Targeting voltage-gated sodium and calcium channels in primary sensory neurons for the development of chronic pain therapeutics
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Type
ThesisThesis type
Doctor of PhilosophyAuthor/s
Munasinghe, NehanAbstract
Chronic pain is a debilitating condition that affects both the emotional and physical well-being of an individual. Current therapeutics for chronic pain are relatively ineffective and produce many adverse side effects including tolerance development and addiction. Therefore, the ...
See moreChronic pain is a debilitating condition that affects both the emotional and physical well-being of an individual. Current therapeutics for chronic pain are relatively ineffective and produce many adverse side effects including tolerance development and addiction. Therefore, the search for novel chronic pain treatments remains a focus of worldwide research. This thesis studied the changes in the function of voltage-gated sodium (NaV) and calcium (CaV) channels in primary sensory neurons located in the dorsal root ganglion (DRG). These neurons are a gateway for sensory processing of nociception. Whole cell patch clamp electrophysiology was conducted on acutely isolated DRG neurons from male Sprague Dawley rats to characterise the activity of a range of compounds. DRG neurons were discriminated based on isolectin-B4 binding (peptidergic neurons do not stain) and cell size (<25 μm= small). The first project aimed to identify the efficacy of two spider toxins, Hs1a and Pn3a that are selective for voltage-gated sodium channel 1.7 (NaV1.7). In humans, the loss function mutation in the SCN9A gene that codes for NaV1.7 cause insensitivity to pain, while the gain of function mutations result in paroxysmal extreme pain disorder, and primary erythromelalgia. NaV1.7, therefore, plays a vital role in pain. In this project, tetrodotoxin (TTX) was utilised as a positive control to inhibit all NaV channel subtypes except NaV1.5 (not expressed in neurons), 1.8, and 1.9. Results revealed that small peptidergic neurons were the most sensitive to TTX, Hs1a, and Pn3a. Since TTX potently blocks NaV1.7 channels, the greater inhibition of sodium current (INa) in small peptidergic neurons corralates with the high level of TTX mediated inhibition. Also, small non-peptidergic DRG neurons had the slowest decay kinetics potentially due to high expression levels of NaV1.8 channels with slower kinetics, which are known to be insensitive to TTX, Hs1a, and Pn3a. In contrast to Hs1a which is equipotent at NaV1.1 and 1.7 channels, Pn3a is at least 40-fold more selective towards NaV1.7 than other NaV channel subtypes. Pn3a inhibited significantly less INa in large peptidergic DRG neurons, which have low levels of NaV1.7 expression compared to small peptidergic neurons. Overall, these results show promise that Pn3a may be able to selectively inhibit NaV1.7 and prevent the generation of action potentials in the DRG neuron to mitigate pain signals from travelling to higher brain regions. μ-Opioid and opioid receptor like receptor 1 (ORL-1) are implicated in pain pathways. However, opioids that mediate their response through μ-opioid receptors are relatively ineffective in chronic pain treatments leading to tolerance development and dependence. Moreover, non-human primates had an analgesic response to intracisternal N/OFQ opening the possibility that ORL-1 receptor-mediated analgesia may be attained in humans despite conflicting outcomes in rodents. Also, both ORL-1 and μ-opioid receptors are located near each other in many neurons throughout the nervous system. Therefore, targeting both μ- and ORL-1 receptors simultaneously has emerged as a novel strategy to treat chronic pain. This synergistic treatment may limit the number of side effects due to reduced activation of a single receptor type. Therefore, a smaller therapeutic dose would be required, and as a result, there would be a decreased probability of tolerance development. In DRG neurons, G-protein coupled receptors linked to ORL-1, and μ-opioid receptors regulate CaV channels. As Ca2+ influx is vital for the regulation of excitability in DRG neurons, a reduction in Ca2+ influx would help decrease the hyperexcitability of sensory neurons and attenuate neurotransmitter release in chronic pain states. Therefore, my second project evaluated the activity of mixed ORL-1 and μ-opioid receptor agonists cebranopadol and [Dmt1]N/OFQ(1-13)-NH2 in DRG neurons of Sprague-Dawley rats that had undergone sciatic nerve ligation to induce chronic pain. Cebranopadol has been tested in multiple models of chronic pain and shown to have analgesic efficacy exceeding opioids with a dose-dependent increase in analgesia, particularly in neuropathic pain models. Previous studies found that [Dmt1]N/OFQ(1-13)-NH2 was similar in potency to N/OFQ in vitro, while in vivo it was approximately 30-fold