Modulating the activity of human nociceptors with a SCN10A promoter-specific viral vector tool.

Despite the high prevalence of chronic pain as a disease in our society, there is a lack of effective treatment options for patients living with this condition. Gene therapies using recombinant AAVs are a direct method to selectively express genes of interest in target cells with the potential of, in the case of nociceptors, reducing neuronal firing in pain conditions. We designed a recombinant AAV vector expressing cargos whose expression was driven by a portion of the SCN10A (NaV1.8) promoter, which is predominantly active in nociceptors. We validated its specificity for nociceptors in mouse and human dorsal root ganglia and showed that it can drive the expression of functional proteins. Our viral vector and promoter package drove the expression of both excitatory or inhibitory DREADDs in primary human DRG cultures and in whole cell electrophysiology experiments, increased or decreased neuronal firing, respectively. Taken together, we present a novel viral tool that drives expression of cargo specifically in human nociceptors. This will allow for future specific studies of human nociceptor properties as well as pave the way for potential future gene therapies for chronic pain.

Investigation of the Mechanisms of Tramadol-Induced Seizures in Overdose in the Rat.

Tramadol overdose is frequently associated with the onset of seizures, usually considered as serotonin syndrome manifestations. Recently, the serotoninergic mechanism of tramadol-attributed seizures has been questioned. This study's aim was to identify the mechanisms involved in tramadol-induced seizures in overdose in rats. The investigations included (1) the effects of specific pretreatments on tramadol-induced seizure onset and brain monoamine concentrations, (2) the interaction between tramadol and γ-aminobutyric acid (GABA)A receptors in vivo in the brain using positron emission tomography (PET) imaging and 11C-flumazenil. Diazepam abolished tramadol-induced seizures, in contrast to naloxone, cyproheptadine and fexofenadine pretreatments. Despite seizure abolishment, diazepam significantly enhanced tramadol-induced increase in the brain serotonin (p < 0.01), histamine (p < 0.01), dopamine (p < 0.05) and norepinephrine (p < 0.05). No displacement of 11C-flumazenil brain kinetics was observed following tramadol administration in contrast to diazepam, suggesting that the observed interaction was not related to a competitive mechanism between tramadol and flumazenil at the benzodiazepine-binding site. Our findings do not support the involvement of serotoninergic, histaminergic, dopaminergic, norepinephrine or opioidergic pathways in tramadol-induced seizures in overdose, but they strongly suggest a tramadol-induced allosteric change of the benzodiazepine-binding site of GABAA receptors. Management of tramadol-poisoned patients should take into account that tramadol-induced seizures are mainly related to a GABAergic pathway.

Is naloxone the best antidote to reverse tramadol-induced neuro-respiratory toxicity in overdose? An experimental investigation in the rat.

CONTEXT: Since the banning of dextropropoxyphene from the market, overdoses, and fatalities attributed to tramadol, a WHO step-2 opioid analgesic, have increased markedly. Tramadol overdose results not only in central nervous system (CNS) depression attributed to its opioid properties but also in seizures, possibly related to non-opioidergic pathways, thus questioning the efficiency of naloxone to reverse tramadol-induced CNS toxicity. OBJECTIVE: To investigate the most efficient antidote to reverse tramadol-induced seizures and respiratory depression in overdose. MATERIALS AND METHODS: Sprague-Dawley rats overdosed with 75 mg/kg intraperitoneal (IP) tramadol were randomized into four groups to receive solvent (control group), diazepam (1.77 mg/kg IP), naloxone (2 mg/kg intravenous bolus followed by 4 mg/kg/h infusion), and diazepam/naloxone combination. Sedation depth, temperature, number of seizures, and intensity, whole-body plethysmography parameters and electroencephalography activity were measured. RESULTS: Naloxone reversed tramadol-induced respiratory depression (p < .05) but significantly increased seizures (p < .01) and prolonged their occurrence time. Diazepam abolished seizures but significantly deepened rat sedation (p < .05) without improving ventilation. Diazepam/naloxone combination completely abolished seizures, significantly improved rat ventilation by reducing inspiratory time (p < .05) but did not worsen sedation. None of these treatments significantly modified rat temperature. CONCLUSIONS: Diazepam/naloxone combination is the most efficient antidote to reverse tramadol-induced CNS toxicity in the rat.

Bifunctional peptide-based opioid agonist/nociceptin antagonist ligand for dual treatment of nociceptive and neuropathic pain.

