Otago Pharmacometrics Group, School of Pharmacy, University of Otago, Dunedin, New Zealand.
Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand.
Br J Pharmacol. 2019 Jul;176(14):2593-2607. doi: 10.1111/bph.14684. Epub 2019 May 21.
Receptor internalisation is by nature kinetic. Application of a standard equilibrium dose response model to describe the properties of a ligand inducing internalisation, while commonly used, are therefore problematic. Here, we propose two quantitative approaches to address this issue-(a) a model-free method and (b) a kinetic modelling approach-and systematically evaluate the performance of these methods against traditional equilibrium methods to characterise the internalisation profiles of cannabinoid CB receptor agonists.
Kinetic internalisation assays were conducted using a concentration series of six CB receptor ligands. Internalisation rate analysis and snapshot equilibrium analysis were performed. A model-free method was developed based on the mean residence time of internalisation. A kinetic internalisation model was developed under the quasi-steady state assumption.
Rates of receptor internalisation depended on both agonist and concentration. Agonist potencies from snapshot equilibrium analysis increased with stimulation time, and there was no single time point at which internalisation profiles could infer agonist properties in a comparative manner. The model-free method yielded a time-invariant measure of potency/efficacy for internalisation. The kinetic model adequately described the internalisation of CB receptors over time and provided robust estimates of both potency and efficacy.
Applying equilibrium analysis to a non-equilibrium pathway cannot provide a reliable estimate of agonist potency. Both the model-free and kinetic modelling approaches characterised the internalisation profiles of CB receptor agonists. The kinetic model provides additional advantages as a method to capture changes in receptor number during other functional assays.
受体内化本质上是动力学的。应用标准平衡剂量反应模型来描述诱导内化的配体的特性，虽然常用，但存在问题。在这里，我们提出了两种定量方法来解决这个问题——（a）无模型方法和（b）动力学建模方法，并系统地评估这些方法对传统平衡方法的性能，以表征大麻素 CB 受体激动剂的内化曲线。
使用六个 CB 受体配体的浓度系列进行了动力学内化测定。进行了内化率分析和快照平衡分析。基于内化的平均停留时间，开发了一种无模型方法。在准稳态假设下，开发了一种动力学内化模型。
受体内化的速度取决于激动剂和浓度。从快照平衡分析得出的激动剂效力随着刺激时间的增加而增加，没有单一的时间点可以以比较的方式推断内化曲线的激动剂性质。无模型方法提供了一种对内化的效力/功效的时不变测量。动力学模型能够很好地描述 CB 受体随时间的内化，并提供了对效力和功效的稳健估计。
将平衡分析应用于非平衡途径不能可靠地估计激动剂的效力。无模型和动力学建模方法都能描述 CB 受体激动剂的内化曲线。动力学模型作为一种在其他功能测定中捕获受体数量变化的方法具有额外的优势。
