The therapeutic use of Δ9-tetrahydrocannabinol (Δ9-THC) and various synthetic cannabinoids for pain treatment has attracted considerable interest.1–3 However, psychoactive effects mediated via central CB1 receptors and insufficient analgesic effects have reduced the enthusiasm about the therapeutic utility of cannabinoids as analgesics.4 Ajulemic acid (AJA, chemical structure in Fig. 1) is a synthetic derivative of Δ9-THC-11-oic acid, a main metabolite of Δ9-THC. AJA induces both anti-inflammatory and analgesic effects in rodent models of acute and neuropathic pain.5–7 Phase 2 clinical studies with AJA showed strong analgesic effects in patients with neuropathic pain but only minor cannabinoid-like psychoactive side effects.8,9 Regarding the anti-inflammatory effects of AJA, the nuclear receptor PPAR-γ was identified as a possible mediating molecule.10,11 While the affinity of AJA to the cannabinoid receptors CB1 and CB2 is supposed to be rather weak,11 we have previously demonstrated that AJA acts as a positive allosteric modulator on the strychnine-sensitive α1 and α1β glycine receptors.12 This modulation of glycine receptors, and in particular, the α3 glycine receptor, has recently been reported to be essential for the analgesic efficacy of several cannabinoids in mice.13,14
Another observed effect of cannabinoids with a potential relevance for analgesia is the inhibition or modulation of voltage-gated sodium channels. Δ9-THC, but also several other endogenous and synthetic cannabinoids, have been demonstrated to directly inhibit sodium channels.15–18 However, studies with a detailed analysis of different sodium channel isoforms are lacking. Furthermore, the molecular mechanisms responsible for cannabinoid-induced inhibition of sodium channels are largely lacking. Theile and Cummins19 reported that the endogenous cannabinoid anandamide very potently inhibits Navβ4 peptide-mediated resurgent currents in Nav1.7 and suggested a molecular mechanism distinct from the local anesthetic binding site. Resurgent currents seem to be enhanced in several inherited sodium channel mutations, causing muscle and neuronal channelopathies,20 and recent reports indicate that resurgent currents can drive pain in humans.21,22
In this study, we investigated the effects of AJA on 6 different α-subunits of sodium channels. To determine whether the effects of AJA are mediated via the local anesthetic binding site, we also tested the local anesthetic-insensitive Nav1.5-mutants N406K and F1760A.23,24 In addition, we questioned whether Navβ4-peptide-mediated resurgent current is inhibited by AJA. We hypothesized that AJA might be an effective modulator of sodium channels with a beneficial pharmacological profile in vivo.
Cell Culture and Transfection Procedures
Stably transfected human embryonic kidney 293 (HEK293) cells expressing the α-subunits Nav1.2, Nav1.3, Nav1.4, Nav1.5, and Nav1.7 were grown under standard conditions as described in previous studies 25. Nav1.2 and Nav1.3 were from rats, and Nav1.4, Nav1.5, and Nav1.7 were of human origin. Briefly, cells were cultured in Dulbecco`s modified Eagle medium (GIBCO-Invitrogen, Karlsruhe, Germany), supplemented with 10% heat-inactivated fetal bovine serum (Biochrom, Berlin, Germany), 1% penicillin/streptomycin (GIBCO-Invitrogen), and 0.4% Zeocin (Nav1.5) or 200 to 400 μg/mL G418 (Nav1.2, 1.3, 1.4 and Nav1.7) (Invitrogen) at 37°C in 5% CO2. As Nav1.8 (from rats) expresses very poorly in HEK 293 cells, we expressed Nav1.8 in the neuroblastoma cell line ND7/23 as previously described.25 The mutant constructs Nav1.5-N406K, and Nav1.5-F1760A were constructed with a mutagenesis kit (Quickchange XL kit, Qiagen GmbH, Hilden) according to the instructions of the manufacturer. Potential mutants were confirmed by DNA sequencing. Transient expression of the mutant constructs (2–3 μg) in HEK-293t cells, and rat Nav1.8 (5 μg) in ND7/23 cells was achieved by means of the calcium phosphate precipitation technique 26. EGFP (1 μg) was cotransfected to allow identification of transfected cells. After incubation for 12 to 15 hours, cells were replated in culture dishes and used for experiments within 2 days.