Drugs able to treat both nociceptive and neuropathic pain effectively without major side effects are lacking. We developed a bifunctional peptide-based hybrid (KGNOP1) that structurally combines a mu-opioid receptor agonist (KGOP1) with antinociceptive activity and a weak nociceptin receptor antagonist (KGNOP3) with anti-neuropathic pain activity. We investigated KGNOP1-related behavioral effects after intravenous administration in rats by assessing thermal nociception, cold hyperalgesia in a model of neuropathic pain induced by chronic constriction injury of the sciatic nerve, and plethysmography parameters including inspiratory time (TI) and minute ventilation (VM) in comparison to the well-known opioid analgesics, tramadol and morphine. Time-course and dose-dependent effects were investigated for all behavioral parameters to determine the effective doses 50% (ED50). Pain-related effects on cold hyperalgesia were markedly increased by KGNOP1 as compared to KGNOP3 and tramadol (ED50: 0.0004, 0.32, and 12.1 µmol/kg, respectively), whereas effects on thermal nociception were significantly higher with KGNOP1 as compared to morphine (ED50: 0.41 and 14.7 µmol/kg, respectively). KGNOP1 and KGOP1 produced a larger increase in TI and deleterious decrease in VM in comparison to morphine and tramadol (ED50(TI): 0.63, 0.52, 12.2, and 50.9 µmol/kg; ED50(VM): 0.57, 0.66, 10.6, and 50.0 µmol/kg, respectively). Interestingly, the calculated ratios of anti-neuropathic pain/antinociceptive to respiratory effects revealed that KGNOP1 was safer than tramadol (ED50 ratio: 5.44 × 10 vs 0.24) and morphine (ED50 ratio: 0.72 vs 1.39). We conclude that KGNOP1 is able to treat both experimental neuropathic and nociceptive pain, more efficiently and safely than tramadol and morphine, respectively, and thus should be a candidate for future clinical developments.

Mechanisms of tramadol-related neurotoxicity in the rat: Does diazepam/tramadol combination play a worsening role in overdose?

Poisoning with opioid analgesics including tramadol represents a challenge. Tramadol may induce respiratory depression, seizures and serotonin syndrome, possibly worsened when in combination to benzodiazepines. Our objectives were to investigate tramadol-related neurotoxicity, consequences of diazepam/tramadol combination, and mechanisms of drug-drug interactions in rats. Median lethal-doses were determined using Dixon-Bruce's up-and-down method. Sedation, seizures, electroencephalography and plethysmography parameters were studied. Concentrations of tramadol and its metabolites were measured using liquid-chromatography-high-resolution-mass-spectrometry. Plasma, platelet and brain monoamines were measured using liquid-chromatography coupled to fluorimetry. Median lethal-doses of tramadol and diazepam/tramadol combination did not significantly differ, although time-to-death was longer with combination (P=0.04). Tramadol induced dose-dependent sedation (P<0.05), early-onset seizures (P<0.001) and increase in inspiratory (P<0.01) and expiratory times (P<0.05). The diazepam/tramadol combination abolished seizures but significantly enhanced sedation (P<0.01) and respiratory depression (P<0.05) by reducing tidal volume (P<0.05) in addition to tramadol-related increase in respiratory times, suggesting a pharmacodynamic mechanism of interaction. Plasma M1 and M5 metabolites were mildly increased, contributing additionally to tramadol-related respiratory depression. Tramadol-induced early-onset increase in brain concentrations of serotonin and norepinephrine was not significantly altered by the diazepam/tramadol combination. Interestingly neither pretreatment with cyproheptadine (a serotonin-receptor antagonist) nor a benserazide/5-hydroxytryptophane combination (enhancing brain serotonin) reduced tramadol-induced seizures. Our study shows that diazepam/tramadol combination does not worsen tramadol-induced fatality risk but alters its toxicity pattern with enhanced respiratory depression but abolished seizures. Drug-drug interaction is mainly pharmacodynamic but increased plasma M1 and M5 metabolites may also contribute to enhancing respiratory depression. Tramadol-induced seizures are independent of brain serotonin.

Bifunctional Peptide-Based Opioid Agonist-Nociceptin Antagonist Ligands for Dual Treatment of Acute and Neuropathic Pain.

Herein, the opioid pharmacophore H-Dmt-d-Arg-Aba-ß-Ala-NH2 (7) was linked to peptide ligands for the nociceptin receptor. Combination of 7 and NOP ligands (e.g., H-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) led to binding affinities in the low nanomolar domain. In vitro, the hybrids behaved as agonists at the opioid receptors and antagonists at the nociceptin receptor. Intravenous administration of hybrid 13a (H-Dmt-d-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) to mice resulted in potent and long lasting antinociception in the tail-flick test, indicating that 13a was able to permeate the BBB. This was further supported by a cell-based BBB model. All hybrids alleviated allodynia and hyperalgesia in neuropathic pain models. Especially with respect to hyperalgesia, they showed to be more effective than the parent compounds. Hybrid 13a did not result in significant respiratory depression, in contrast to an equipotent analgesic dose of morphine. These hybrids hence represent a promising avenue toward analgesics for the dual treatment of acute and neuropathic pain.