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In the present study, we examined whether LDK1258, which produces strong CB receptor allosteric effects in in vitro assays, would elicit in vivo effects consistent with allosteric activity. In initial studies, LDK1258 reduced food consumption and elicited delayed antinociceptive effects in the chronic constrictive injury of the sciatic nerve (CCI) model of neuropathic pain, which unexpectedly emerged 4 h post-injection. UPLC-MS/MS analysis quantified significant levels of LDK1258 in both blood and brain tissue at 30 min post-administration that remained stable up to 4 h. The observation that LDK1258 also produced respective antinociceptive and anorectic effects in rimonabant-treated wild type mice and CB (-/-) mice suggests an off-target mechanism of action. Likewise, LDK1258 produced a partial array of common cannabimimetic effects in the tetrad assay, which were not CB receptor mediated. Additionally, LDK1258 did not substitute for the CB receptor orthosteric agonists CP55,940 or anandamide in the drug discrimination paradigm. In other in vivo assays sensitive to CB receptor allosteric modulators, LDK1258 failed to shift the dose-response curves of either CP55,940 or anandamide in producing thermal antinociception, catalepsy, or hypothermia, and did not alter the generalization curve of either drug in the drug discrimination assay. Thus, this battery of tests yielded results demonstrating that LDK1258 produces antinociceptive effects in the CCI model of neuropathic pain, anorectic effects, and other in vivo pharmacological effects in a manner inconsistent with CB receptor allosterism. More generally, this study offers a straightforward screening assay to determine whether newly synthesized CB receptor allosteric modulators translate to the whole animal.
Learn More >The anterior cingulate cortex (ACC) is a critical region of the brain for the emotional and affective components of pain in rodents and humans. Hyperactivity in this region has been observed in neuropathic pain states in both patients and animal models and ablation of this region from cingulotomy, or inhibition with genetics or pharmacology can diminish pain and anxiety. Two adenylyl cyclases (AC), AC1 and AC8 play an important role in regulating nociception and anxiety-like behaviors through an action in the ACC, as genetic and pharmacological targeting of these enzymes reduces mechanical hypersensitivity and anxiety-like behavior, respectively. However, the distribution of these ACs in the ACC has not been studied in the context of neuropathic pain. To address this gap in knowledge, we conducted RNAscope hybridization to assess AC1 and AC8 mRNA distribution in mice with spared nerve injury (SNI). Given the key role of AC1 in nociception in neuropathic, inflammatory and visceral pain animal models, we hypothesized that AC1 would be upregulated in the ACC of mice following nerve injury. This hypothesis was also founded on data showing increased AC1 expression in the ACC of mice with zymosan-induced visceral inflammation. We found that AC1 and AC8 are widely expressed in many regions of the mouse brain including the hippocampus, ACC, medial prefrontal cortex and midbrain regions, but AC1 is more highly expressed. Contrary to our hypothesis, SNI causes an increase in AC8 mRNA expression in NMDAR-2B (Nr2b) positive neurons in the contralateral ACC but does not affect AC1 mRNA expression. Our findings show that changes in mRNA expression in the ACC are insufficient to explain the important role of this AC in mechanical hypersensitivity in mice following nerve injury and suggest a potential unappreciated role of AC8 in regulation of ACC synaptic changes after nerve injury.
Learn More >Tetrodotoxin (TTX) is a highly specific voltage-gated sodium channel (VGSC) blocker in clinical evaluation as a peripheral-acting analgesic for chronic pain. This study presents the first published results of the safety including cardiac liability of TTX at therapeutic-relevant concentrations in twenty-five healthy adults. Randomized, double-blind, placebo-, and positive- (moxifloxacin) controlled study evaluated single ascending doses of 15 µg, 30 µg, and 45 µg TTX over 3 periods with a 7-day washout between each period. Subcutaneous injections of TTX were readily absorbed, reaching maximum plasma concentration (C) within 1.5 h. Both extent of exposure (AUC) and C increased in proportion to dose. No QT prolongation was identified by concentration-QTc analysis and the upper bounds of the two-sided 90% confidence interval of predicted maximum baseline and placebo corrected QTcF (ΔΔQTcF) value did not exceed 10 ms for all tetrodotoxin doses, thereby meeting the criteria of a negative QT study. Safety assessments showed no clinically relevant changes with values similar between all groups and no subject withdrawing due to adverse events. Paresthesia, oral-paresthesia, headache, dizziness, nausea, and myalgia were the most common TEAEs (overall occurrence ≥5%) in the TTX treatment groups. TTX doses investigated in this study are safe, well-tolerated, and lack proarrhythmic proclivity.
