Long-term exposure to DEHP during PN increased free radical activity in vivo.

The clozapine-induced agranulocytosis could be due to the formation of a reactive intermediate formed in polymorphonuclear neutrophils and granulocyte precursors with the myeloperoxidase-hydrogen peroxide system. On the contrary, no case of agranulocytosis has been described for loxapine, an other neuroleptic drug with a very close structural analogy. We have compared the clozapine and loxapine interaction with the oxidative burst and particularly with this enzymatic complex. On the one hand, the assay of the oxidative species demonstrated a different impact for the two neuroleptics. The 50% inhibitory concentration was 92 microM for hydrogen peroxide and 40 microM for hypochlorous acid for loxapine. The loxapine target is located before the myeloperoxidase-hydrogen peroxide system in the oxidative stream, whereas clozapine diverts the chlorination pathway of the enzyme. On the other hand, the in vitro metabolism of drugs by the myeloperoxidase-hydrogen peroxide system has been investigated by mass spectrometry. Loxapine remains inert but clozapine undergoes the oxidation. The glutathione or ascorbate addition in the medium leads to a removal of the oxidation. Glutathione is able to trap the toxic intermediate and could avoid its formation.

The use of clozapine, an unique antipsychotic drug, raises the real problem of drug-induced polymorphonuclear neutrophil cytotoxicity. Clozapine prescription has been restricted due to a 1-2% incidence of drug-induced agranulocytosis. The exact mechanism of this adverse effect is not yet known. The myeloperoxidase-hydrogen peroxide system could play a key role in the initiation of agranulocytosis. Therefore, we have investigated the clozapine effects on hydrogen peroxide and hypochlorous acid, evaluated the peroxidase-mediated metabolism of clozapine by mass spectrometry analysis because myeloperoxidase uses hydrogen peroxide and chloride producing hypochlorous acid in its chlorination cycle, and thus could oxidise clozapine in its peroxidation cycle. First, evidence for inhibition of hypochlorous acid production and scavenging of hydrogen peroxide by clozapine were demonstrated in vitro, in different cell-free and cellular systems. Results are consistent with an inhibition of the myeloperoxidase chlorination cycle when clozapine is oxidised in the peroxidation cycle. Secondly, ion-spray mass spectrometry analysis allowed us to confirm clozapine oxidation by the myeloperoxidase system. Actually, clozapine N-oxide with a m/z at 343 was formed. It could be the final step of the metabolisation of clozapine via two successive univalent oxidations mediated by peroxidase. We suggest that generation of a free cation radical, CLZ(o+), was the initial step. CLZ(o+) is a very reactive species and may play an important role in the onset of agranulocytosis either by direct toxicity or via an immunological mechanism. However, this assumption does not exclude the possible role of other metabolic ways involving, in particular, N-desmethylclozapine.

SUMMARYClozapine is an antipsychotic drug with few extrapyramidal motor side-effects, used to treat schizophrenia which is resistant to classical neuroleptic therapy. This report shows that norclozapine but not clozapine-N-oxide has the same D2 receptor affinity as clozapine. Assay results suggest a bimodal distribution which may be explained by CYF'IA2 polymorphism. Extensive metabolizers could produce other active metabolites, probably other hydroxy-clozapine derivatives.