Commentary on “Morphine‐induced respiratory depression is independent of β‐arrestin2 signalling”

Opioid abuse, addiction, and overdose present a major public health crisis internationally. Now more than ever, there is a critical need for safer effective painkillers with lower abuse potential. Researchers from academia and industry have turned their attention to the concept of biased agonism, which describes the phenomenon where a ligand stabilises a subset of receptor active conformations, giving rise to differential or preferential activation of different cellular responses when compared to a reference agonist and reference pathway. For the μ-opioid receptor, the aim is to develop novel agonists that stimulate analgesia with the exclusion of adverse effects, ultimately to offer an improved therapeutic index when compared to commonly used μ-opioid receptor agonists such as morphine and fentanyl. A prevailing hypothesis in the field has been that μ-opioid receptor signalling via β-arrestin2 mediates the adverse effects including respiratory depression and constipation. The study from Kliewer et al. (2020) presents a highly collaborative effort to convincingly demonstrate that β-arrestin2 knockout mice retain the phenotype for morphineand fentanyl-induced respiratory depression and constipation. These findings contradict an earlier report (Raehal, Walker, & Bohn, 2005), challenging the paradigm that μ-opioid receptor signalling linked to adverse, as distinct from therapeutic, effects occurs through β-arrestin and G protein respectively, which has major implications for discovery efforts focused on biased μ-opioid receptor agonists as novel analgesics. A major strength of the study from Kliewer et al. (2020) is the independent replication of respiratory depression experiments from laboratories spanning three different countries. The authors in each location use different methods to measure respiration in mice. All consistently showed that complete knockout of β-arrestin2 in C57BL/6J mice had no effect on morphine-induced respiratory depression. The study design reported here is particularly strong with three independent cohorts of animals used and experimenters blinded to both genotype and treatment. The authors are unable to pinpoint why the outcomes here did not replicate the original report (Raehal, Walker, & Bohn, 2005). However, their hypothesis that differences in breeding and the use of mice with mixed genetic backgrounds in the original study likely contributes to these differences is highly probable. In this respect, the study highlights some of the inherent limitations of using knockout animals to dissect the mechanistic basis of drug action. Namely, the potential effects of background strain and inbreeding on the functional response/s under investigation or possibly compensatory mechanisms masking the effects of knocking out a single protein. This study demonstrates the importance of transparency in animal research, including recognition of the need to publish research outcomes that fail to confirm a hypothesis, as well as independent replication studies. Additional evidence by Kliewer et al. (2020) supports their conclusions regarding the role of β-arrestin2 in opioid-induced adverse effects. Fentanyl caused a similar degree of respiratory depression in β-arrestin2 knockout mice compared to WT littermates (Kliewer et al., 2020). In addition to respiratory depression, both morphine and fentanyl induced constipation in WT and β-arrestin2 knockout mice to a similar degree. These data are consistent with μ-opioid receptor knock-in mice that contain phosphorylation-deficient mutations (that display decreased β-arrestin2 recruitment) also retaining constipation and respiratory depression phenotypes in response to fentanyl and morphine (Kliewer et al., 2019). This is particularly noteworthy in light of the recent report that, relative to DAMGO, a synthetic μ-opioid receptor agonist, fentanyl is generally biased towards β-arrestin2 recruitment over GTPγS binding. However, the reported relative efficacy of fentanyl is very much dependent on species (rat vs. human), response or assay used to quantify the effect (Rivero et al., 2012; Schmid et al., 2017). Kliewer et al. (2020) did not explore other possible mechanisms for morphineor fentanyl-induced respiratory depression and constipation, although a compensatory increase in β-arrestin1 expression in the β-arrestin2 knockout mice was ruled out. Multiple independent studies support the conclusion that knockout or knockdown of β-arrestin2 enhances morphine analgesia and prevents tolerance development (reviewed in Raehal, Schmid, Groer, & Bohn, 2011). Collectively, these studies support the notion that other, different, mechanisms contribute to μ-opioid receptor-mediated cellular responses that give rise to the various side effect liabilities Received: 14 February 2020 Revised: 30 March 2020 Accepted: 23 April 2020