Chemicals and Solutions
AJA was provided by Prof. Burstein, University of Massachusetts Medical School. AJA was prepared as 100 mmol/L stock solution in dimethylsulfoxide (Fluka, Steinheim, Germany), light-protected and stored in glass vessels at 4°C. The AJA stock was directly dissolved in bath solution to reach the final drug concentration immediately before the experiments. A stock solution of tetrodotoxin (TTX, 1 mmol/L in water) was directly dissolved in the bath solution to a final concentration of 300 nmol/L. TTX was used in all experiments on Nav1.8 to inhibit endogenous TTX-sensitive Na+ channels in ND7/23 cells. Test solutions were applied via a gravity-driven application system with a common outlet positioned approximately 100 μm from the cell. The bath solution contained (mmol/L) 140 mmol/L NaCl, 3 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 10 mmol/L HEPES (adjusted to pH 7.4 with tetramethylammonium hydroxide). The pipette solution contained (mmol/L) 140 mmol/L CsF, 10 mmol/L NaCl, 1 mmol/L EGTA, and 10 mmol/L HEPES (adjusted to pH 7.3 with CsOH). The osmolarity of all solutions was adjusted to 290 to 300 mosm/L. For measuring resurgent currents of Nav1.5, the Navβ-4 peptide (KKLITFILKKTREK, JPT Peptide Technologies, Berlin, Germany) was included into the pipette solution with a final concentration of 100 μmol/L as has been described in previous reports.19
Standard whole-cell voltage-clamp experiments were performed at room temperature. For data acquisition and further analysis, we used the EPC9 digitally controlled amplifier in combination with Pulse and Pulse Fit software (HEKA Electronics, Lambrecht, Germany). The data were filtered at 5 kHz and sampled at 20 kHz. Patch pipettes fabricated from borosilicate glass tubes (GB150EFT-10; Science Products, Germany) were pulled to a resistance of 1.0 to 2.0 MΩ after heat polishing. The series resistance was compensated by 60% to 80% to minimize voltage errors, and the capacitance artefacts were cancelled using the automatic subtraction of the EPC9 amplifier. Linear leak subtraction, based on resistance estimates from 4 hyperpolarizing pulses applied before the test pulse, was performed for all experiments.
To obtain concentration–response relationships, at least 5 independent experiments were performed. The experiments started with a control recording in the presence of extracellular solution, followed by the test recording where increasing concentrations of AJA were applied sequentially and finally concluded with the washout by extracellular solution.
The residual sodium current (I/Imax) in the presence of AJA, with respect to the current elicited with the same protocol in the respective control recording, was plotted against the applied concentration of the drug (C).
All averaged data were fitted using the Hill equation, Eq. 1, yielding the concentration for half-maximum channel blockade (IC50).
Equation (Uncited)Image Tools
The voltage dependence of fast and slow inactivation was assessed by applying a double-pulse protocol. Currents elicited by test pulses (Itest), after prepulses at varying potentials, normalized to the current elicited at the most hyperpolarized prepotential, represent the relative fraction of channels that have not been inactivated during the inactivating prepulse. Boltzmann fits to the resulting current-voltage plots yield the membrane potential at half-maximum channel availability (V0.5).
Equation (Uncited)Image Tools
Drug effects on the peak current amplitude were investigated at a holding potential close to the resting potential in physiological conditions (−70 mV) or at a hyperpolarized membrane potential (−150 mV). Due to its distinct inactivation properties, Nav1.8 channels were examined after a holding potential of −40 mV instead of −70 mV.26
Use-dependent block was assessed by 60 test pulses to 0 mV applied at 10 Hz. Steady-state fast inactivation was examined in cells held at −150 mV and induced by 100-millisecond long prepulses from −150 to −40 mV in intervals of 5 mV. Slow inactivation was induced by 10-second long prepulses from −120 to −10 mV in steps of 10 mV, followed by a 100-millisecond long pulse at −120 mV, allowing recovery from fast inactivation.
Resurgent currents were recorded by a protocol consisting of a 20-millisecond prepulse to +30 mV, followed by 50-millisecond long test pulses from −110 to 0 mV in steps of 10 mV.