more potent than N/OFQ and produced longer lasting analgesia. Given the analgesic potential of these compounds, it was of value to evaluate their activity in DRG neurons. Results revealed that both cebranopadol and [Dmt1]N/OFQ(1-13)-NH2 did not show a major difference in the inhibition of Ca2+ current (ICa) between sham surgery and nerve ligated rats. However, reversal of [Dmt1]N/OFQ(1-13)-NH2 mediated inhibition was more substantial when μ- and ORL-1 receptor antagonists were co-applied in comparison to a single antagonist. Thus, it was evident that [Dmt1]N/OFQ(1-13)-NH2 mediated ICa inhibition in DRG neurons was a result of mixed μ- and ORL-1 receptor activity. Results further revealed that the lack of difference between ICa inhibition of sham and nerve-injured rats might be a result of increased ORL-1 receptor-mediated constitutive inhibition of ICa in nerve-injured rats which does not alter based on the time after neuronal isolation. Despite the promising in vitro results of Pn3a, in other studies, it did not show strong analgesia in vivo. Intrathecal Pn3a did not reverse mechanical or thermal allodynia (painful response to a stimulus that is not deemed noxious) in complete Freund's adjuvant treated rats. However, other studies also indicated that NaV1.7 inhibition upregulates endogenous opioids. Therefore, it was of interest to test whether there would be any synergistic effect when an opioid was co-applied with Pn3a in DRG neurons. Results revealed that Pn3a co-applied with μ-opioid receptor agonist [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO) produced a greater ICa inhibition than DAMGO alone. However, Pn3a also inhibited ICa independent of opioid receptors. Therefore, the increased ICa inhibition when DAMGO and Pn3a were co-applied must be a result of direct ICa inhibition by Pn3a coupled with G-protein mediated ICa inhibition by DAMGO. Further study in the presence of G-protein inhibitors is required to clarify if Pn3a mediates its ICa inhibition through a G-protein pathway. Overall, these results indicate that the in vivo synergistic analgesia observed in the presence of Pn3a and opioids does not occur within DRG neurons. Nevertheless, the direct inhibition of ICa mediated by Pn3a in DRG neurons may be analgesic as decreased Ca2+ influx could downregulate neurotransmitter release in ascending pain pathways. In general, results of this thesis suggest that future analgesic development needs to focus on combination therapies that show potential to produce more significant pain relief in chronic pain states with fewer side effects and tolerance development.
See less
See moreChronic pain is a debilitating condition that affects both the emotional and physical well-being of an individual. Current therapeutics for chronic pain are relatively ineffective and produce many adverse side effects including tolerance development and addiction. Therefore, the search for novel chronic pain treatments remains a focus of worldwide research. This thesis studied the changes in the function of voltage-gated sodium (NaV) and calcium (CaV) channels in primary sensory neurons located in the dorsal root ganglion (DRG). These neurons are a gateway for sensory processing of nociception. Whole cell patch clamp electrophysiology was conducted on acutely isolated DRG neurons from male Sprague Dawley rats to characterise the activity of a range of compounds. DRG neurons were discriminated based on isolectin-B4 binding (peptidergic neurons do not stain) and cell size (<25 μm= small). The first project aimed to identify the efficacy of two spider toxins, Hs1a and Pn3a that are selective for voltage-gated sodium channel 1.7 (NaV1.7). In humans, the loss function mutation in the SCN9A gene that codes for NaV1.7 cause insensitivity to pain, while the gain of function mutations result in paroxysmal extreme pain disorder, and primary erythromelalgia. NaV1.7, therefore, plays a vital role in pain. In this project, tetrodotoxin (TTX) was utilised as a positive control to inhibit all NaV channel subtypes except NaV1.5 (not expressed in neurons), 1.8, and 1.9. Results revealed that small peptidergic neurons were the most sensitive to TTX, Hs1a, and Pn3a. Since TTX potently blocks NaV1.7 channels, the greater inhibition of sodium current (INa) in small peptidergic neurons corralates with the high level of TTX mediated inhibition. Also, small non-peptidergic DRG neurons had the slowest decay kinetics potentially due to high expression levels of NaV1.8 channels with slower kinetics, which are known to be insensitive to TTX, Hs1a, and Pn3a. In contrast to Hs1a which is equipotent at NaV1.1 and 1.7 channels, Pn3a is at least 40-fold more selective towards NaV1.7 than other NaV channel subtypes. Pn3a inhibited significantly less INa in large peptidergic DRG neurons, which have low levels of NaV1.7 expression compared to small peptidergic neurons. Overall, these