Damiana Cavallo

University of Bristol

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Interaction With the Lipid Membrane Influences Fentanyl Pharmacology

Advances in Drug and Alcohol Research (2022)

Katy J. Sutcliffe Robin A. Corey Norah Alhosan Damiana Cavallo Sam Groom

Overdose deaths from fentanyl have reached epidemic proportions in the USA and are increasing worldwide. Fentanyl is a potent opioid agonist that is less well reversed by naloxone than morphine. Due to fentanyl's high lipophilicity and elongated structure we hypothesised that its unusual pharmacology may be explained by its interactions with the lipid membrane on route to binding to the μ-opioid receptor (MOPr). Through coarse-grained molecular dynamics simulations, electrophysiological recordings and cell signalling assays, we determined how fentanyl and morphine access the orthosteric pocket of MOPr. Morphine accesses MOPr via the aqueous pathway; first binding to an extracellular vestibule, then diffusing into the orthosteric pocket. In contrast, fentanyl may take a novel route; first partitioning into the membrane, before accessing the orthosteric site by diffusing through a ligand-induced gap between the transmembrane helices. In electrophysiological recordings fentanyl-induced currents returned after washout, suggesting fentanyl deposits in the lipid membrane. However, mutation of residues forming the potential MOPr transmembrane access site did not alter fentanyl's pharmacological profile in vitro. A high local concentration of fentanyl in the lipid membrane, possibly in combination with a novel lipophilic binding route, may explain the high potency and lower susceptibility of fentanyl to reversal by naloxone.

10.3389/adar.2022.10280

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Citations (19)

Neuroprotective effects of mGluR5 activation through the PI3K/Akt pathway and the molecular switch of AMPA receptors

Neuropharmacology (2019)

Damiana Cavallo Elisa Landucci Elisabetta Gerace Daniele Lana Filippo Ugolini

Astrogliosis

Excitotoxicity

10.1016/j.neuropharm.2019.107810

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Citations (20)

Comparison of the effects of fentanyls and other μ opioid receptor agonists on the electrical activity of respiratory muscles in the rat

Frontiers in Pharmacology (2023)

Damiana Cavallo Eamonn Kelly Graeme Henderson Ana P. Abdala

Introduction: Deaths due to overdose of fentanyls result primarily from depression of respiration. These potent opioids can also produce muscle rigidity in the diaphragm and the chest muscles, a phenomenon known as Wooden Chest Syndrome, which further limits ventilation. Methods: We have compared the depression of ventilation by fentanyl and morphine by directly measuring their ability to induce muscle rigidity using EMG recording from diaphragm and external and internal intercostal muscles, in the rat working heart-brainstem preparation. Results: At equipotent bradypnea-inducing concentrations fentanyl produced a greater increase in expiratory EMG amplitude than morphine in all three muscles examined. In order to understand whether this effect of fentanyl was a unique property of the phenylpiperidine chemical structure, or due to fentanyl’s high agonist intrinsic efficacy or its lipophilicity, we compared a variety of agonists with different properties at concentrations that were equipotent at producing bradypnea. We compared carfentanil and alfentanil (phenylpiperidines with relatively high efficacy and high to medium lipophilicity, respectively), norbuprenorphine (orvinolmorphinan with high efficacy and lipophilicity) and levorphanol (morphinan with relatively low efficacy and high lipophilicity). Discussion: We observed that, agonists with higher intrinsic efficacy were more likely to increase expiratory EMG amplitude (i.e., produce chest rigidity) than agonists with lower efficacy. Whereas lipophilicity and chemical structure did not appear to correlate with the ability to induce chest rigidity.

Lipophilicity

Diaphragm (acoustics)

10.3389/fphar.2023.1277248

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Citations (4)

Potency, dissociation kinetics and reversibility of fentanyls and nitazenes by naloxone at the μ opioid receptor

Authorea (Authorea) (2024)

Norah Alhosan Damiana Cavallo Marina Santiago Eamonn Kelly Graeme Henderson

Background and Purpose: Fentanyls and nitazenes are μ opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we compared the potency, dissociation kinetics and antagonism by naloxone at the μ receptor of several fentanyl and nitazene analogues and compared them to morphine and DAMGO. Experimental Approach: In vitro assays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing μ receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing μ opioid receptors, to assess Gi protein activation and β-arrestin 2 recruitment. Key Results: The rate of agonist dissociation from the μ receptor varied, with morphine, DAMGO, alfentanil and fentanyl dissociating rapidly whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow rate of dissociation was not due to G protein receptor kinase-mediated arrestin recruitment as its rate of dissociation was not affected by inhibition of GRKs with Compound 101. The in vitro relative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed in in vivo experiments. Conclusions and Implications: With fentanyls and nitazenes, that slowly dissociate from the opioid receptor, antagonism by naloxone is pseudo competitive. In overdoses involving fentanyls and nitazenes higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose.

