MOLECULAR MECHANISMS OF CLOZAPINE & QUETIAPINE CYTOTOXICITY
Creators: Rashid, Md Harunur
Publisher: name: University of Alberta Library
Year: 2024

<p>Clozapine (dibenzodiazepine) is the only evidence-based medication for treatment-resistant schizophrenia and is superior in the reduction of suicide. Another important structurally related atypical antipsychotic drug, quetiapine (dibenzothiazepine), is currently used for the treatment of schizophrenia. However, both drugs are associated with idiosyncratic drug reactions (such as agranulocytosis or neutropenia), and it is believed that quinone-like reactive metabolites are responsible for their toxicity. The neutrophilic myeloperoxidase (MPO) metabolism of clozapine is known, but quetiapine metabolism by MPO is not well-studied, although quetiapine is metabolized by cytochrome P450 (CYP). Moreover, two important drug-detoxifying enzymes, NAD(P)H: quinone oxidoreductase-1 (NQO1) and NRH: quinone oxidoreductase-2 (NQO2), have been previously proposed to be indirectly connected with the clozapine-induced agranulocytosis but no direct connection has been made. So, the overall goal of this thesis is to compare the cellular responses and metabolic differences of clozapine and quetiapine as well as possible protection mechanisms against toxicities caused by reactive metabolites of these drugs involving MPO, CYP, NQO1 and NQO2. In the first study, we hypothesized that MPO could metabolize quetiapine to form reactive metabolites like clozapine, and there is an overlap between CYP and MPO metabolism of quetiapine as quetiapine and clozapine have structural similarities. The enzymatic interaction of quetiapine and clozapine with MPO was determined using UV−vis spectrophotometry via an enzymatic activities assay. MPO chlorination activity was also determined using UV−vis spectrophotometry. The metabolites were characterized using liquid chromatography-mass spectrometry (LC-MS). The electron paramagnetic resonance (EPR) spectroscopy technique was applied to detect drug-catalyzed glutathione oxidation. We found that increasing quetiapine concentrations resulted in hanging up the catalytic cycle of MPO in compound II with no change in the UV−vis spectral and a decrease in MPO chlorination activity. In contrast, the opposite results were found in the case of clozapine. Although different metabolites of both quetiapine and clozapine were observed, surprisingly, the N-dealkylated quetiapine metabolite was observed in the presence of MPO that overlaps between MPO and CYP. Also, quetiapine metabolites were generally less reactive than clozapine. In the second study, it was hypothesized that clozapine and quetiapine reactive metabolites caused by MPO are responsible for the induction of NQO1 &amp; NQO2 via the Nrf2 signalling pathway. HL-60 cells were used to test this hypothesis as they are similar to granulocytes/neutrophils and contain enzymes to metabolize clozapine. THP-1 cells are used to make comparative studies. A cell viability test was done to select concentrations using the Cell Counting Kit-8 (CCK-8) assay. A UV-Vis spectrophotometric assay was performed to measure NQO1&#39;s and NQO2&#39;s enzymatic activity using selective substrates and inhibitors. Western blotting was applied to determine NQO1, NQO2, and Nrf2 protein expression. NQO1 and NQO2 gene expressions were investigated using reverse transcription polymerase chain reaction (RT-PCR). Clozapine (but not quetiapine) was found to upregulate NQO1 and NQO2 proteins, genes, and their enzymatic activities, which correlated with the translocation of Nrf2 from the cytoplasm to the nucleus. The findings suggest the direct involvement of NQO1 and NQO2 in the clozapine toxicity mechanisms caused by quinone-like reactive metabolites. In the third study, we hypothesized that an antioxidant-generating phytochemical, sulforaphane, induces a protective response through upregulating NQO1 and NQO2 levels; what remaining clozapine levels are as both NQO1 and NQO2 are directly involved in the clozapine toxicity mechanisms caused by reactive metabolites like quinone. For testing this hypothesis, sulforaphane and clozapine cell viability were measured to select appropriate treatment concentrations using a CCK-8 kit. Immunoblotting was applied to investigate NQO1 and NQO2 protein expression and protection against clozapine-protein binding. Glutathione (GSH) was determined by microtiter plates, and LCMS was used to quantify clozapine metabolites. UV-Vis spectrophotometric assay was performed to analyze MPO activity. Sulforaphane increased MPO activity and showed protection against clozapine toxicity via induction of NQO1 &amp; NQO2 proteins, attenuation of clozapine cytotoxicity, enhanced clozapine oxidative metabolites, glutathione upregulation, and attenuation of clozapine-protein covalent binding. In conclusion, these studies have uncovered the potential roles of NQO1 and NQO2 proteins as a response to clozapine reactive metabolites and indicate that sulforaphane imparts significant protective mechanisms against clozapine cytotoxicity caused by quinone-like reactive metabolites in the presence of MPO. Also, the unexpected finding in this study was the N-dealkylated quetiapine metabolite, which overlaps between MPO and CYP. </p>