Predicting drug-induced agranulocytosis: characterizing neutrophil-generated metabolites of a model compound, DMP 406, and assessing the relevance of an in vitro apoptosis assay for identifying drugs that may cause agranulocytosis

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Abstract

DMP 406 is a clozapine analogue developed by Dupont-Pharma for the treatment of schizophrenia. Unfortunately it caused agranulocytosis in dogs during preclinical studies. Clozapine also causes agranulocytosis and this is believed to be due to a reactive nitrenium ion metabolite produced by neutrophils. We studied the oxidation of DMP 406 by activated neutrophils and found that the major reactive species that is produced is not a nitrenium ion but rather an imine. This metabolite is similar to the reactive metabolite that has been proposed to be responsible for mianserin-induced agranulocytosis. Therefore we also studied the oxidation of mianserin by activated neutrophils and found that, although the major species is an iminium ion, it also bears a lactam moiety in the piperazine ring resulting from further oxidation. We usually find that HOCl is a good model system for the production of reactive metabolites of drugs that are formed by activated neutrophils, but in the case of both DMP 406 and mianserin, the products produced were significantly different than those formed by activated neutrophils. In contrast, the combination of horseradish peroxidase and hydrogen peroxide (HRP/H2O2) formed a very similar pattern of products, and this system was used to produce sufficient quantities of metabolites to allow for identification. The reactive metabolites of both DMP 406 and mianserin reacted with a range of nucleophiles, but in many cases the reaction was reversible. The best nucleophile for trapping these reactive metabolites was cyanide. It has been demonstrated that the products of clozapine oxidation by HRP/H2O2, presumably the nitrenium ion, induced apoptosis in neutrophils at therapeutic concentrations of clozapine. It has been suggested that this process is involved in the mechanism of clozapine-induced agranulocytosis. We tested DMP 406 and mianserin in this system to see if the ability of a reactive metabolite of a drug to cause apoptosis could predict the ability of that drug to cause agranulocytosis. We used clozapine as a positive control and we also tested olanzapine, a drug that forms a reactive metabolite similar to that of clozapine but is given at a lower dose and does not cause agranulocytosis. We found that DMP 406 did not increase apoptosis at concentrations below 50 μM, and although mianserin did increase apoptosis at 10 μM this is above the therapeutic concentration. Olanzapine caused an increase in apoptosis at the same concentration as clozapine (1 μM), but because its therapeutic concentration is lower, this concentration was above the pharmacological range. There was no increase in apoptosis with any drug in the absence of HRP/H2O2. These results indicate that this assay is unable to reliably predict the ability of different types of drugs to cause agranulocytosis. This is not a surprising result given that different drugs may induce agranulocytosis by different mechanisms.

Introduction

The atypical antipsychotics, clozapine and olanzapine (Fig. 1), do not usually cause the disabling motor side effects that are commonly associated with the conventional neuroleptic drugs. As such, these drugs are increasingly being used as first-line treatment of schizophrenia [1]. Clozapine is arguably the most effective atypical antipsychotic to date; however, its use is often superceded by other atypical agents, such as olanzapine, due to its propensity to cause idiosyncratic agranulocytosis in approximately 0.5% of patients [2], [3]. Agranulocytosis is a life-threatening syndrome that is defined by a neutrophil count of <500 cells/μl blood. Although olanzapine has a similar chemical structure, no clear-cut cases of agranulocytosis have been seen even though several million patients have been treated with the drug [4], [5].

The mechanism of drug-induced agranulocytosis is unknown. It is generally believed that most idiosyncratic drug reactions are due to reactive metabolites that act as haptens and induce an immune response [6], [7]. In view of the fact that the target of agranulocytosis is neutrophils or neutrophil precursors in the bone marrow, it is likely that the reactive metabolite must be formed in these cells. Clozapine is oxidized to a reactive species by activated neutrophils, and it is likely that this reactive metabolite is responsible for clozapine-induced agranulocytosis (Fig. 2). When neutrophils are activated, significant quantities of hydrogen peroxide and myeloperoxidase are released [8]. Peroxidases are known to catalyze one-electron oxidations of aromatic amines, leading to free radicals [9], [10]. In addition, myeloperoxidase, along with the cofactors hydrogen peroxide and chloride ion, produce hypochlorous acid-a powerful 2-electron oxidant (Fig. 2) [8].

