Similarly, no EG-induced currents were observed in recordings from oocytes expressing GlyR α1, α2, or α3 homopentameric receptors, although glycine and even sarcosine-induced currents were readily detectable (Fig. 2A, B, D-F). To analyze whether EG acts as a (partial) antagonist on the glycine receptor, glycine and EG were coapplied to oocytes expressing the respective homopentameric receptor. Here, in none of the recordings, significant effects of EG coapplication on the current amplitude as compared with the currents observed after application of glycine alone were observed, demonstrating that EG does not function as a partial antagonist concerning any of these proteins (Fig. 2G-I). At central nervous system (CNS) synapses, the majority of GlyRs is not present as GlyR α homopentameric receptors but as heteropentamers together with the GlyR β.21 To exclude that EG acts specifically on these heteropentameric receptors, the effects of EG and/or sarcosine were also analyzed in oocytes expressing the respective heteromers. Here, no significant differences to oocytes expressing the respective GlyR α homoreceptor were observed (Fig. 2C-I), confirming that EG acts neither in an agonistic nor antagonistic fashion on GlyRs.
Similar to our experiments on GlyT2 and GlyR receptors, effects of EG on NMDAR (NR1/NR2a) currents were analyzed (Fig. 3). Although NMDAR-mediated currents were readily detectable after application of glycine together with glutamate (Fig. 3A-C, E), no substance-induced currents were observed when glutamate or glycine was applied alone. Application of EG either alone, or in combination with glycine or glutamate, did not produce significant substance-induced currents (Fig. 3A, D). To determine whether EG inhibits NMDAR-mediated currents induced by glutamate and glycine, all 3 substances were applied on NMDAR-expressing oocytes. Similar to our observations in GlyT2- and GlyR-expressing oocytes, the observed current amplitude after application of all 3 substances was indistinguishable from that observed after the application of glutamate and glycine alone (Fig. 3E), indicating that EG does not function as an inhibitor at the NMDAR.
The application of any of the aforementioned substances had no effect on currents in noninjected (wild-type) oocytes (data not shown). Taken together, these data show that EG acts only as a substrate for GlyT1 but has no agonistic or antagonistic function at the other major glycine-responsive proteins within the CNS.
3.2. Systemic application of EG efficiently ameliorates inflammatory hyperalgesia
To test whether the lidocaine metabolite EG can directly exert an antinociceptive effect on inflammatory pain in vivo, CFA was injected into the left hind paw of adult male C57BL/6J mice (n = 8 per group). Testing of mechanical sensitivity using von Frey filaments revealed a profound allodynia that developed in the treated paw 72 hours after injection of CFA, whereas mechanical sensitivity of the untreated paw was not altered. Subcutaneous bolus application of EG (200 mg/kg) or sarcosine (200 mg/kg) increased withdrawal thresholds observed in the CFA-treated paw within 1 hour after application, indicating a reduction of mechanical hyperalgesia. After a plateau of 4 hours, the apparent beneficial effect of EG and sarcosine slowly declined, and after 24 hours, the mechanical sensitivity was back to baseline levels (Fig. 4A). In contrast, mechanical sensitivity of the untreated contralateral hind paw did not change at any point during the experiment (Fig. 4B). The antihyperalgesic effect was fully reproducible when repeating the treatment after 48 hours (Fig. 4A). When repeating EG applications at 12-hour intervals for 3 consecutive days, however, no additional beneficial effects in the sense of a delayed therapeutic response were observed when evaluating 12 hours after injection (data not shown).
To test whether this antihyperalgesic effect results from an anti-inflammatory effect of EG or sarcosine, the degree of inflammation-induced edema was determined on the basis of paw thickness. Here, thickness of the paw that received the CFA injection (left paw) was approximately 40% increased as compared with the untreated control side, confirming, in addition to the behavioral data, the successful induction of the inflammation. At 24 hours after treatment with sarcosine or EG, however, no change in the inflammation-induced edema was observed, suggesting that, at least in the current experimental setting, neither test drug shows any anti-inflammatory potential (Fig. 4C).
To determine the dose dependency of EG in this model of inflammatory pain, inflammation-induced hyperalgesia was induced by CFA injection in the left hind paw of the animals. After successful verification that a pronounced allodynia had developed, various concentrations of EG were injected subcutaneously and the withdrawal threshold after mechanical stimulation tested 2 hours after injection (n = 8 per group). Half-logarithmic plotting of the data revealed an EC50 of 98 mg/kg (Fig. 5A).