All data are presented as mean ± SEM. or fitted value ± SE of the fit. Sample sizes were ≥5 as shown in Tables 1 and 2. When pairs of groups were compared, statistical analysis was performed by use of the Student t test. When there were >2 groups, analysis of variance (ANOVA) followed by Tukey Honestly Significant Difference (HSD) tests were used. P < 0.05 was considered statistically significant.
Throughout all investigated isoforms, the onset of drug effect developed rapidly. A steady state of sodium channel block at a given concentration of AJA was obtained during 40 to 60 seconds of drug application. During washout, currents reached 50% to 70 % of the control current value. The blocking effect remained stable and was left unchanged even when the respective concentration of AJA was applied longer than 10 minutes.
We first examined the effect of AJA on Nav1.7. Tonic block was investigated on resting (Fig. 2A) and inactivated channels (Fig. 2B). AJA induced a concentration-dependent tonic block of Nav1.7, yielding IC50 values of 5.0 ± 0.7 (n = 13) for resting channels and 2.7 ± 0.3 μmol/L (n = 9) for inactivated channels (Table 1). Tonic block was significantly different compared with the other channel isoforms except for Nav1.8 (P = 0.7); see section below (statistical results of ANOVA).
We next investigated further effects of 3 μmol/L AJA on Nav1.7; this concentration induced a robust inhibition of resting channels. As demonstrated in Figure 2D, 3 μmol/L AJA induced a small and not significant use-dependent block at 10 Hz (the last 20 of the applied repetitive pulses were compared by ANOVA P = 0.065; n = 6). We did not observe any effect of 3 μmol/L AJA on the current-voltage curve of Nav1.7 (n = 6) (Fig. 2F). In contrast, 3 μmol/L AJA induced robust effects on both fast (Fig. 2G) and slow inactivation (Fig. 2H). AJA 3 μmol/L induced a prominent hyperpolarizing shift of the steady-state inactivation curve from V0.5 −71 ± 1 mV in control solution to V0.5 −90 ± 1 mV in 3 μmol/L AJA (fast inactivation) (n = 8) (Table 2). Regarding slow inactivation, 3 μmol/L AJA induced a shift of the midpoint (V0.5) from −63 ± 1 mV in control solution to −73 ± 1 mV (n = 9) (Fig. 2H).
As is illustrated in Figure 3, A–J, AJA exhibited a tonic block on all investigated subunits. The IC50 values obtained by the Hill fit are shown in Table 1. Except for Nav1.8, all subunits were investigated as described above for Nav1.7. Statistical analysis generally yielded significant differences in IC50 values for tonic block of the investigated channel isoforms Nav1.2, 1.3, 1.4, 1.5, 1.7, and 1.8 (ANOVA, F(5, 49) = 69.051, P < 0.0001; for post hoc analysis, the Tukey HSD test was used). According to the post hoc test, IC50 values for block of resting channels by AJA were significantly lower in Nav1.4, 1.5, 1.7, 1.8 compared with Nav1.2 and 1.3 (P was 0.0001 for Nav1.4, 1.5, 1.7, and 1.8, respectively, in comparison with both Nav1.2 and Nav1.3, while P was 0.07 for Nav1.2 and 1.3 when comparing these 2 isoforms). IC50 values for Nav1.4 and Nav1.5 did not significantly differ (P = 0.99), the same as for Nav1.7 and Nav1.8 (P = 0.7). In comparison to the rest of the investigated isoforms, the respective IC50 value of all these 4 channels were significantly different (P < 0.001).
In the overall view, in all investigated channel isoforms, AJA exhibited half-maximal inhibition of sodium currents at low micromolar concentrations (approximately 2–9 μmol/L, as shown in Table 1). AJA 3 μmol/L induced a strong hyperpolarizing shift of the steady-state inactivation on Nav1.2, Nav1.4, Nav1.5, 1.5NN406K, 1.5F1760A, and 1.7 (Table 2).