results show promise that Pn3a may be able to selectively inhibit NaV1.7 and prevent the generation of action potentials in the DRG neuron to mitigate pain signals from travelling to higher brain regions. μ-Opioid and opioid receptor like receptor 1 (ORL-1) are implicated in pain pathways. However, opioids that mediate their response through μ-opioid receptors are relatively ineffective in chronic pain treatments leading to tolerance development and dependence. Moreover, non-human primates had an analgesic response to intracisternal N/OFQ opening the possibility that ORL-1 receptor-mediated analgesia may be attained in humans despite conflicting outcomes in rodents. Also, both ORL-1 and μ-opioid receptors are located near each other in many neurons throughout the nervous system. Therefore, targeting both μ- and ORL-1 receptors simultaneously has emerged as a novel strategy to treat chronic pain. This synergistic treatment may limit the number of side effects due to reduced activation of a single receptor type. Therefore, a smaller therapeutic dose would be required, and as a result, there would be a decreased probability of tolerance development. In DRG neurons, G-protein coupled receptors linked to ORL-1, and μ-opioid receptors regulate CaV channels. As Ca2+ influx is vital for the regulation of excitability in DRG neurons, a reduction in Ca2+ influx would help decrease the hyperexcitability of sensory neurons and attenuate neurotransmitter release in chronic pain states. Therefore, my second project evaluated the activity of mixed ORL-1 and μ-opioid receptor agonists cebranopadol and [Dmt1]N/OFQ(1-13)-NH2 in DRG neurons of Sprague-Dawley rats that had undergone sciatic nerve ligation to induce chronic pain. Cebranopadol has been tested in multiple models of chronic pain and shown to have analgesic efficacy exceeding opioids with a dose-dependent increase in analgesia, particularly in neuropathic pain models. Previous studies found that [Dmt1]N/OFQ(1-13)-NH2 was similar in potency to N/OFQ in vitro, while in vivo it was approximately 30-fold more potent than N/OFQ and produced longer lasting analgesia. Given the analgesic potential of these compounds, it was of value to evaluate their activity in DRG neurons. Results revealed that both cebranopadol and [Dmt1]N/OFQ(1-13)-NH2 did not show a major difference in the inhibition of Ca2+ current (ICa) between sham surgery and nerve ligated rats. However, reversal of [Dmt1]N/OFQ(1-13)-NH2 mediated inhibition was more substantial when μ- and ORL-1 receptor antagonists were co-applied in comparison to a single antagonist. Thus, it was evident that [Dmt1]N/OFQ(1-13)-NH2 mediated ICa inhibition in DRG neurons was a result of mixed μ- and ORL-1 receptor activity. Results further revealed that the lack of difference between ICa inhibition of sham and nerve-injured rats might be a result of increased ORL-1 receptor-mediated constitutive inhibition of ICa in nerve-injured rats which does not alter based on the time after neuronal isolation. Despite the promising in vitro results of Pn3a, in other studies, it did not show strong analgesia in vivo. Intrathecal Pn3a did not reverse mechanical or thermal allodynia (painful response to a stimulus that is not deemed noxious) in complete Freund's adjuvant treated rats. However, other studies also indicated that NaV1.7 inhibition upregulates endogenous opioids. Therefore, it was of interest to test whether there would be any synergistic effect when an opioid was co-applied with Pn3a in DRG neurons. Results revealed that Pn3a co-applied with μ-opioid receptor agonist [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO) produced a greater ICa inhibition than DAMGO alone. However, Pn3a also inhibited ICa independent of opioid receptors. Therefore, the increased ICa inhibition when DAMGO and Pn3a were co-applied must be a result of direct ICa inhibition by Pn3a coupled with G-protein mediated ICa inhibition by DAMGO. Further study in the presence of G-protein inhibitors is required to clarify if Pn3a mediates its ICa inhibition through a G-protein pathway. Overall, these results indicate that the in vivo synergistic analgesia observed in the presence of Pn3a and opioids does not occur within DRG neurons. Nevertheless, the direct inhibition of ICa mediated by Pn3a in DRG neurons may be analgesic as decreased Ca2+ influx could downregulate neurotransmitter release in ascending pain pathways. In general, results of this thesis suggest that future analgesic development needs to focus on combination therapies that show potential to produce more significant pain relief in chronic pain states with fewer side effects and tolerance development.
See less
Date
2018-02-28Licence
The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.Faculty/School
Faculty of Medicine and Health, Sydney Pharmacy SchoolAwarding institution
The University of SydneyShare