Effect of Iboga Alkaloids on µ-Opioid Receptor-Coupled G Protein Activation

ObjectiveThe iboga alkaloids are a class of small molecules defined structurally on the basis of a common ibogamine skeleton, some of which modify opioid withdrawal and drug self-administration in humans and preclinical models. These compounds may represent an innovative approach to neurobiological investigation and development of addiction pharmacotherapy. In particular, the use of the prototypic iboga alkaloid ibogaine for opioid detoxification in humans raises the question of whether its effect is mediated by an opioid agonist action, or if it represents alternative and possibly novel mechanism of action. The aim of this study was to independently replicate and extend evidence regarding the activation of μ-opioid receptor (MOR)-related G proteins by iboga alkaloids. MethodsIbogaine, its major metabolite noribogaine, and 18-methoxycoronaridine (18-MC), a synthetic congener, were evaluated by agonist-stimulated guanosine-5´-O-(γ-thio)-triphosphate ([35S]GTPγS) binding in cells overexpressing the recombinant MOR, in rat thalamic membranes, and autoradiography in rat brain slices. Results And SignificanceIn rat thalamic membranes ibogaine, noribogaine and 18-MC were MOR antagonists with functional Ke values ranging from 3 uM (ibogaine) to 13 uM (noribogaine and 18MC). Noribogaine and 18-MC did not stimulate [35S]GTPγS binding in Chinese hamster ovary cells expressing human or rat MORs, and had only limited partial agonist effects in human embryonic kidney cells expressing mouse MORs. Ibogaine did not did not stimulate [35S]GTPγS binding in any MOR expressing cells. Noribogaine did not stimulate [35S]GTPγS binding in brain slices using autoradiography. An MOR agonist action does not appear to account for the effect of these iboga alkaloids on opioid withdrawal. Taken together with existing evidence that their mechanism of action also differs from that of other non-opioids with clinical effects on opioid tolerance and withdrawal, these findings suggest a novel mechanism of action, and further justify the search for alternative targets of iboga alkaloids.

**研究目的**
伊波加生物碱（iboga alkaloids）是一类以共同的伊波加胺骨架（ibogamine skeleton）为结构特征的小分子化合物，其中部分化合物可在人体及临床前模型中调节阿片类戒断反应与药物自身给药行为。此类化合物有望为神经生物学研究以及成瘾治疗药物的开发提供全新思路。其中，原型伊波加生物碱伊波加因（ibogaine）用于人类阿片类戒断脱毒治疗的应用，引发了关键问题：其药理作用是否由阿片受体激动介导，亦或是存在独特且全新的作用机制。本研究旨在独立重复并拓展相关证据，验证伊波加生物碱激活μ阿片受体（μ-opioid receptor, MOR）相关G蛋白的可能性。

**实验方法**
本研究选取伊波加因、其主要代谢产物去甲伊波加因（noribogaine）以及合成同系物18-甲氧基坎地拉明（18-methoxycoronaridine, 18-MC）作为研究对象，通过三种实验体系开展检测：一是在过表达重组μ阿片受体的细胞中进行激动剂刺激的鸟苷-5'-O-(γ-硫代)三磷酸([³⁵S]GTPγS)结合实验；二是在大鼠丘脑膜中开展上述结合实验；三是在大鼠脑切片中进行放射自显影检测。

**结果与意义**
在大鼠丘脑膜中，伊波加因、去甲伊波加因与18-MC均表现为μ阿片受体拮抗剂，其功能平衡解离常数（functional Ke values）范围为3 μM（伊波加因）至13 μM（去甲伊波加因与18-MC）。在表达人源或大鼠μ阿片受体的中国仓鼠卵巢细胞中，去甲伊波加因与18-MC未对[³⁵S]GTPγS结合产生刺激作用；在表达小鼠μ阿片受体的人胚肾细胞中，二者仅表现出微弱的部分激动剂活性。伊波加因在所有表达μ阿片受体的细胞系中均未激活[³⁵S]GTPγS结合。通过放射自显影实验发现，去甲伊波加因在大鼠脑切片中同样未刺激[³⁵S]GTPγS结合。综上，伊波加生物碱的阿片类戒断调节作用并非由μ阿片受体激动介导。结合现有研究证据——此类化合物的作用机制与其他对阿片类耐受及戒断具有临床疗效的非阿片类药物均存在差异——本研究结果提示伊波加生物碱存在全新的作用机制，同时也为进一步探索其潜在替代靶点提供了科学依据。