Tonic block of resting channels by the local-anesthetic insensitive Nav1.5-mutants N406K and F1760A revealed IC50 values of 3 ± 0.1 μmol/L (n = 9) for N406K (Fig. 4, A and B) and 4 ± 0.3 μmol/L (n = 8) for F1760A (Fig. 4, C and D, Table 1). Thus, both mutants displayed a preserved sensitivity to block by AJA. Statistical analysis by ANOVA (F(2,21) = 5.8267, P < 0.01) followed by the Tukey HSD post hoc test yielded significant differences in IC50 when Nav1.5 (wild-type) was compared with the mutant F1760A (P = 0.009); however, no significant difference in IC50 values was determined comparing the wild-type channel Nav1.5 with the mutant N406K (P = 0.7).
Moreover, we found that 3 μmol/L AJA induced a prominent shift of steady-state fast inactivation of N406K (−87 ± 1 mV in control to −120 ± 1 mV in 3 μmol/L AJA, n = 10) (Fig. 4F) and of F1760A (−76 ± 1 mV in control to −118 ± 1 mV in 3 μmol/L AJA, n = 9) (Fig. 4G, Table 2).
We observed robust resurgent currents by including 100 μmol/L of the Navβ4-peptide into the pipette solution (Fig. 5, A and B). As is demonstrated in Figure 4C, 1 μmol/L AJA induced a robust inhibition of the resurgent current at all potentials. The inhibition of the peak current amplitude was 24 ± 4 % (n = 7). Tonic block of resting Nav1.5 channels by 1 μmol/L AJA was significantly less pronounced (6.6% ± 1%, n = 9) (P = 0.002, unpaired Student t test), indicating that AJA is indeed a potent inhibitor of resurgent currents.
This in vitro study identified the synthetic cannabinoid AJA as a potent blocker of voltage-gated sodium channels. Despite the fact that AJA does not induce cardiac or central nervous side effects in human volunteers, we found that AJA nonspecifically blocked all investigated α-subunits in a low micromolar concentration range.9 The molecular mechanism mediating this property is distinct from that used by classical local anesthetics. Thus, AJA might target a yet unknown binding site of α-subunits of sodium channels. Although speculative, it is possible that substances targeting this binding site can suppress pathological excitability in sensory neurons while leaving normal membrane excitability largely unaffected.
It is generally acknowledged that the redistribution and altered functional expression of voltage-gated sodium channels are implicated in chronic painful neuropathies that can arise from peripheral nerve injury.27,28 Among the α-subunits investigated in this study, primarily Nav1.3, Nav1.7, and Nav1.8 are expressed in sensory neurons. There is strong evidence that Nav1.3 and Nav1.8 have an impact on neuropathic pain-like behavior after nerve injury in rodents, and Nav1.7 has emerged as a key molecule for peripheral pain processing and for certain painful neuropathies in humans.27,29 Although the Nav1.9 channel is thought to play a prominent role in nociception and has the most divergent amino acid sequence among all Nav channel isoforms, it was not studied because it cannot be reliably expressed in heterologous expression systems.30
We found that AJA is an unspecific blocker of investigated α-subunits, excluding the possibility that analgesia induced by this compound involves a selective interaction with 1 sodium channel isoform. Although we did not have any direct reasons to hypothesize this property for AJA, the reported lack of cardiac and central nervous side effects in humans receiving AJA did not indicate an unselective inhibition of cardiac and neuronal α-subunits.8 Batista et al.31 reported that the plasma-concentration of AJA in patients receiving AJA at analgesic effective doses can be up to 2 μmol/. A fact that hinders the direct comparison between the plasma concentration in patients and the effective concentrations in our in vitro experiments is the plasma protein binding of AJA. From animal studies and unpublished phase I studies it is known that AJA is highly bound to plasma proteins in dog (99.5%–99.9%), rat (98.5%–99.8%), and human (97.0%–99.9%) samples (unpublished results of pharmacokinetic studies). Considering our experiments on Nav1.5 (heart) and Nav1.2 (central nervous system), one would definitely expect side effects to occur at these concentrations if the drug were all present in the free state and not bound to plasma proteins.
