Binding properties of clozapine and related compounds on native D2 dopamine receptors in normal and oxidative medium

Despite the presence of several side effects, clozapine remains a valuable drug in treating schizophrenia. The presence of haematological toxicity was proposed to be related to the formation of nitrenium species (1). Indeed, nitrogen derivatives like clozapine or olanzapine are very sensitive to oxidation while oxygen or sulphur isosteres such as loxapine, clothiapine and JL13, possess a very low sensitivity to oxidation (2-4).
In the present study, we explore the impact of oxidation conditions on the binding of these drugs on native rat dopamine D2 receptors.
Rats brains were quickly removed after cerebral dislocation and dissected on ice to get striata. After weighting, tissues were homogenized in buffer and washed three times by centrifugation. The final pellet was dispersed in the appropriate volume of incubation buffer (Tris 50 mM, MgCl2 5 mM, EDTA (Na2) 1 mM buffered at pH 7.4 with 4N HCl) depending on the experimental conditions. In oxidative conditions, horseradish peroxidase (1.25 µg/tube) and H2O2 (50 µM) were added to the incubation buffer. [3H]-Spiperone was used as radioligand and the non specific binding was determined in the presence of haloperidol (10 µM). Incubation temperature and time were 27°C and 60 min respectively. Competition experiments were done with different molecules such as haloperidol and various tricyclic derivatives.
In our previous studies (2,3), we had observed that compounds like clozapine or olanzapine are significantly affected by oxidative conditions. In the present report, we show that this sensitivity is also associated with a dramatic decrease of binding affinity. Unlike such diazepine analogues, the binding of loxapine and JL13, two oxygen isosteres is slightly affected in oxidative conditions. Firstly, these results show that the distal nitrogen is not affected by the oxidative conditions. Secondly, the nitrenium formation (1) might lead to a tridimensional change that would reduce the interactions in the binding pocket. The oxidative effect of HRP/H2O2 mixture can be prevented by addition of ascorbic acid to the incubation medium.
Thus depending on the oxidant character of the extracellular medium in physiological or pathophysiological conditions, interaction of molecules with different targets can be modified significantly.