Assessment of cellular environments on radioligand binding - An application to opioid receptor PET imaging

Imaging non-invasively release of endogenous opioid peptides following pharmacological challenge would vastly increase our understanding of the opioid system in substance abuse and pain. The imaging radioligands for the opioid system, [11C]diprenorphine [1] and [11C]carfentanil [2] are known to be susceptible to release of endogenous opioid peptides during physiological challenge studies. Internalisation processes are suggested to cause a reduction in Binding Potential (BP) during imaging studies [3]. The amount that opioid receptor internalisation processes might contribute to these changes in BP and the ability of opioid receptor ligands to bind sub-cellular receptors has not been assessed. We report the effect of different cellular environments on the binding of two widely used opioid receptor PET radioligands, [11C]diprenorphine and [11C]carfentanil to aid the translation and interpretation of endogenous release imaging paradigms to the opioid system. The ability of opioid receptor ligands to bind in the extracellular (Extra), intracellular (Intra) and endosomal (Endo) environment of the cell were investigated. Rat whole brain homogenate binding assays were performed using [3H]diprenorphine and [11C]carfentanil. Saturation studies were performed in the presence of three buffers representative of different cellular compartments: - Extra - 50 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 1.5 mM CaCl2, pH 7.4, 37°C; - Intra - 50mM Tris-HCl, 10 mM NaCl, 140 mM KCl, 0.5 mM MgCl2, pH 7.0, 37°C; - Endo - 20 mM MES, 10 mM NaCl, 140 mM KCl, 0.5 mM MgCl2, 0.003 mM CaCl2, pH 6.0, 37°C. Naloxone (10μM) was used to determine specific binding. Assays using tritium were incubated for 90 minutes, and 30 minutes for carbon-11 (37°C). Changes in cellular environments caused a significant decrease in receptor availability (Bmax) in the Endosomal condition compared to the Extracellular for both [11C]diprenorphine and [11C]carfentanil (P < 0.05). No significant effect on the binding affinity (Kd) for either of the radioligands tested was observed with a change in environments. However, a clear trend was seen for a reduction in affinity in the Intracellular and Endosomal environment compared to the Extracellular for [11C]carfentanil. In vitro studies with short-lived PET radioisotopes such as carbon-11 (t1/2 ∼ 20 minutes) are more prone to variability than studies with tritiated counterparts. The lack of any significant difference for [11C]carfentanil in terms of affinity or BP in the Intracellular and Endosomal compared to Extracellular may be a result of this phenomena. When these in vitro data are converted to BP values (BP = Bmax/Kd) it is possible to conclude that a decrease in binding signal in vivo could support an internalisation hypothesis. These changes in BP would primarily be driven by a decrease in receptor availability in the Endosomal condition with [3H]diprenorophine and [11C]carfentanil. However, for [11C]carfentanil this change may also be driven by a reduction in affinity in the endosomal compartment. (Table presented).