10.22541/au.171906096.68090393/v1

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The anomalous pharmacology of fentanyl

British Journal of Pharmacology (2021)

Eamonn Kelly Katy J. Sutcliffe Damiana Cavallo Nokomis Ramos‐Gonzalez Norah Alhosan

Fentanyl is a key therapeutic, used in anaesthesia and pain management. It is also increasingly used illicitly and is responsible for a large and growing number of opioid overdose deaths, especially in North America. A number of factors have been suggested to contribute to fentanyl's lethality, including rapid onset of action, in vivo potency, ligand bias, induction of muscle rigidity and reduced sensitivity to reversal by naloxone. Some of these factors can be considered to represent ‘anomalous’ pharmacological properties of fentanyl when compared with prototypical opioid agonists such as morphine. In this review, we examine the nature of fentanyl's ‘anomalous’ properties, to determine whether there is really a pharmacological basis to support the existence of such properties, and also discuss whether such properties are likely to contribute to overdose deaths involving fentanyls. LINKED ARTICLES This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc

Opioid Overdose

10.1111/bph.15573

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Citations (87)

784 TOLL-LIKE RECEPTOR-2 SIGNALLING IS A KEY PLAYER IN INSULIN RESISTANCE OCCURRENCE DURING OBESITY

Journal of Hepatology (2010)

Paola Brun Damiana Cavallo S. Dagnolo S Signori Carlos Eduardo Anselmi

Signalling

10.1016/s0168-8278(10)60786-6

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The ability of fentanyls and other opioids to increase EMG amplitude in respiratory muscles correlates with their agonist efficacy

The FASEB Journal (2021)

Damiana Cavallo Eamonn Kelly Graeme Henderson Ana Abdala Sheikh

Acute overdose deaths involving fentanyls have been dramatically increasing in the USA since 2013. These potent opioids depress respiratory drive and produce rigidity in the intercostal muscles and diaphragm (resulting in what is referred to as Wooden Chest Syndrome) as well as in glottic and supraglottic muscles which further reduces the ability to breathe. In this study we have sought to characterise the ability of various opioid agonists to induce respiratory muscle rigidity using electromyographic (EMG) recording. EMG amplitude was recorded simultaneously from diaphragm, external and internal intercostal muscles of P21 male Wistar rats, using an in situ decerebrated and arterially perfused preparation. First, the effects of fentanyl and morphine on respiratory muscle EMG were studied using a dose of each agonist that depressed respiratory rate by approximately 40%. Fentanyl produced a marked increase in EMG amplitude during expiration in all three respiratory muscles whereas the effect of morphine was very slight. In order to understand whether the difference between fentanyl and morphine related to differences in chemical structure, lipid solubility or agonist intrinsic efficacy we studied the effects of a range of opioids that differed in these characteristics – carfentanil and fentanyl (piperidine derivatives, high lipid solubility, high efficacy), alfentanil (piperidine derivative, moderate lipid solubility, high efficacy) norbuprenorphine (thebaine derivative, high lipid solubility, high efficacy), levorphanol (morphinan, high lipid solubility, low efficacy), morphine and hydromorphone (morphine derivatives, low lipid solubility, low efficacy). With doses of each that produced around 40% depression of respiratory rate we observed that the rank order of ability to increase internal and external intercostal muscle EMG amplitude was carfentanil = fentanyl = norbuprenorphine > alfentanil = hydromorphone > morphine = levorphanol but for the diaphragm the rank order was fentanyl > alfentanil = norbuprenorphine ≥ hydromorphone > carfentanil ≥ morphine = levorphanol. These results suggest that the ability of opioid agonists to increase respiratory muscle EMG amplitude and therefore respiratory muscle rigidity does not depend solely on either chemical structure or lipid solubility of the agonist. For the intercostal muscles there is a better correlation with agonist efficacy. Why the effect of carfentanil on the diaphragm is weak remains to be elucidated.

