Targeting Nitric Oxide Production in Microglia with Novel Imidazodiazepines for Nonsedative Pain Treatment

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 EC(50) of 260 nM and 1940 nM, respectively. Human microglia expressed high levels of the γ-aminobutyric acid type A receptor (GABA(A)R) α(3) subunit, which can assemble with β(1) and γ(2)/δ subunits to form functional GABA(A)Rs. Mouse microglia contained α(2), α(3) and α(5), in addition to β(1–3), γ(1–2) and δ, mRNA, enabling a more diverse array of GABA(A)Rs than human microglia. Benzodiazepines are well-established modulators of GABA(A)R 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 GABA(A)R antagonists flumazenil, picrotoxin, and bicuculline demonstrated that GABA(A)Rs 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.