The state-dependent inhibition of sodium channels found for AJA in this study closely resembles the properties of local anesthetics, that is, our data strongly suggested that AJA also interacts with the local anesthetic binding site. However, both analyzed local anesthetic-insensitive Nav1.5-mutants (N406K and F1760A) displayed a preserved AJA sensitivity. These data more or less exclude a relevant interaction of AJA with the local anesthetics binding site and indicate that AJA targets a yet unknown site to block sodium channels. A more critical analysis of our data indeed reveals further properties of AJA that support this notion: Use-dependent block by 3 μmol/L AJA was minimal and probably not relevant. Use-dependent block is likely to arise from a higher affinity of a blocker to the open and inactivated states, and is a prototypical effect for most sodium channel blockers interacting with the local anesthetic binding site.32 Blockers with a high cardiotoxic potential, such as bupivacaine and amitriptyline, generally induce a very strong use-dependent block.23,33 It is possible that this property is predisposed for a high cardiac toxicity, and that the obvious lack of cardiotoxicity of AJA is due to its failure to induce use-dependent block. However, use-dependent inhibition may also be an important mechanism when sodium channel blockers are applied for treatment of neuropathic pain.34 Another remarkable property of AJA observed in this study was an impressive shift of the voltage dependency of steady-state fast inactivation. Even though this shift is also induced by local anesthetics and the opiod methadone,35 we are not aware of any substance inducing such a strong shift at low micromolar concentrations (approximately 20 mV by 3 μmol/L). For local anesthetics, this effect is due to an interaction with the local anesthetic site since it is abbreviated in local anesthetic channel mutants.32 However, the shift induced by AJA is perfectly preserved on the mutants Nav1.5—N406K and F1760A, and thus, this effect appears independent of the local anesthetic binding site.
Considering the lipophilic structure and chemical properties of AJA, interactions in the lipid bilayer of the cell membrane could be one possible site of action as suggested for halothane.36 AJA shares some features with volatile anesthetics. Both AJA and volatile anesthetics such as sevoflurane, desflurane, or isoflurane show a hyperpolarizing shift of the voltage-dependency of fast inactivation as well as a voltage-dependent block of sodium currents among the different sodium channel subunits.37–39 Unlike AJA, volatile anesthetics induce significant use-dependent inhibition, whose extent varies due to substance-specific differences in block among different volatile anesthetics.37
AJA’s LogP value (octanol:water partition coefficient) is approximately 7.55. Its lipophilicity might facilitate effects on binding sites in the lipid bilayer to shift the channel gating. But also an interaction with a hydrophobic pocket within the channel protein itself is hypothetically conceivable. Thus, further studies are needed to explore the detailed molecular mechanism of this drug’s action.
A comparable effect on steady-state fast inactivation observed for AJA in this study was recently demonstrated for the endogenous cannabinoid anandamide on Nav1.7.19 The authors of this study found that this property correlates with the ability of the drug to inhibit resurgent currents, and postulated that substances inducing a preferential block of resurgent currents might be useful therapeutic tools. It is interesting to note that this inhibitory property was suggested to be independent from the local anesthetic binding site.19 In our study, AJA inhibited Navβ4-peptide-mediated resurgent Nav1.5-currents more potently than transient currents generated by wild-type Nav1.5. Wild-type Nav1.5 was chosen because it was previously demonstrated to generate large Navβ4 peptide-mediated resurgent currents.40 In contrast, wild-type Nav1.7 hardly generates any detectable resurgent currents.19 As resurgent currents of different α-subunits have been suggested to drive different pain syndromes in humans,21,22 it is possible that the analgesic efficacy of AJA is in part due to inhibition of resurgent currents.
Limitations of Our Study
We have studied the effects of AJA in vitro in sodium channel α-subunits. Alpha-subunits of sodium channels show normal gating characteristics (with respect to experiments in native tissue) when expressed in a mammalian cell line in the absence of the β-subunit.41,42 In HEK293 cells mRNA encoding, the β1α splicing of the putative regulatory sodium channel subunit is abundantly expressed.43
Irrespective of the role that altered expression of β-subunits may have in the development of pain states in vivo,44 concerns have been raised that exogenous introduction of β1 in HEK cells in vitro may lead to an overexpression of β-subunits which in turn might have unpredictable effects on the pharmacologic interaction between the sodium channel α-subunit and channel blockers.43
The advantage of this experimental approach is that drug interaction with the sodium channel can be studied without confounding factors in this model since it is generally accepted that the receptor sites for pharmacological agents interfering with voltage-gated sodium channels are located on the α-subunits.45
In summary, our in vitro study identified AJA as an unselective inhibitor of voltage-dependent sodium channels. This inhibition is concentration- and state-dependent, properties that AJA shares with other cannabinoids. It is assumed that these inhibitory effects of AJA are mediated by mechanisms distinct from the local anesthetic binding site. This could be one of the mechanisms by which AJA exerts analgesic effects in patients with neuropathic pain. Thus, AJA is not only an interesting drug with implications for pain treatment but also as a tool to further explore a yet poorly defined route to inhibit voltage-dependent sodium channels.