To test for possible side effects of EG that might result from a general increase of the glycinergic tone or from other routes of action, eg, acute toxic effects, the general activity of mice after treatment with 400 mg/kg EG was analyzed and compared with mice injected with 40 mg/kg of the parent substance lidocaine (n = 8 per group). Although in all lidocaine-treated animals, severe adverse effects like irregular movements or (respiratory) distress were seen within the first 8 minutes after injection, such effects were not observed in any of the mice treated with the lidocaine metabolite EG (Fig. 5B). To determine whether EG treatment causes subtle adverse effects, that result from, for example, a general increase in the glycinergic tone, the motor performance of mice was analyzed with rotarod assay before and 2 hours after EG treatment and compared with that of sarcosine- or saline-treated animals (Fig. 5C; n = 8 per group). Both, before and after EG treatment, the performance of the animals in all 3 groups was indistinguishable, suggesting that short-term treatment with EG does not produce major side effects in mice. Consistent with these findings, no change in the paw withdrawal threshold to heat exposure using the Hargreaves test was observed after application of EG to naive animals at any time point analyzed (Fig. 5D; n = 8 per group), indicating that responses to acute painful stimuli were not altered after EG treatment. The effect of EG application on general activity and explorative behavior was subsequently determined in an open-field experiment. Here, neither the general activity of EG-treated mice (200 mg/kg, subcutaneously) as determined by the total distance traveled nor their explorative behavior as determined by the regional preference for the central quadrant was distinguishable from that of mice that received saline injections only (Fig. 5E-G; n = 6-7 per group). Taken together, these data demonstrate that systemic application of EG, similar to sarcosine, efficiently ameliorates inflammatory hyperalgesia, whereas acute pain sensitivity is not affected. Moreover, these data reveal no major additional effects of acute EG treatment on the general activity of mice.
3.3. Systemic application of EG reduces allodynia and hyperalgesia in mice with neuropathic pain
Subsequently, we analyzed whether EG has an antinociceptive effect only in inflammatory pain or whether this phenomenon can also be observed in an animal model of neuropathic pain. To induce neuropathy, we made use of the CCI model of the sciatic nerve. The establishment of CCI-induced allodynia and hyperalgesia was determined by mechanical and thermal testing of withdrawal thresholds. All animals analyzed (n = 8 per group) developed pronounced mechanical allodynia and thermal hyperalgesia within 72 hours after the operation at the ipsilateral side (Fig. 6), whereas no changes in pain sensitivity were observed at the contralateral side (data not shown). Upon subcutaneous injection of EG, a significant reduction of both CCI-induced mechanical (Fig. 6A) and thermal hyperalgesia (Fig. 6B) was observed within 2 hours after injection. Similar to our findings for inflammatory pain, the observed reduction in hyperalgesia was only transient with a maximal effect observed after 2 to 4 hours. Pain sensitivity was indistinguishable from that of saline-treated mice at 24 hours after injection (Fig. 6).
3.4. Systemic application of EG leads to an increase in serum and cerebrospinal fluid glycine concentrations
Based on its action on GlyT1 as an artificial substrate, we hypothesized that EG might compete with glycine for the uptake into the surrounding tissue of glycinergic synapses and thus cause an increase in the extracellular glycine concentration at least within the CNS. Therefore, we investigated both the concentration of EG and glycine in the serum and CSF in EG-treated animals. As the obtained volume, especially of CSF, was not sufficient for HPLC-based analysis when isolated from mice, we performed these experiments in rats (n = 4 per group). In the serum of untreated rats, a basal glycine concentration of 207 ± 37 μM was observed. Interestingly, EG also was already found in low concentrations (43 ± 6 μM). After subcutaneous injection of EG, a rapid increase in the EG serum concentration was observed that peaked with 384 ± 34 μM only 1 hour after injection. After a 1-hour plateau, a steady decline of the serum EG concentration was observed, and after 24 hours, the serum concentration of EG reached the basal level. During the whole observation period, however, there was no significant change in the serum glycine concentration (Fig. 7A). Based on these measurements, we calculated a volume of distribution for EG of approximately 1.2 L or 4.8 L/kg. Thus, a theoretical dose of 98 mg/kg, estimated as EC50 in our behavioral dose–response experiments (Fig. 5A), would result in a serum concentration of 20.4 mg/L (198 μM) 2 hours after injection.
