Animal Studies

The goal of this research is the identification of new treatments for neuropathic pain. We characterized the GABAergic system of immortalized mouse and human microglia using electrophysiology and qRT-PCR. Cells from both species exhibited membrane current changes in response to γ-aminobutyric acid, with an EC50 of 260 nM and 1940 nM, respectively. Human microglia expressed high levels of the γ-aminobutyric acid type A receptor (GABAAR) α3 subunit, which can assemble with β1 and γ2/δ subunits to form functional GABAARs. Mouse microglia contained α2, α3 and α5, in addition to β1-3, γ1-2 and δ, mRNA, enabling a more diverse array of GABAARs than human microglia. Benzodiazepines are well-established modulators of GABAAR activity, prompting a screen of a library of diverse benzodiazepines in microglia for cellular effects. Several active compounds were identified by reduction of nitric oxide (NO) in interferon gamma and lipopolysaccharide activated microglia. However, further investigation with GABAAR antagonists flumazenil, picrotoxin, and bicuculline demonstrated that GABAARs were not linked to the NO response. A screen of 48 receptors identified the κ-opioid receptor and to a lesser extent the µ-opioid receptor as molecular targets, with opioid receptor antagonist norbinaltorphimine reversing benzodiazepine induced reduction of microglial NO. Functional assays identified the downregulation of inducible NO synthase as the mode of action of imidazodiazepines MP-IV-010 and GL-IV-03. Like other κ-opioid receptor agonists, GL-IV-03 reduced the agitation response in both phases of the formalin nociception test. However, unlike other κ-opioid receptor agonists, MP-IV-010 and GL-IV-03 did not impair sensorimotor coordination in mice. Thus, MP-IV-010 and GL-IV-03 represent a new class of non-sedative drug candidates for inflammatory pain.

The role of voltage-gated sodium (Na) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms Na1.8 and Na1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat Na1.8 and mouse Na1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant Na isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human Na1.8 and Na1.9, and lacked analgesic efficacy in a murine model of inflammatory pain.

Management of chronic pain presents a major challenge, since many currently available treatments lack efficacy and have problems such as addiction and tolerance. Loss of function mutations in the SCN9A gene lead to a congenital inability to feel pain, with no other sensory deficits aside from anosmia. SCN9A encodes the voltage-gated sodium (Na) channel 1.7 (Na1.7), which has been identified as a primary pain target. However, in developing Na1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other Na subtypes that are critical for survival. Since spider venoms are an excellent source of Na channel modulators, we screened a panel of spider venoms to identify selective Na1.7 inhibitors. This led to identification of two novel Na modulating venom peptides (β/μ-theraphotoxin-Pe1a and β/μ-theraphotoxin-Pe1b (Pe1b) from the arboreal tarantula Phormingochilus everetti. A third peptide isolated from the tarantula Bumba pulcherrimaklaasi was identical to the well-known ProTx-I (β/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the Na1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and Na channel subtype selectivity. Several analogues had improved potency against Na1.7, and altered specificity against other Na channels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of Na1.7.

Recent studies have shown that the endogenous opioid system is considerably affected by early life stress such as child abuse. Here, we investigated whether early life stress changes the endogenous opioid receptors and their peptides, and if such stress impacts morphine antinociception. We used mice affected by maternal separation and social isolation (MSSI) as an early life stress model. In the tail-flick test, 10-week-old MSSI mice showed a significant decrease in morphine antinociception compared to age-matched control mice. The number of c-Fos-positive cells increased in the periaqueductal gray (PAG), nucleus accumbens, and thalamus of control mice after the morphine injections, whereas hardly any positive cells were detected in the same areas of MSSI mice. The expression of μ- and κ-opioid receptor (MOR and KOR, respectively) messenger RNA (mRNA) was significantly decreased in the PAG of MSSI mice, whereas KOR expression was significantly increased in the amygdala of MSSI mice. The expression of δ-opioid receptor (DOR) mRNA was significantly reduced in the PAG and rostral ventromedial medulla of MSSI mice compared to control mice. Moreover, the lack of morphine antinociception was observed in 18-week-old MSSI mice. Our findings suggest that the supraspinal opioid system may be affected by early life stress exposure, and that this exposure may impact morphine antinociception.

Mechano growth factor (MGF) is an alternatively spliced form of insulin-like growth factor-1 (IGF-1) that has shown to be neuroprotective against 6-hydroxydopamine toxicity and ischemic injury in the brain. MGF also induces neural stem cell proliferation in the hippocampus and preserves olfactory function in aging mice. Cisplatin is a chemotherapy drug that induces peripheral neuropathy in 30-40% of treated patients. Our studies were designed to see if MGF would protect dorsal root ganglion (DRG) neurons from cisplatin-induced neurotoxicity and to identify potential mechanisms that may be involved. Expression of endogenous MGF in adult DRG neurons in vivo ameliorated cisplatin-induced thermal hyperalgesia. Exogenous MGF and MGF with a cysteine added to the N-terminus (CMGF) also protected embryonic DRG neurons from cisplatin-induced cell death in vitro. Mass spectroscopy analysis of proteins bound to MGF showed that nucleolin is a key-binding partner. Antibodies against nucleolin prevented the neuroprotective effect of MGF and CMGF in culture. Both nucleolin and MGF are located in the nucleolus of DRG neurons. RNAseq of RNA associated with MGF indicated that MGF may be involved in RNA processing, protein targeting and transcription/translation. Nucleolin is an RNA binding protein that is readily shuttled between the nucleus, cytoplasm and plasma membrane. Nucleolin and MGF may work together to prevent cisplatin-induced neurotoxicity. Exploring the known mechanisms of nucleolin may help us better understand the mechanisms of cisplatin toxicity and how MGF protects DRG neurons.

NSAIDs are the drugs most commonly used to alleviate pain. Despite being a heterogeneous group of compounds, all of them share a mechanism of action based on blockade of COXs enzymes, which confers them anti-inflammatory and analgesic properties. Diclofenac is a NSAID with preferred activity on COX-2 isozymes, but additionally, other targets may be implicated in its analgesic activity. Among them, diclofenac may facilitate the activity of K7 channels, that have been previously recognized as potential therapeutic targets in analgesia. In this study, the antinociceptive actions of diclofenac acting at the spinal level and the role of K7 channels in its effects were evaluated. Electrophysiological recordings of spinal reflexes and responses of dorsal horn neurons were obtained using in vitro spinal cord preparations from neonatal mice. Diclofenac, applied at clinically relevant concentrations to the entire preparation, depressed wind-up of spinal reflexes with a pattern similar to that of flupirtine, an analgesic with activity as K7 channel opener. Depressant actions of both compounds were strongly reduced after K7 channel blockade with XE-991, indicating the implication of these channels in the observed effects. Flupirtine, but not diclofenac, also reduced action potential firing of dorsal horn neurons in response to electrical activation of nociceptive afferents, suggesting differences in the actions of both compounds on K7 channel configurations present in sensory areas of the cord. Results demonstrate previously unknown central actions of diclofenac on K7 channels located in spinal circuits, expanding the knowledge about its pharmacological actions.