Name: Nilufar Foadi, MD.
Contribution: Nilufar Foadi designed experiments, acquired, analyzed, interpreted data, and participated in writing the manuscript.
Attestation: Nilufar Foadi read and approved the final manuscript and is the archival author.
Name: Christian Berger, MD.
Contribution: Christian Berger performed experiments and acquired, analyzed, and interpreted data.
Attestation: Christian Berger read and approved the final manuscript.
Name: Igor Pilawski, VMD.
Contribution: Igor Pilawski performed experiments and acquired, analyzed, and interpreted data.
Attestation: Igor Pilawski read and approved the final manuscript.
Name: Carsten Stoetzer, MD.
Contribution: Carsten Stoetzer performed experiments and acquired, analyzed, and interpreted data.
Attestation: Carsten Stoetzer read and approved the final manuscript.
Name: Matthias Karst, MD.
Contribution: Matthias Karst participated in design of experiments, contributed to the methods and illustrations, and edited the manuscript.
Attestation: Matthias Karst read and approved the final manuscript.
Name: Gertrud Haeseler, MD.
Contribution: Gertrud Haeseler participated in design of experiments, contributed to the methods and illustrations, and edited the manuscript.
Attestation: Gertrud Haeseler read and approved the final manuscript.
Name: Florian Wegner, MD.
Contribution: Florian Wegner participated in design of experiments, contributed to the methods and illustrations, and edited the manuscript.
Attestation: Florian Wegner read and approved the final manuscript.
Name: Andreas Leffler, MD.
Contribution: Andreas Leffler mentored, conceived, designed, coordinated the study, and drafted the manuscript.
Attestation: Andreas Leffler read and approved the final manuscript.
Name: Jörg Ahrens, MD.
Contribution: Jörg Ahrens mentored, conceived, designed, coordinated the study, and drafted the manuscript.
Attestation: Jörg Ahrens read and approved the final manuscript.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
We are indebted to Prof. Frank Lehmann-Horn (Ulm, Germany) for providing us with transfected cells, Andreas Niesel (Hannover, Germany) for technical support and Prof. Sumner Burstein, (Boston) for his kind supply of AJA.
1. Costa B. On the pharmacological properties of Delta9-tetrahydrocannabinol (THC). Chem Biodivers. 2007;4:1664–77
2. Burns TL, Ineck JR. Cannabinoid analgesia as a potential new therapeutic option in the treatment of chronic pain. Ann Pharmacother. 2006;40:251–60
3. Karst M, Wippermann S, Ahrens J. Role of cannabinoids in the treatment of pain and (painful) spasticity. Drugs. 2010;70:2409–38
4. McCarberg BH, Barkin RL. The future of cannabinoids as analgesic agents: a pharmacologic, pharmacokinetic, and pharmacodynamic overview. Am J Ther. 2007;14:475–83
5. Burstein S. Ajulemic acid (IP-751): synthesis, proof of principle, toxicity studies, and clinical trials. AAPS J. 2005;7:E143–8
6. Burstein SH, Karst M, Schneider U, Zurier RB. Ajulemic acid: A novel cannabinoid produces analgesia without a “high”. Life Sci. 2004;75:1513–22
7. Dyson A, Peacock M, Chen A, Courade JP, Yaqoob M, Groarke A, Brain C, Loong Y, Fox A. Antihyperalgesic properties of the cannabinoid CT-3 in chronic neuropathic and inflammatory pain states in the rat. Pain. 2005;116:129–37
