The mechanism of drug-induced agranulocytosis is unknown, but is believed to have many components, with reactive metabolites being a major one. We previously have determined that aminoglutethimide and procainamide, both of which contain aniline substructures, produce protein free radicals upon oxidation by myeloperoxidase. In addition, it was found that both drugs generated a phenyl radical metabolite. The relationship between the phenyl radical metabolite and protein radical formation is unknown. Moreover, the capacity for aniline compounds to induce MPO• formation has also not been known previously. We thus undertook this study to identify the physico-chemical parameters that may be involved in protein free radical formation, and to determine whether phenyl radical metabolites could be implicated.
We have now derived QSARs that demonstrate a relationship between two electronic parameters (σ, IP-P) of aniline compounds and their EC2
values for protein free radical formation. Aniline lipophilicity was not related, suggesting that hydrophobic binding is not important for protein free radical formation. It has been shown previously that σ was inversely related to the rate constants for HRP Compound I reduction (Job and Dunford, 1976
) We derived an equation from this data but instead of the ratio of substituted aniline to aniline, we simply used the log of the rate constant (k
X) and correlated this with σ:
Simply, this equation shows that anilines which reduce HRP Compound I the fastest (i.e., the best HRP substrates) have more negative values for σ. In our study, the derived QSAR equations showed a similar relationship, such that lower EC2
values (more potent MPO•
inducers) were obtained from anilines that had more negative σ values. This implies that better substrates were better at generating MPO free radical formation.
In our experiments, however, we found that there were two groups of substrates that did not induce MPO free radical formation. One group, p-anisidine and 3,4-, and 2,4-dimethoxyaniline, are highly efficient donor substrates for peroxidases; p-anisidine has a σ = −0.28, 3,4-dimethoxyaniline σ = −0.17, and 2,4-dimethoxyaniline σ = −0.28. Interestingly, ptoluidine (σ = −0.14) and aminoglutethimide (σ = −0.15) both induced MPO free radical formation with great efficacy. This suggests that MPO• formation is not favoured by the most highly efficient peroxidase substrates. On the other hand, relatively inefficient peroxidase substrates appeared to be capable of generating MPO•, e.g., dichloroanilines and nitroanilines. However, they did so with much less efficacy as indicated by their relatively high EC2 values. Electronic factors may not solely be responsible for the low efficacy of MPO• generation, since we could not determine EC2 values for 2- and 3-nitroaniline (σ = 1.72, 0.74, respectively) but we could for 4-nitroaniline (σ = 0.78). The proximity of the nitro group to the amine could prevent generation of a reactive metabolite that could oxidize MPO.
Our initial studies on drug induced MPO• formation involved the peroxidase metabolism of aminoglutethimide and procainamide, which are both aniline-based drugs. Having derived QSAR equations to describe the physicochemical relationship between aniline compounds and MPO formation, we wished to determine whether these equations would reasonably predict the EC2 values of the drugs. The drugs were not used to derive the QSAR equations and therefore represented a small test set. Interestingly, equations using σ did not accurately predict the EC2 for procainamide, but did so for aminoglutethimide. One reason for this disparity could be that the σ values for the drugs are not known (we used σ values from structurally similar para substituents). Another reason may be that the group in the para position for procainamide is electron withdrawing (-CONH2CH2R, σ=0.36), and would be predicted to be a weak protein radical inducer, and a relatively poor peroxidase substrate. However, when the QSAR equation using IP was applied (Eqn. 6 & 7), a significantly better prediction was obtained. This suggests that σ may not be the best predictor of the ability to induce MPO• formation.
Identification of the presumed free radical metabolite that is responsible for generating the MPO• is technically challenging because it is difficult to differentiate the effects of the nitrogen-centered cation radical from those of the carbon-centered phenyl radical. Since we previously determined that aminoglutethimide and procainamide formed phenyl radicals, we wished to investigate whether the anilines used in this study also formed phenyl radicals. In addition, we wished to determine the relationship between the formation of a phenyl radical metabolites and MPO•. With two exceptions out of 26 aniline derivatives, it appeared that there was a qualitative relationship between phenyl radical metabolite formation and MPO•. More importantly, the aniline compounds for which we were unable to detect phenyl radicals did not form MPO•. This suggests that the phenyl radical is the metabolite responsible for MPO• formation.
The mechanisms of drug induced agranulocytosis are still unresolved, and are considered to be multifactoral, i.e., a particular alignment of many parameters must be present for the adverse drug reaction to occur. In the absence of suitable models, investigations of such parameters have been hindered. It has been proposed for some time that leukocyte (myeloperoxidase)-generated reactive metabolites may be involved in the etiology of drug-induced agranulocytosis (Uetrecht, 1989a
); a role for myeloperoxidase-generated free radical metabolites has also been proposed (Fischer et al., 1991
). We have focused on the physicochemical side of these reactions by evaluating the tendency of aromatic amines that generate phenyl radical metabolites to induce MPO•
. This study established that the substitution on the aniline ring can determine the outcome of protein free radical formation on MPO. In the future, this research needs to establish the significance of MPO•
in the etiology of agranulocytosis and identify potential in vivo pathways of inducing and altering this reaction.