyres.2008.09.006 © 2008 Elsevier B.V. All rights reserved. | |
|
FULL TEXT00277-5/journalimage?img=PIIS0920121108002775.gr1.lrg.gif&fig=fig1&kwhquery=&issn=0920-1211&ishighres=false&allhighres=false&free=yes','journalimage');)
00277-5/journalimage?img=PIIS0920121108002775.gr2.lrg.gif&fig=fig2&kwhquery=&issn=0920-1211&ishighres=false&allhighres=false&free=yes','journalimage');)
00277-5/journalimage?img=PIIS0920121108002775.gr3.lrg.gif&fig=fig3&kwhquery=&issn=0920-1211&ishighres=false&allhighres=false&free=yes','journalimage');)
00277-5/journalimage?img=PIIS0920121108002775.gr4.lrg.gif&fig=fig4&kwhquery=&issn=0920-1211&ishighres=false&allhighres=false&free=yes','journalimage');)
00277-5/journalimage?img=PIIS0920121108002775.gr5.lrg.gif&fig=fig5&kwhquery=&issn=0920-1211&ishighres=false&allhighres=false&free=yes','journalimage');)