8. Karst M, Salim K, Burstein S, Conrad I, Hoy L, Schneider U. Analgesic effect of the synthetic cannabinoid CT-3 on chronic neuropathic pain: a randomized controlled trial. JAMA. 2003;290:1757–62
9. Salim K, Schneider U, Burstein S, Hoy L, Karst M. Pain measurements and side effect profile of the novel cannabinoid ajulemic acid. Neuropharmacology. 2005;48:1164–71
10. Ambrosio AL, Dias SM, Polikarpov I, Zurier RB, Burstein SH, Garratt RC. Ajulemic acid, a synthetic nonpsychoactive cannabinoid acid, bound to the ligand binding domain of the human peroxisome proliferator-activated receptor gamma. J Biol Chem. 2007;282:18625–33
11. Liu J, Li H, Burstein SH, Zurier RB, Chen JD. Activation and binding of peroxisome proliferator-activated receptor gamma by synthetic cannabinoid ajulemic acid. Mol Pharmacol. 2003;63:983–92
12. Ahrens J, Leuwer M, Demir R, Krampfl K, de la Roche J, Foadi N, Karst M, Haeseler G. Positive allosteric modulatory effects of ajulemic acid at strychnine-sensitive glycine alpha1- and alpha1beta-receptors. Naunyn Schmiedebergs Arch Pharmacol. 2009;379:371–8
13. Xiong W, Cheng K, Cui T, Godlewski G, Rice KC, Xu Y, Zhang L. Cannabinoid potentiation of glycine receptors contributes to cannabis-induced analgesia. Nat Chem Biol. 2011;7:296–303
14. Xiong W, Cui T, Cheng K, Yang F, Chen SR, Willenbring D, Guan Y, Pan HL, Ren K, Xu Y, Zhang L. Cannabinoids suppress inflammatory and neuropathic pain by targeting α3 glycine receptors. J Exp Med. 2012;209:1121–34
15. Strichartz GR, Chiu SY, Ritchie JM. The effect of delta9-tetrahydrocannabinol on the activation of sodium conductance in node of Ranvier. J Pharmacol Exp Ther. 1978;207:801–9
16. Turkanis SA, Partlow LM, Karler R. Delta-9-tetrahydrocannabinol depresses inward sodium current in mouse neuroblastoma cells. Neuropharmacology. 1991;30:73–7
17. Kim HI, Kim TH, Shin YK, Lee CS, Park M, Song JH. Anandamide suppression of Na+ currents in rat dorsal root ganglion neurons. Brain Res. 2005;1062:39–47
18. Fu H, Xiao JM, Cao XH, Ming ZY, Liu LJ. Effects of WIN55,212-2 on voltage-gated sodium channels in trigeminal ganglion neurons of rats. Neurol Res. 2008;30:85–91
19. Theile JW, Cummins TR. Inhibition of Navβ4 peptide-mediated resurgent sodium currents in Nav1.7 channels by carbamazepine, riluzole, and anandamide. Mol Pharmacol. 2011;80:724–34
20. Jarecki BW, Piekarz AD, Jackson JO 2nd, Cummins TR. Human voltage-gated sodium channel mutations that cause inherited neuronal and muscle channelopathies increase resurgent sodium currents. J Clin Invest. 2010;120:369–78
21. Klinger AB, Eberhardt M, Link AS, Namer B, Kutsche LK, Schuy ET, Sittl R, Hoffmann T, Alzheimer C, Huth T, Carr RW, Lampert A. Sea-anemone toxin ATX-II elicits A-fiber-dependent pain and enhances resurgent and persistent sodium currents in large sensory neurons. Mol Pain. 2012;8:69
22. Sittl R, Lampert A, Huth T, Schuy ET, Link AS, Fleckenstein J, Alzheimer C, Grafe P, Carr RW. Anticancer drug oxaliplatin induces acute cooling-aggravated neuropathy via sodium channel subtype Na(V)1.6-resurgent and persistent current. Proc Natl Acad Sci U S A. 2012;109:6704–9
23. Nau C, Wang SY, Strichartz GR, Wang GK. Block of human heart hH1 sodium channels by the enantiomers of bupivacaine. Anesthesiology. 2000;93:1022–33
24. O’Reilly AO, Eberhardt E, Weidner C, Alzheimer C, Wallace BA, Lampert A. Bisphenol A binds to the local anesthetic receptor site to block the human cardiac sodium channel. PLoS One. 2012;7:e41667