Most of the characteristics of clozapine-induced agranulocytosis suggest that it is immune-mediated. However, we failed to find evidence of T cells that recognized clozapine-modified neutrophils or their bone marrow precursors from patients with a recent history of clozapine-induced agranulocytosis [11]. More importantly, when patients with a history of clozapine-induced agranulocytosis were retreated with the drug, it took just as long (about 6 weeks) before the onset of agranulocytosis [12]. Such a long delay is inconsistent with an amnestic response of the immune system. Although this does not preclude an immune-mediated reaction, it makes this hypothesis less attractive [6]. Alternatively, clozapine-induced agranulocytosis could be due to direct toxicity. This is supported by the in vitro data of Williams et al. who found that the reactive metabolite of clozapine accelerated the apoptosis of neutrophils [13]. However, it is not clear that the conditions of such experiments occur in vivo nor how drug-induced apoptosis would lead to a 6 week delay between the start of clozapine therapy and the onset of agranulocytosis. In short, the characteristics of clozapine-induced agranulocytosis do not easily fit either an immune-mediated or direct toxicity model. A valid animal model would be extremely useful for the study of drug-induced agranulocytosis. Unfortunately, the syndrome is just as idiosyncratic in animals as it is in people; therefore, it is very difficult to find such a model.

Dupont-Pharma discovered DMP 406 (Fig. 1), a chemical and pharmacological analogue of clozapine and olanzapine that had potential as an antipsychotic agent. A three-month preclinical toxicity study in beagles revealed DMP 406-induced agranulocytosis in 4/7 female beagles [14]. Male dogs given the same dose developed only mild bone marrow changes but no significant changes in peripheral neutrophil counts and this makes it appear idiosyncratic in nature. Although the development of this drug was discontinued at Dupont-Pharma, the syndrome that it caused in beagles provided us with a possible animal model in which to study idiosyncratic drug-induced agranulocytosis. We attempted to reproduce this model in a small pilot study where two female beagles were dosed with 30 mg/kg/day of DMP 406 over a 14 week period. Weekly complete blood cell counts, differentials and monthly bone marrow biopsies were performed. Similar to the effects of high doses of clozapine in rats (unpublished data), the peripheral blood neutrophil counts in our DMP 406-treated beagles remained within the normal range despite the appearance of a slight left-shifted, mildly hyperplastic bone marrow in both animals (unpublished data). The limited availability of DMP 406 prevented us from proceeding to studies involving large numbers of animals.

Although we were not able to easily use the agent for an animal model, the structural similarities between DMP 406, clozapine and olanzapine compelled us to characterize the neutrophil-mediated oxidation of DMP 406. Preliminary studies indicated that the metabolites were significantly different than those of clozapine and more like those proposed for mianserin (Fig. 1), another drug associated with agranulocytosis. This led us to further study the neutrophil oxidation of mianserin. We then compared the ability of the reactive metabolites of each of these drugs to induce neutrophil apoptosis to determine if their potency in this respect would predict the parent drug's risk of causing agranulocytosis analogous to the previous experiments with clozapine.

Section snippets

Chemicals

DMP406, clozapine and olanzapine were generous gifts from Dupont-Pharma (Wilmington, DW), Novartis (Basel, Switzerland) and Eli Lilly (Indianapolis, IN), respectively. Horseradish peroxidase (HRP, Type VI-A), potassium cyanide, glutathione (GSH), hypochlorous acid (HOCl), bovine serum albumin (BSA), mianserin and phorbol 12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO). Sterile phosphate buffered saline (PBS) and Hanks’ Balanced Salt Solution (HBSS) were prepared

Oxidation of DMP 406

Preliminary oxidation experiments, using HOCl or HRP/H2O2 as the oxidizing agent, showed that DMP 406 (MH+=m/z 322) underwent the equivalent of a two-electron oxidation to yield a product with a MH+ of m/z 320 and hydrates with a MH+ of m/z 338. During adduct isolation when methanol was used as a solvent, a methoxy adduct was also detected (MH+=m/z 352). Mass spectral analyses of incubations that contained GSH revealed the presence of GSH adduct species (MH+=m/z 627). GSH was either present

Discussion

Dupont-Pharma discovered that its candidate drug, DMP 406, caused agranulocytosis in beagles that appeared to be idiosyncratic in that only 4 of 7 beagles developed the syndrome. Since it is believed that reactive metabolites of drugs are involved in the pathogenic mechanism of drug-induced idiosyncratic drug reactions including agranulocytosis, the neutrophil-mediated oxidation of DMP 406 was investigated. The neutrophil-generated metabolites of DMP 406 were compared to those of the chemically

Acknowledgements

This research was supported by a grant from the Canadian Institutes of Medical Research. J.P. Uetrecht would like to acknowledge the support given by a Canadian Reserve Chair in Adverse Drug Reactions. We would also like to thank Bruce Car of DuPont-Pharma for his advice in attempting to develop the dog model.

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