In contrast to our findings on the serum concentrations of EG and glycine, much lower basal concentrations were found in CSF samples of untreated animals (16.5 ± 0.5 μM and 5.5 ± 0.9 μM for glycine and EG, respectively). In samples from animals that received EG injections, the concentration of EG almost doubled within 2 hours after injection, followed by a slow decline. Corroborating our serum concentration findings, the EG CSF levels of rats that received EG injections 24 hours before testing were indistinguishable from those of untreated control animals. Analysis of the CSF glycine concentration revealed that EG injection resulted in a 25% increase in extracellular glycine concentration (to 20.5 ± 1.9 μM). The maximal increase was observed 2 hours after injection of EG. Subsequently, the CSF glycine concentration slowly declined and in rats that received the EG injection 24 hours before CSF sampling, glycine concentrations were comparable with those of untreated animals (Fig. 7B).
Taken together, these findings demonstrate that after systemic application, EG reaches the CSF and has an effect on the extracellular glycine concentration that is consistent with a transient reduction of the GlyT1-mediated glycine transport capacity.
3.5. EG affects the pain processing circuitry by inhibiting the inflammation-induced hyperexcitability of dorsal horn neurons
To investigate the effects of EG on the evoked responses of dorsal horn neurons, we determined the number of action potentials elicited by electrical stimulation in recordings from WDR neurons of the deep laminae in the rat spinal cord dorsal horn. After recording baseline responses after stimulation of the corresponding receptive field at the glabrous surface of the hind paw, an inflammatory response followed by neuronal hyperexcitability was induced by injection of 1% carrageenan solution into the hind paw. Individual action potentials were attributed to the respective fiber types according to their latency (Aβ fibers: 0-20 milliseconds, Aδ fibers: 20-90 milliseconds, and C fibers: 90-350 milliseconds). Here, evaluation of electrical stimulus–evoked responses revealed that the majority of detected action potentials had a stimulus–response latency that corresponded to C fibers (90-350 milliseconds), while neuronal input from Aβ and Aδ fibers was less abundant (see Fig. 8A and Supplemental Digital Content 1 [available online at http://links.lww.com/PAIN/A93], which contains examples of responses of single neurons to electrical stimulation at baseline, after carrageenan injection and subsequent EG treatment). After carrageenan injection, a significant increase in the firing rate in response to Aβ-, Aδ-, and C-fiber activity was observed after 3 hours. Subsequently, EG was applied directly onto the dorsal surface of the spinal cord (n = 4 per group). Further recordings 30 minutes after application revealed a dose-dependent reduction of detected action potentials in response to electrical stimulation reaching complete suppression of neuronal hyperexcitability at concentrations higher than 100 μM (Fig. 8B). As individual neuron responses varied considerably, normalization to maximal responses (3 hours after carrageenan injection) was performed. To determine whether this dose-dependent reduction in the firing rate of dorsal horn neurons was also observed in reaction to physiological stimuli, the respective receptive field at the glabrous surface of the hind paw was stimulated thermally (Fig. 8C; constant water jet 40°C or 48°C) or mechanically (Fig. 8D; brush, 8 or 60g hair). Here, consistent with our findings after electrical stimulation, we observed a strong facilitation of the stimulus-induced firing rate after carrageenan with innocuous stimuli being affected more strongly. After treatment with increasing concentrations of EG, the carrageenan-induced hyperexcitability was dose-dependently reduced. Here, also a stronger effect was observed, when normally innocuous stimuli (8g hair, 40°C water jet) were applied. These findings demonstrate that EG affects the pain processing circuitry by inhibiting the inflammation-induced hyperexcitability of dorsal horn neurons, thus confirming the antihyperalgesic action of EG independently of behavioral approaches.
Treatment of chronic pain is still one of the most challenging clinical endeavors. Here, classical analgesics like opioids and nonsteroidal antirheumatics usually have limited efficacy. Despite continuing research, no substantial improvement in therapeutic outcome has emerged.4,29
For more than 50 years, it has been known that systemic long-term application of lidocaine results in a long-lasting general antinociceptive effect,33 although the mechanism mediating these effects remained up to now enigmatic. The relative late onset of the therapeutic benefit, as well as the extremely low levels of the plasma lidocaine concentration required, argue against a direct function of lidocaine on the principal target of this class of substances ie, voltage-gated sodium channels.47 Previous studies have demonstrated that in addition to the general analgesic effect, long-term treatment with lidocaine has an anti-inflammatory potential31 through an up-to-now unknown mechanism, which might contribute to the long-lasting beneficial effects. In this study, we provide evidence that the antinociceptive effect seen after long-term treatment with lidocaine can be mimicked by systemic application of the lidocaine metabolite EG, which acts as an artificial substrate specifically on glycine transporters of the GlyT1 subtype, suggesting a novel additional route of action for systemically applied lidocaine. Systemic application of EG results in a significant elevation of the extracellular glycine concentration within the CSF and thereby inhibition of inflammation-induced hyperexcitability of WDR neurons in the deep lamina of the dorsal horn.