25. Haeseler G, Foadi N, Wiegand E, Ahrens J, Krampfl K, Dengler R, Leuwer M. Endotoxin reduces availability of voltage-gated human skeletal muscle sodium channels at depolarized membrane potentials. Crit Care Med. 2008;36:1239–47
26. Leffler A, Reiprich A, Mohapatra DP, Nau C. Use-dependent block by lidocaine but not amitriptyline is more pronounced in tetrodotoxin (TTX)-Resistant Nav1.8 than in TTX-sensitive Na+ channels. J Pharmacol Exp Ther. 2007;320:354–64
27. Liu M, Wood JN. The roles of sodium channels in nociception: implications for mechanisms of neuropathic pain. Pain Med. 2011;12(Suppl 3):S93–9
28. Theile JW, Cummins TR. Recent developments regarding voltage-gated sodium channel blockers for the treatment of inherited and acquired neuropathic pain syndromes. Front Pharmacol. 2011;2:54
29. Dib-Hajj SD, Black JA, Waxman SG. Voltage-gated sodium channels: therapeutic targets for pain. Pain Med. 2009;10:1260–9
30. Waxman SG, Estacion M. Nav1.9, G-proteins, and nociceptors. J Physiol. 2008;586:917–8
31. Batista C, Berisha M, Karst M, Salim K, Schneider U, Brenneisen R. Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2005;820:77–82
32. Nau C, Wang GK. Interactions of local anesthetics with voltage-gated Na+ channels. J Membr Biol. 2004;201:1–8
33. Nau C, Seaver M, Wang SY, Wang GK. Block of human heart hH1 sodium channels by amitriptyline. J Pharmacol Exp Ther. 2000;292:1015–23
34. Ritter AM, Ritchie C, Martin WJ. Relationship between the firing frequency of injured peripheral neurons and inhibition of firing by sodium channel blockers. J Pain. 2007;8:287–95
35. Schulze V, Stoetzer C, O’Reilly AO, Eberhardt E, Foadi N, Ahrens J, Wegner F, Lampert A, de la Roche J, Leffler A. The opioid methadone induces a local anaesthetic-like inhibition of the cardiac Na+
channel, Na(v)1.5. Br J Pharmacol. 2014;171:427–37
36. Vemparala S, Saiz L, Eckenhoff RG, Klein ML. Partitioning of anesthetics into a lipid bilayer and their interaction with membrane-bound peptide bundles. Biophys J. 2006;91:2815–25
37. Ouyang W, Herold KF, Hemmings HC Jr. Comparative effects of halogenated inhaled anesthetics on voltage-gated Na+ channel function. Anesthesiology. 2009;110:582–90
38. Haydon DA, Urban BW. The effects of some inhalation anaesthetics on the sodium current of the squid giant axon. J Physiol. 1983;341:429–39
39. Herold KF, Hemmings HC Jr. Sodium channels as targets for volatile anesthetics. Front Pharmacol. 2012;3:50
40. Wang GK, Edrich T, Wang SY. Time-dependent block and resurgent tail currents induced by mouse beta4(154-167) peptide in cardiac Na+ channels. J Gen Physiol. 2006;127:277–89
41. Sarkar SN, Adhikari A, Sikdar SK. Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line. J Physiol. 1995;488 (Pt 3):633–45
42. Chahine M, Bennett PB, George AL Jr, Horn R. Functional expression and properties of the human skeletal muscle sodium channel. Pflugers Arch. 1994;427:136–42
43. Moran O, Nizzari M, Conti F. Endogenous expression of the beta1A sodium channel subunit in HEK-293 cells. FEBS Lett. 2000;473:132–4
44. Takahashi N, Kikuchi S, Dai Y, Kobayashi K, Fukuoka T, Noguchi K. Expression of auxiliary beta subunits of sodium channels in primary afferent neurons and the effect of nerve injury. Neuroscience. 2003;121:441–50
45. Catterall W, Epstein PN. Ion channels. Diabetologia. 1992;35(Suppl 2):S23–33