Learn More >This report examines the association between tetrahydrocannabinol (THC) plasma levels and pain response in a secondary analysis of data from a recent diabetic neuropathy study that demonstrated a dose-dependent reduction in spontaneous and elicited pain at specific time points. A randomized, double-blinded, placebo-controlled crossover study was conducted in sixteen patients with painful diabetic peripheral neuropathy. Subjects participated in four sessions, separated by 2 weeks, during each of which they were exposed to one of four conditions: placebo, or 1%, 4%, or 7% THC dose of cannabis. Baseline assessments of spontaneous and evoked pain were performed. Subjects were then administered aerosolized cannabis or placebo and pain intensity and cognitive testing at specific time points for 4 hours. A blood sample was drawn from the left antecubital vein for plasma assay of total THC at 0, 15, 30, 45, 60, 150, and 240 minutes. Associations were made between pain intensity, cognitive impairment and THC plasma levels in this secondary analysis. Results suggested a U-shaped relation whereby pain ratings are greatest at extreme (low and high) levels of THC. The therapeutic window appeared to fall between 16 ng/mL and 31 ng/mL THC plasma level. There was a significant linear effect of THC on only one out of the three cognitive tests. These findings stress the importance of measuring cannabinoid plasma levels when performing future research. Perspective: This analysis correlating plasma THC levels and pain reduction in diabetic neuropathy suggest a therapeutic window. Low and high THC levels had a negative association (no reduction) and THC levels within the window had a positive association (reduction). There was a minor negative linear effect of THC on cognitive function.
Learn More >Naltrexone reversibly blocks the effects of opioids and has been shown to decrease placebo analgesia. However, it is not clear (1) to what extent naltrexone affects pain modulation in a nontreatment context, for example, in response to pain cues or (2) how naltrexone given prior to pain-cue learning shapes pain responses. In a double-blind procedure prior to pain-cue conditioning, 30 healthy participants were randomized to receive an oral dose of naltrexone (50 mg) or inert pill. During functional magnetic resonance imaging, high and low pain pressures were paired with two different visual cues: a high pain cue and a low pain cue (learning sequence). During a test sequence, medium levels of pressure were used for both cues and the difference in subjective pain ratings following high and low pain cues was calculated. Results showed significant conditioned pain responses across groups (<.001); however, no significant difference between participants receiving naltrexone or inert pill (=.193). There was a significant correlation between the difference in high and low pain ratings during the learning sequence and the effect of high and low pain cues during the test sequence (r = .575, =.002). Functional magnetic resonance imaging analyses revealed no significant difference in brain activation between groups. Here, we demonstrate comparable learning of pain responses in participants treated with naltrexone or inert pill. The results point to the possibility that associative learning, and conditional responding to pain cues, is not dependent on endogenous opioids. Our results, using pain-cue conditioning to create reduced pain responses, contrast previous studies where opioid antagonists significantly reduced the placebo effect in treatment of pain.