In the mature nervous system, the extracellular glycine concentration is synergistically regulated by GlyT1 and GlyT215 with GlyT2 being essential for replenishing the presynaptic glycine pool. Loss of the GlyT2 activity results in a pronounced disinhibition of motor neurons and consequently leads to severe neuromotor deficits in both rodents and humans.16,20,38 In contrast, the major function of glial GlyT1 is the maintenance of low extracellular glycine concentrations.19 Inactivation of GlyT1 expression in glial cells of neonatal animals results in severe hypotonia, respiratory depression and premature death. At later developmental stages, however, a loss of glial GlyT1 seems to be well tolerated.17 Additionally, neuronal expression of GlyT1 has been shown to be an important modulator of glutamatergic neurotransmission through glutamate receptors of the NMDAR subtype.50 Therefore, GlyT1 inhibitors are currently in phase 3 trials for the treatments of diseases associated with NMDAR dysfunction like schizophrenia (see, eg, ClinicalTrials.gov Identifier: NCT01192867).
There is an increasing body of evidence, in addition to this study, that suggests both inhibition of GlyT1 and GlyT2 are suitable strategies for the treatment of chronic pain conditions including inflammatory and neuropathic pain.25 The interpretation of these findings has in many cases been hampered by the fact that the currently available inhibitors are poorly characterized and/or are proven to cross-react with other components of glycine-dependent neurotransmission. For example, the GlyT2 inhibitor ALX1393 also inhibits GlyT1 at low micromolar concentrations,34 while the GlyT1 substrate sarcosine was shown to act also as a partial agonist on GlyR53 and NMDAR.52 In contrast, we found EG to be specific for GlyT1 without any effects on GlyT2, GlyRs, or NMDARs in vitro. This high specificity for GlyT1 might facilitate better predictability of the expected effects in vivo and therefore prevent undesired secondary effects.
Our behavioral experiments show that systemic EG is dose-dependently antinociceptive in chronic but not acute pain. Interestingly, EG diminished pain states induced by either inflammation or CCI in the same manner, although the spinal cord pathophysiology has been shown to be different. In contrast to neuropathic pain,26,28 inflammation-induced pain requires an increase in the production of the pronociceptive prostaglandin PGE2 in the spinal cord, which diminishes glycinergic inhibition in the dorsal horn by phosphorylation of α3 subunit containing GlyR.1,24 Thus, it appears that the reduction of the glycine uptake capacity of GlyT1 might constitute a general treatment strategy for diseases associated with a diminished inhibitory neurotransmission in caudal regions of the CNS like chronic pain with central sensitization.
The time course of the antinociceptive effect elicited by EG was similar in CFA- and CCI-induced pain; withdrawal thresholds were already significantly increased within 1 hour after application, and this increase lasted for 4 to 6 hours. Thus, onset of antinociceptive effects was significantly faster than what was observed with GlyT1 inhibitors previously.5,35 Whether this discrepancy in the kinetics of action results from differences in the pharmacokinetics or from a different mechanism (eg, inhibitor vs artificial substrate) is not clear at present.
Measurements of serum concentrations revealed that after subcutaneous injection, serum EG concentrations increased quickly and remained elevated for 2 hours at levels slightly below 400 μM while serum glycine concentration remained unchanged. These findings support the idea that EG specifically acts on GlyT1 because the plasma concentration of glycine has been suggested to be controlled by other (low affinity) transporters such as System A.32 Interestingly, EG concentrations found after subcutaneous EG injection were similar to those reported in the plasma of patients receiving continuous systemic or epidural applications of lidocaine (range: 3-65 mg/L, ie, 30-650 μM).39 By measuring the spinal fluid EG and glycine levels, we could show that EG crosses the blood–brain barrier and leads to a short-term increase in spinal fluid glycine concentration. This further supports the hypothesis that EG functions through a reduction of the glycine transport capacity of GlyT1. It has to be noted, however, that the concentration measurements performed here only allow for an evaluation of extrasynaptic glycine levels that may differ from those at synaptic sites. Taken together, these data suggest that after continuous application of lidocaine, EG can accumulate and reach plasma concentrations that are sufficient for a significant glycine transport inhibition in vitro and antinociceptive effects in vivo. These results strongly suggest that the antinociceptive effects of systemic lidocaine are caused by EG accumulation that results in elevated glycine levels at synapses of spinal inhibitory circuits.
