Tolerance to Endogenous Opioid Tone Causes Reinstatement of Persistent Hypersensitivity

Abstract

There is a large unmet need for effective analgesic strategies to manage persistent pain. Although opioid analgesics including the prototypical drug, morphine, are currently the most effective analgesic strategy for managing severe-acute pain, their long-term use is compromised by the development of side effects including respiratory depression, constipation, tolerance and a paradoxical enhancement in pain sensitivity (hyperalgesia). In addition, opioids induce euphoria, a property that may lead to addiction, dependence and diversion towards illicit use. Therefore, the search for analgesics that are as effective as morphine at alleviating pain but are devoid of detrimental side effects remains a significant challenge for managing chronic pain.

An emerging paradigm in opioid pharmacology that has dominated the search for superior opioid analgesics is the idea of biased agonism of the µ receptor. The search for G protein biased agonists, which preferentially activate inhibitory G proteins while avoiding the recruitment of β-arrestin2, stems from early observations in β-arrestin2 knockout mice (β-arrestin2-/-) which demonstrated that morphine antinociception is enhanced while the development of respiratory depression, constipation and analgesic tolerance is reduced. This search led to the development of PZM21 and TRV130, reportedly biased µ receptor agonists that stimulate negligible β-arrestin2 recruitment in vitro, while producing antinociception comparable to morphine but fewer side effects in vivo. However, recent evidence casts doubt on the interpretation of earlier studies and challenges evidence for the existence of bias. Instead, biased agonists may be lower efficacy and retain the capacity to promote detrimental effects including tolerance.

My research examines the relationship between µ receptor availability and the appearance of biased agonism for a panel of µ agonists, including TRV130, which was recently approved by the Food and Drug Administration for intravenous use. My findings demonstrate that overexpression of µ receptors gives rise to the presence of spare receptors that enhances the apparent efficacies of µ receptor agonists in assays of G protein activation. By taking measures to deplete receptor availability, by exposing cells to the µ receptor irreversible antagonist, β-funaltrexamine, I demonstrate that reportedly biased agonists exhibit partial efficacy. Furthermore, in the absence of spare receptors, the efficacies of µ receptor agonists to inhibit the accumulation of cAMP (a measure of G protein activation) correlates with their efficacies to recruit β-arrestin2. Therefore, my findings suggest that the presence of spare receptors has led to the misclassification of partial agonists as biased.

Using a tail withdrawal assay I demonstrate that TRV130 causes dose-dependent antinociception in wild type C57BL/6 mice without causing tolerance, while in µ+/- mice, which have negligible spare receptors, TRV130 causes tolerance similar to that seen with morphine in wild type mice. This implies that, even though TRV130 is a µ receptor partial agonist, it has the capacity to cause detrimental side effects. Therefore, my findings cast doubt on the existence of biased agonism and the effectiveness of using this approach to develop analgesics that may be superior for treating chronic pain.

Rather than developing new opioid analgesics, which has been attempted without success, an alternative approach is to improve existing opioid analgesics by developing strategies that enhance the function of innate opioid signalling mechanisms. While endogenous analgesic mechanisms provide powerful antinociception to compensate for the development of acute pain, endogenous opioid tone declines over time, possibly through tolerance, giving rise to persistent pain. Approaches that reverse tolerance to endogenous opioid tone may be an effective strategy to provide sustained antinociception to treat chronic pain. I set out to test this hypothesis using the complete Freund’s adjuvant model of persistent inflammation and a variety of approaches that modulate opioid tolerance. I demonstrate that approaches which enhance opioid tolerance (daily morphine, escalating morphine doses or µ+/- mice) cause reinstatement of persistent mechanical hypersensitivity while approaches that diminish opioid tolerance (β-arrestin2-/- mice, δ-/- mice or c-Src inhibition) prevent reinstatement of mechanical hypersensitivity.

I additionally applied some of these approaches to a model of early life adversity (ELA) evoked by a limited bedding paradigm in mice, which prior observations from the lab demonstrate causes enhanced tolerance. I demonstrate that ELA enhances endogenous opioid tone and causes reinstatement of persistent mechanical hypersensitivity in wild type mice whereas approaches that diminish opioid tolerance (β-arrestin2-/- and δ-/- mice) similarly prevented reinstatement of mechanical hypersensitivity. My findings suggest that tolerance to endogenous opioid tone facilitates the transition from acute to persistent hypersensitivity. These findings may be used as a platform to develop analgesic strategies to treat chronic pain by mitigating the impact of tolerance to endogenous opioid tone.

Date of Award	2022
Original language	English
Awarding Institution	University of Dundee
Sponsors	National Institute of Academic Anaesthesia
Supervisor	Tim Hales (Supervisor) & Jeremy Lambert (Supervisor)