Learn More >Memantine (MEM) is one of the important clinical medications in treating moderate to severe Alzheimer disease. The effect of MEM on preventing or treating punctate allodynia has been thoroughly studied but not on the induction of dynamic allodynia. The aim of this study is to investigate whether MEM could prevent the induction of dynamic allodynia and its underlying spinal mechanisms. 1)in vivo SNI pain model, pretreatment with MEM at a lower dose (10nmol, i.t.; MEM-10) selectively prevented the induction of dynamic allodynia, but not the punctate allodynia. 2) Pretreatment with either MK801-10 (MK801-10nmol, i.t.) or higher dose of MEM (30nmol, i.t.; MEM-30) prevented the induction of both dynamic and punctate allodynia. 3) MEM-10 showed significant effect on the inhibition of the SNI induced overactivation of microglia in spinal dorsal horn. 4) In contrast, in CFA model, MEM-10 neither affected the CFA injection induced activation of microglia in spinal dorsal horn nor the induction of dynamic allodynia. 5) Immunohistology studies showed Kir2.1 channel distributed widely and co-localized with microglia in the spinal dorsal horn of mice. 6) Pretreatment with either minocycline, a microglia inhibitor, or ML133, a Kir2.1 inhibitor, both selectively prevented the overactivation of microglia in spinal dorsal horn, and the induction of dynamic allodynia following SNI. The selectively inhibitory effect on the induction of dynamic allodynia in SNI model by low dose of the memantine (MEM-10) was tightly correlated with the blockade of microglia Kir2.1 channel to suppress the microglia activation.
Learn More >Vincristine, oxaliplatin, and cisplatin are commonly prescribed chemotherapeutic agents for the treatment of many tumors. However, a main side-effect is chemotherapy-induced peripheral neuropathy (CIPN), which may lead to changes in chemotherapeutic treatment. Although symptoms associated with CIPN are recapitulated by mouse models, there is limited knowledge of how these drugs affect the expression of genes in sensory neurons. The present study carried out a transcriptomic analysis of dorsal root ganglia (DRG) following vincristine, oxaliplatin, and cisplatin treatment with a view to gain insight into the comparative pathophysiological mechanisms of CIPN. RNA-Seq revealed 368, 295 and 256 differential expressed genes (DEGs) induced by treatment with vincristine, oxaliplatin and cisplatin, respectively and only five shared genes were dysregulated in all three groups. Cell type enrichment analysis and gene set enrichment analysis showed predominant effects on genes associated with the immune system after treatment with vincristine, while oxaliplatin treatment affected mainly neuronal genes. Treatment with cisplatin resulted in a mixed gene expression signature. Perspective: These results provide insight into the recruitment of immune responses to DRG and indicate enhanced neuro-inflammatory processes following administration of vincristine, oxaliplatin, and cisplatin. These gene expression signatures may provide insight into novel drug targets for treatment of CIPN.
Learn More >Chemotherapy-induced painful peripheral neuropathy (CIPN) is a significant clinical problem that is associated with widely used chemotherapeutics. Unfortunately, the molecular mechanisms by which CIPN develops has remained elusive. The proteasome inhibitor, bortezomib, has been shown to induce aerobic glycolysis in sensory neurons. This altered metabolic phenotype leads to the extrusion of metabolites which sensitize primary afferents and cause pain. Hypoxia-inducible factor alpha (HIF1A) is a transcription factor that is known to reprogram cellular metabolism. Furthermore, HIF1A protein is constantly synthesized and undergoes proteasomal degradation in normal conditions. However, metabolic stress or hypoxia stabilize the expression of HIF1A leading to the transcription of genes that reprogram cellular metabolism. This study demonstrates that treatment of mice with bortezomib stabilize the expression of HIF1A. Moreover, knockdown of HIF1A, inhibition of HIF1A binding to its response element or limiting its translation by using metformin prevent the development of bortezomib-induced neuropathic pain. Strikingly, the blockade of HIF1A expression does not attenuate mechanical allodynia in mice with existing bortezomib-induced neuropathic pain. These results establish the stabilization of HIF1A expression as the molecular mechanism by which bortezomib initiates CIPN. Crucially these findings reveal that the initiation and maintenance of bortezomib-induced neuropathic pain are regulated by distinct mechanisms.
Learn More >To investigate the region-specific effects of painful trigeminal capsaicin stimulation in healthy participants.
Learn More >Management of bone cancer pain (BCP) is difficult because of its complex mechanisms, which has a major impact on the quality of patients' daily life. Recent studies have indicated that endoplasmic reticulum (ER) stress is involved in many neurological and inflammatory pathways associated with pain. However, the factors that contribute to ER stress and its causes in bone cancer pain are still unknown. In this study, we examined whether the ER stress response is involved in caspase signaling pathway-dependent apoptosis in neurons in the spinal dorsal horn of tumor-38 bearing rats and whether it thereby induces bone cancer pain.
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