Neuroimmunopharmacology of opioids

Opioids are an extremely important part of medical practice, and for thousands of years, continue to provide relief from severe acute and chronic pain. Intriguingly, however, the use of opioids activates endogenous counter-regulatory mechanisms resulting in the release of proinflammatory mediators from central immune cells that facilitates a wide-range of effects on opioid pharmacodynamics including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid- induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. Until recently, the counter regulation of opioid-induced analgesia had been had been attributed to neuronal receptors where the beneficial and detrimental actions of opioids were thought to be inseparable. It is now apparent from molecular and rodent data that opioids have non-neuronal, non-classic, non-stereoselective sites of action. Therefore, the purpose of this thesis was to further identify this non-classical, non-stereoselective site of action and examine the kinetics of opioid binding. The first study examined, using in vivo, in vitro, and in silico techniques, the potential involvement of toll-like receptor (TLR) 2 and TLR4 in the pharmacodynamic actions of opioids. TLR4-/- animals demonstrated significantly altered analgesia, tolerance, withdrawal responses to opioids compared with wildtype mice. A range of saturation, displacement, and kinetic binding experiments were subsequently conducted to assess the non-stereoselective binding of opioids to TLR4. However, at concentrations required for classical μ receptor binding, no TLR4 binding of classical opioid antagonists was observed. The second study assessed if many of the key drugs that target opioid induced immune activation have any direct action at opioid receptors or whether they can modify opioid receptor activation. Classical binding and functional studies were conducted in cells overexpressing the human μ receptor and in rat brain membranes. Of all the tested ligands, only amitriptyline and WZ811 (CXCR4 antagonist) were shown to have direct binding at sites defined by the classical antagonists diprenorphine and (-)-naloxone. All of the tested ligands had no detectable efficacy for the m receptor or any allosteric modulatory effects. This study confirms that ligands that alter in vivo opioid pharmacodynamic responses (increase opioid analgesia and reduction of tolerance) do not do so via modifying μ-opioid receptor activity. The final study employed the use of [3H](+)-naloxone, a stereoisomer of (-)-naloxone that has been shown to have extensive effects on classical opioid pharmacodynamics. Mouse brain membranes and HEK293 cells overexpression systems were used in order to unmask any non-stereoselective (+)-opioid isomer binding sites. However, using traditional [3H] binding experiments, (+)-naloxone had no detectable binding at sites at low nM concentrations. Due to the lipophilicy of (+)-naloxone and the subsequent high level of nonspecific binding, no specific binding was detectable at μM concentrations. In sum, this thesis provides additional evidence that TLR4 plays a significant role in the pharmacodynamic responses to opioids. The kinetic assessment of TLR4 as a nonstereoselective opioid site of action reveals that this site does not bind opioids at nM concentrations. As such, this does not rule out the direct involvement of TLR4 in opioid binding, but rather suggest that TLR4 has disparate binding characteristics compared with classical μ-opioid receptor binding. Further assessment of this complex system is required, particularly at higher concentrations. However, the present finding can assist with the future discovery of a non-classical opioid binding site that is involved in opioid induced central inflammatory events.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, Adelaide Medical School, 2017