In previous studies, the beneficial effect of GlyT inhibitors was observed only when a fraction of the total GlyT activity was blocked. Strong inhibition of GlyT1 activity caused severe adverse effects like pronociceptive effects, neuromotor disturbances, or respiratory depression.27,35,37 Such negative consequences were not observed in animals treated with even twice the antinociceptive dose of EG. This possibly could be explained by EG's only moderate affinity for GlyT1 and the fact that EG, in contrast to previously evaluated substances, constitutes an artificial GlyT1 substrate and does not act as an inhibitor of GlyT1.
Interestingly, previous studies have shown that long-term blockade of GlyT1 by the irreversible GlyT1 inhibitor ALX5407 appears not only to ameliorate pathological pain symptoms but also to reverse some of the neuroplastic changes involved in the manifestation of neuropathic pain such as an increase of NMDAR subunit expression NR1 within the spinal cord.5 This might result from the previously described internalization of NMDAR after sustained saturation of the NMDAR glycine binding.36 These effects might facilitate the beneficial effects of GlyT1 in the treatment of pathological pain and explain some of the long-lasting effects of even a single dose application of GlyT1 inhibitors.35 In this study, using the GlyT1 substrate EG, no long-lasting antihyperalgesic effect was observed, most likely due to the relative short treatment period and the fact that here we used not a GlyT1 inhibitor but an artificial substrate. Whether injection of higher doses or continuous application of EG might result in sustained or long-lasting effects on nociception remains to be elucidated in further experiments.
Using in vivo extracellular recordings at dorsal horn neurons in the deep laminae of the rat lumbar spinal cord, we identified strong inhibitory effects of EG on inflammation-induced neuronal hyperexcitability in WDR neurons. Here, only intrathecal concentrations of more than 100 μM EG led to complete suppression of neuronal hyperexcitability, although significant effects were observed using 30 μM EG. This finding most likely results from inhibition of glycine uptake capacity and the subsequent accumulation of extracellular glycine. Support for the hypothesis that this is the most likely mode of action also comes from our HPLC experiments, which show that CSF concentrations of up to 20 μM EG can be reached by systemic application of EG at a dose that has been shown to be effective in behavioral experiments (200 mg/kg). However, it has to be considered that all in vivo electrophysiological experiments were performed under general anesthesia and thus a direct correlation with behavioral data is difficult.
In conclusion, our findings suggest that EG, as a substrate of GlyT1, mediates at least in part the antinociceptive effect of systemically applied lidocaine. Furthermore, our results demonstrate that competitive inhibition of GlyT1 by application of GlyT1 substrates like EG is a promising strategy for the therapy of chronic pain conditions. Future investigations are needed to determine the precise molecular mechanisms behind the antinociceptive action, eg, to what extent altered spinal and supraspinal glycinergic and glutamatergic neurotransmission contributes to this phenomenon or whether low-affinity glycine transport by systems A, ASC, etc., is sensitive to EG. Finally, whether these effects can be confirmed in clinical studies remains to be elucidated.
Conflict of interest statement
The authors have no conflicts of interest to declare.
This work was supported by the German Research Foundation (DFG We4860/1-1 to R.W. and EU110/3-1 to V.E.), the Wellcome Trust funded London Pain Consortium (to J.N.W.), and the Interdisciplinary Center for Clinical Research (IZKF) of the University Hospital Erlangen (TP E15 to V.E.).
The authors would like to gratefully acknowledge Dr Carolyn Hyde (Bioanalysis Manager, University College London, United Kingdom) for technical guidance in high-pressure liquid chromatography and Dr Christoph Korbmacher (Institute for Physiology, University of Erlangen-Nürnberg) for providing Xenopus laevis oocytes. The excellent technical assistance of Marina Wenzel (Institute of Biochemistry, University of Erlangen-Nürnberg) is gratefully acknowledged.
Supplemental Digital Content
Supplemental Digital Content associated with this article can be found online at http://links.lww.com/PAIN/A93.
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Neuropathic pain; Inflammatory pain; N-ethylglycine; Glycine transporter; Glycinergic inhibition; Lidocaine metabolites
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