P.1.030 Antagonist dissociation from dopamine D2 receptors: studies using a time-resolved ion channel activation assay

Abstract

Purpose of the study: Antipsychotic medication is often associated with adverse effects such as extrapyramidal symptoms (EPS) and increased serum prolactin. There is evidence that the lower liability to produce EPS and increased prolactin attributed to newer, so-called atypical antipsychotics, is correlated with their faster rates of dissociation from the dopamine D2 receptor [1]. Recent studies have indicated that the novel D2 receptor ligands, ACR16 and (−)-OSU6162, initially described as “dopamine stabilizers,” act as antagonists with similarly high dissociation rates [2,3]. However, these previous studies of antagonist unbinding rates measured either dissociation of radiolabeled ligand from membrane preparations [1] or used modified G proteins to study receptor activation-induced calcium release in living cells [2,3]. We wanted to examine the relative kinetics of antagonist dissociation in living cells, using an assay based an activation of G protein-coupled potassium (GIRK) channels. This assay uses native G proteins and has higher temporal resolution than previously used assays. Methods used: Xenopus oocytes were injected with cRNA encoding the human dopamine D2S receptor, Regulator of G protein Signalling (RGS)-4, and GIRK1/4 channel subunits. GIRK current responses to dopamine receptor activation were recorded at −80mV using twoelectrode voltage clamp. First, dopamine (100 nM) was applied, resulting in a “baseline” GIRK response. Next, a maximally effective concentration of antagonist was washed in, in the continued presence of dopamine. After achieving steady-state response inhibition the antagonist was washed out, still in the presence of dopamine. Response recovery was recorded over six minutes, and the recovery time-course and the amplitude of the (pseudo)steady state current relative to the baseline response were taken as measures of antagonist dissociation. Summary of results: Significant differences (P< 0.05; Student’s t-test) in response recovery T1/2 (time to half-maximal recovery) and recovery amplitudes were observed between the different D2 receptor antagonists: In experiments with haloperidol, risperidone, paliperidone, aripiprazole, and bifeprunox, no response recovery was detected. With clozapine, quetiapine, and N-desmethylclozapine, similar recovery time courses were observed (T1/2 = 48±5.5 s, 60±2.2 s, and 46±3.7 s, respectively). With clozapine and quetiapine, about 50% response recovery was seen, whereas N-desmethylclozapine washout allowed 80% recovery. The “stabilizer” compounds ACR16 and (−)-OSU6162, along with the structurally related experimental antipsychotic, (−)-3-PPP, behaved as antagonists, lacking detectable efficacy in the assay. These compounds washed out with similar time courses (T1/2 = 8.2±1.8 s, 6.1±0.44 s, and 7.8±0.87 s, respectively); significantly faster than the other antagonists in the study, and allowed near-complete response recovery. Conclusions: The present data support the idea that the atypical antipsychotics, clozapine and quetiapine, dissociate faster from D2 receptors than the typical antipsychotic haloperidol, but also faster than several other atypical drugs in the study, including risperidone, paliperidone, and aripiprazole. Furthermore, the “dopamine stabilizers” ACR16 and (−)-OSU6162 appear to dissociate faster than clozapine and quetiapine, a finding which has not been reported earlier. Such very rapid dissociation might be relevant to the low incidence of side effects reported from clinical trials with these compounds.