Alhosan Norah, Cavallo Damiana, Santiago Marina, Kelly Eamonn, Henderson Graeme
School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK.
College of Pharmacy, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.
Br J Pharmacol. 2025 Feb;182(4):969-987. doi: 10.1111/bph.17376. Epub 2024 Oct 22.
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.
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.
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.
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.
芬太尼类和硝氮烯类是μ-阿片受体激动剂，导致大量阿片类药物过量致死事件。在此，我们测定了几种芬太尼和硝氮烯类似物在μ受体上的效力、解离动力学以及纳洛酮的拮抗作用，并与吗啡和DAMGO进行比较。
进行了G蛋白激活、信号传导和阻遏蛋白募集的体外测定。用膜电位染料加载表达μ受体的AtT20细胞，并利用荧光变化来确定激动剂效力、解离动力学以及对纳洛酮拮抗作用的敏感性。在表达μ受体的HEK293T细胞中进行BRET实验，以评估Gi蛋白激活和β-阻遏蛋白2募集情况。
激动剂从μ受体的表观解离速率各不相同：吗啡、DAMGO、阿芬太尼和芬太尼解离迅速，而异硝氮烯、依托尼嗪、奥芬太尼和卡芬太尼解离缓慢。解离缓慢的激动剂对纳洛酮的拮抗作用更具抗性。对于卡芬太尼，其缓慢的表观解离速率并非由于G蛋白受体激酶介导的阻遏蛋白募集，因为用化合物101抑制G蛋白偶联受体激酶（GRKs）后，其表观解离速率并未增加。与吗啡相比，芬太尼类和硝氮烯类的体外相对效力远低于先前在体内实验中观察到的效力。
对于从μ受体缓慢解离的芬太尼类和硝氮烯类药物，纳洛酮的拮抗作用是伪竞争性的。在涉及芬太尼类和硝氮烯类药物的过量用药中，可能需要比通常用于逆转海洛因过量更高剂量的纳洛酮来进行逆转。
