Toxicological and clinical investigations of metamizole-associated neutropenia

Metamizole is a non-opioid analgesic, antipyretic, and pasmolytic prodrug, which is widely prescribed in certain countries due to its good efficacy and low gastrointestinal toxicity. Despite the favorable safety profile overall, metamizole has been banned in several countries due to reports of metamizole-associated neutropenia, a severe and potentially fatal decrease of circulating neutrophil granulocytes. In Switzerland and Germany, metamizole use has increased over the last fifteen years even though it has been restricted to narrow indications.
The aim of this PhD project was to elucidate the underlying toxicological mechanisms of metamizole-associated neutropenia. The gained knowledge could lead to a better understanding of the cellular mechanisms of metamizole-associated neutropenia and improve the safety of metamizole treatment. Hence, this thesis is composed of toxicological in vitro investigations of direct metamizole metabolite toxicity on neutrophils and neutrophil progenitor cells as well as of clinical investigations composed of an observational case-control study.
For the in vitro part of this thesis, I investigated the cytotoxicity of metamizole and its main metabolites on the promyelocytic cell line HL60 in comparison with mature neutrophils. To form potentially cytotoxic secondary metabolites, I combined the metamizole metabolites with components of the neutrophil antioxidative system. Furthermore, I assessed potential formation of cytotoxic metabolites after combination of metamizole metabolites with various iron compounds found in neutrophils and blood. None of the tested metamizole metabolites was toxic in any cell line. The main metamizole metabolite N-methyl-4-aminoantipyrine (MAA) even reduced cytotoxicity of the myeloperoxidase substrate hydrogen peroxide at low concentrations (< 50 μM), but increased cytotoxicity at a concentration of 100 μM hydrogen peroxide. In contrast, neutrophil granulocytes were resistant to any tested hydrogen peroxide concentration and MAA. Furthermore, MAA did not increase the toxicity of the iron compounds lactoferrin, hemoglobin or methemoglobin in HL60 cells. However, the hemoglobin degradation product hemin was toxic on HL60 cells and cytotoxicity was increased by MAA. The radical scavengers N-acetylcysteine and glutathione as well as the iron chelator ethylenediaminetetraacetic acid(EDTA) were able to reduce the toxicity of hemin and MAA-hemin. The interaction between the trivalent iron ion of hemin and MAA was displayed in the absorption spectrum of hemin. The spectrum changed concentration-dependently after addition of MAA, suggesting an interaction between hemin iron and MAA. The corresponding NMR of the combination MAA and hemin revealed the formation of a stable MAA reaction product with a reaction pathway involving the formation of an electrophilic intermediate. Hence, the main metamizole metabolite MAA increased the cytotoxicity of hemin by a reaction involving the formation of an electrophilic metabolite, which elicits apoptosis in promyelocytic HL60 cells but not in mature neutrophil granulocytes. These results suggest that the cellular antioxidative defense system and/or heme-metabolizing capacity changes during neutrophil differentiation, rendering mature cells less susceptible to the reactive MAA intermediate. Thus, I assessed the toxicity of hemin and MAA on differentiating HL60 cells that were differentiated into mature neutrophils over 5 days. After 3 days differentiation, the cell population was predominantly metamyelocytic and resistant against MAA-hemin, whereas hemin alone was still cytotoxic. At 5 days of differentiation, when the cell population consisted mainly of mature neutrophils, hemin was not toxic anymore. These results were compared with immature myeloid cells from umbilical cord blood, representing early neutrophil precursor cells, which were differentiated over 14 days into the neutrophil lineage. Similarly to promyelocytic HL60 cells, MAA-hemin was more toxic than hemin alone on immature myeloid cells from umbilical cord blood. However, no cytotoxicity was observed on freshly isolated human neutrophils. During differentiation of HL60 cells, the protein expression of enzymes responsible for hemin metabolism increased. Inhibition of the heme-metabolizing enzymes heme oxygenase-1 or cytochrome P450 reductase increased hemin and MAA-hemin toxicity in undifferentiated HL60 cells. In differentiated HL60 cells, only hemin was cytotoxic. Furthermore, protein expression of enzymes involved in defense against superoxide radicals and hydrogen peroxide degradation increased with HL60 cell differentiation. Accordingly, the cellular glutathione pool, which represents the non-enzymatic antioxidative defense system, increased in parallel with HL60 cell differentiation. Hence, the resistance of differentiated HL60 cells is associated with the development of heme metabolism and of the antioxidative defense system including the cellular glutathione pool.
The observational case-control study aimed to identify possible risk factors for the development of neutropenia associated with metamizole use. Therefore, 48 patients with metamizole-associated neutropenia treated at the University Hospitals Basel and Bern (2005-2017) were characterized and compared with 39 tolerant controls who took metamizole for at least 28 days perpetually without developing neutropenia. Neutropenia patients were subdivided into inpatient or outpatient cases who had developed neutropenia after metamizole treatment in the hospital or at home, respectively. Hence, outpatient cases were compared in more detail with tolerant control patients who had also received metamizole in an outpatient treatment setting. Due to the similar treatment circumstances, it was possible to analyze risk factors in a regression-based model. The main finding of this study was the increased frequency of acute infections among neutropenia patients before neutropenia diagnosis compared to tolerant control patients. There was no association between the development of neutropenia after metamizole treatment and nonmyelotoxic and non-immunosuppressive co-medication (p=0.6627), history of drug allergy (p=0.1304), or preexisting auto-immune diseases (p=0.2313). Thus, acute infections may increase the risk to develop neutropenia during metamizole treatment. But since it cannot be distinguished for all case patients, whether this is a consequence of or a risk factor for neutropenia, further investigation is needed.
In conclusion, the main metamizole metabolite MAA can form an electrophilic intermediate in presence of hemin and potentially also in presence of other highly oxidative compounds. Thus, weak antioxidative defense, low heme-metabolizing capacity, or increased generation of free heme or other highly oxidative compounds, as it might occur during infections, may render cells more susceptible to MAA toxicity and therefore facilitate neutropenia development.