Respiratory Rate

Hydromorphone

10.1096/fasebj.2021.35.s1.03977

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Citations (1)

Slow dissociation kinetics of fentanyls and nitazenes correlates with reduced sensitivity to naloxone reversal at the μ‐opioid receptor

British Journal of Pharmacology (2024)

Norah Alhosan Damiana Cavallo Marina Santiago Eamonn Kelly Graeme Henderson

Abstract Background and Purpose Fentanyls and nitazenes are μ‐opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we determined the potency, dissociation kinetics and antagonism by naloxone at the μ receptor of several fentanyl and nitazene analogues, compared to morphine and DAMGO. Experimental Approach In vitro assays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing μ receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing μ receptors to assess Gi protein activation and β‐arrestin 2 recruitment. Key Results The apparent rate of agonist dissociation from the μ receptor varied: morphine, DAMGO, alfentanil and fentanyl dissociated rapidly, whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow apparent rate of dissociation was not because of G protein receptor kinase‐mediated arrestin recruitment as its apparent rate of dissociation was not increased by inhibition of G protein‐coupled receptor kinases (GRKs) with Compound 101. The in vitro relative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed in in vivo experiments. Conclusions and Implications With fentanyls and nitazenes that slowly dissociate from the μ receptor, antagonism by naloxone is pseudo‐competitive. In overdoses involving fentanyls and nitazenes, higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose.

DAMGO

μ-opioid receptor

10.1111/bph.17376

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Changes in excitatory and inhibitory receptor expression and network activity during induction and establishment of epilepsy in the rat Reduced Intensity Status Epilepticus (RISE) model

Neuropharmacology (2019)

Hope I. Needs Benjamin S. Henley Damiana Cavallo Sonam Gurung Tamara Modebadze

The RISE model is an effective system to study the underlying molecular and cellular mechanisms involved in the initiation and maintenance of epilepsy in vivo. Here we profiled the expression of excitatory and inhibitory neurotransmitter receptor subunits and synaptic scaffolding proteins in the hippocampus and temporal lobe and compared these changes with alterations in network activity at specific timepoints during epileptogenesis. Significant changes occurred in all of the ionotropic glutamate receptor subunits tested during epilepsy induction and progression and the profile of these changes differed between the hippocampus and temporal lobe. Notably, AMPAR subunits were dramatically decreased during the latent phase of epilepsy induction, matched by a profound decrease in the network response to kainate application in the hippocampus. Moreover, decreases in the GABAAβ3 subunit are consistent with a loss of inhibitory input contributing to the perturbation of excitatory/inhibitory balance and seizure generation. These data highlight the synaptic reorganisation that mediates the relative hypoexcitability prior to the manifestation of seizures and subsequent hyperexcitability when spontaneous seizures develop. These patterns of changes give new insight into the mechanisms underpinning epilepsy and provide a platform for future investigations targeting particular receptor subunits to reduce or prevent seizures.

Kainate receptor

Ionotropic glutamate receptor

10.1016/j.neuropharm.2019.107728

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Citations (20)

Potency, dissociation kinetics and reversibility of fentanyls and nitazenes by naloxone at the μ opioid receptor

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Norah Alhosan Damiana Cavallo Marina Santiago Eamonn Kelly Graeme Henderson

Abstract Background and Purpose Fentanyls and nitazenes are μ opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we compared the potency, dissociation kinetics and antagonism by naloxone at the μ receptor of several fentanyl and nitazene analogues and compared them to morphine and DAMGO. Experimental Approach In vitro assays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing μ receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing μ opioid receptors, to assess Gi protein activation and β-arrestin 2 recruitment. Key Results The rate of agonist dissociation from the μ receptor varied, with morphine, DAMGO, alfentanil and fentanyl dissociating rapidly whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow rate of dissociation was not due to G protein receptor kinase-mediated arrestin recruitment as its rate of dissociation was not affected by inhibition of GRKs with Compound 101. The in vitro relative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed in in vivo experiments. Conclusions and Implications With fentanyls and nitazenes, that slowly dissociate from the μ opioid receptor, antagonism by naloxone is pseudo competitive. In overdoses involving fentanyls and nitazenes higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose. What is already known “Fentanyls” and “nitazenes” are potent agonists at the μ opioid receptor. What does this study add Some fentanyls and nitazenes dissociate slowly and are less sensitive to naloxone antagonism What is the clinical significance More naloxone may be required to reverse overdoses involving fentanyls and nitazenes

10.1101/2024.06.04.597322

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