The Role of Myeloperoxidase in Clozapine-Induced Inflammation: A Mechanistic Update for Idiosyncratic Drug-Induced Agranulocytosis

Abstract

The risk of idiosyncratic drug-induced agranulocytosis (IDIAG) markedly constrains the use of clozapine, a neuroleptic with unparalleled efficacy. Most clozapine patients experience an early inflammatory response, likely a necessary step in IDIAG onset. However, most patients do not progress to IDIAG, presumably because of the requirement of specific human leukocyte antigen (HLA) haplotypes, T cell receptors, and other unknown factors. We established that clozapine activates inflammasomes and that myeloperoxidase bioactivation of clozapine generates neoantigens, but the connection between these early mechanistic events remained unknown and, thus, was the aim of this work. We found that the myeloperoxidase inhibitor PF-1355 attenuated myeloperoxidase activity in phorbol myristate acetate (PMA)-differentiated THP-1 macrophages, and it also attenuated clozapine-induced release of inflammatory mediators (e.g., IL-1β, CXCL1, and C-reactive protein). In vivo, pretreatment of Sprague Dawley rats with PF-1355 significantly attenuated clozapine-induced increases in neutrophil mobilization from the bone marrow to the blood and spleen, as determined using differential blood counts and flow cytometry. Moreover, the clozapine-triggered release of inflammatory mediators (e.g., IL-1β, calprotectin, CXCL1, and α-1-acid glycoprotein) from the liver, spleen, and bone marrow was dampened by myeloperoxidase inhibition. These data support the working hypothesis that oxidation of clozapine to a reactive metabolite by myeloperoxidase is critical for induction of the inflammatory response to clozapine. Ultimately, a better mechanistic understanding of the early events involved in the immune response to clozapine may elucidate ways to prevent IDIAG, enabling safer, more frequent therapeutic use of this and potentially other highly efficacious drugs.

Involvement of Extracellular Vesicles in the Proinflammatory Response to Clozapine: Implications for Clozapine-Induced Agranulocytosis

“…Extracellular vesicles in clozapine-induced inflammation 11 Uetrecht, 2014a;Sernoskie et al, 2022Sernoskie et al, , 2023 that this model recapitulates immune changes observed during the first month of clozapine treatment in patients (Pollmacher et al, 1996;Pollmächer et al, 1997;Löffler et al, 2010;Blackman et al, 2021;Sernoskie et al, 2021). Rats were euthanized by CO 2 inhalation and exsanguination at 3 hours post-injection (n = 6/group).…”

“…Extracellular vesicles in clozapine-induced inflammation 7 et Sernoskie et al, 2022Sernoskie et al, , 2023. Unlike clozapine, the structural analogues olanzapine and fluperlapine did not activate inflammasomes in vitro using a THP-1 macrophage model, as indicated by a lack of caspase-1 activation and release of IL-1β (Sernoskie et al, 2022(Sernoskie et al, , 2023.…”

“…Extracellular vesicles in clozapine-induced inflammation 7 et Sernoskie et al, 2022Sernoskie et al, , 2023. Unlike clozapine, the structural analogues olanzapine and fluperlapine did not activate inflammasomes in vitro using a THP-1 macrophage model, as indicated by a lack of caspase-1 activation and release of IL-1β (Sernoskie et al, 2022(Sernoskie et al, , 2023. Moreover, in vivo using a female Sprague Dawley rat model, we observed that although fluperlapine caused a similar decrease in circulating lymphocytes compared to clozapine, no increases in circulating neutrophils or serum CXCL1 levels were detected, indicating that fluperlapine did not induce a similar innate immune response (Sernoskie et al, 2022).…”

“…Using preclinical models, we have also demonstrated that myeloperoxidase plays a key role in the induction of this inflammatory response, specifically contributing to the release of soluble DAMPs and inflammatory mediators, as well as the chemotaxis of immune cells including neutrophils (Sernoskie et al, 2023). Yet, recent studies have also suggested that drugmodified proteins, DAMPs, and other signaling molecules, can also be released encapsulated in extracellular vesicles (EVs) as opposed to directly into the extracellular space or into circulation.…”

“…Direct drug incubations with clozapine (10 μg/mL; 30.6 μM), olanzapine (10 μg/mL; 32 μM) or drug vehicle (0.1% DMSO) were employed as controls. Cells were incubated under each condition for 24 hours, prior to the collection of conditioned media for cytokine analysis or the assessment of cell viability using a CCK-8 assay (GLPBio, Montclair, CA, USA), as described (Sernoskie et al, 2023).…”