Drug-protein conjugates--XIV. Mechanisms of formation of protein-arylating intermediates from amodiaquine, a myelotoxin and hepatotoxin in man.

The enzymic and non-enzymic formation of protein-arylating intermediates from amodiaquine (AQ,7-chloro-4-(3'-diethylamino-4'-hydroxyanilino) quinoline), an anti-malarial associated with agranulocytosis and liver damage in man, was studied in vitro. [14C]AQ in phosphate buffer, pH 7.4, under air was autoxidized to a reactive derivative(s) which possessed characteristics indicative of a semiquinone/quinone imine: reduction by NADPH and ascorbic acid, conjugation with thiols and irreversible binding to microsomal and soluble proteins. Cysteinyl SH groups were major sites of arylation. Radiolabelled material irreversibly bound to HSA after 24 hr and to human liver microsomes after 4 hr represented 26.5 +/- 1.8% and 31.4 +/- 0.6% (means +/- SD, N = 3) of incubated [14C]AQ (10 microM), respectively. The quinone imine of AQ(AQQI) was synthesized, and displayed the same oxidative and electrophilic reactions as the product(s) of AQ's autoxidation. A water-soluble product formed in buffered solutions of AQ and N-acetylcysteine was identified as an AQ mercapturate by comparison with an adduct prepared from synthetic AQQI. Irreversible binding of [14C]AQ was inhibited by a radical scavenger; this indicated that the semiquinone imine contributed to the binding. Although AQ was extensively de-ethylated by human liver microsomes, oxidation by cytochrome P-450 did not appear to be principally responsible for its activation and irreversible binding in microsomal incubations. AQ was oxidized to protein-arylating intermediates by horseradish peroxidase. It also formed reactive derivatives, possibly N-chloro compounds, in chlorine solutions. These findings indicated that AQ can give rise to chemically reactive species by at least three distinct mechanisms, viz. autoxidation in neutral solution under air, peroxidase-catalyzed oxidation and N-chlorination. Formation of such species in liver and myeloid cells might be responsible for the adverse reactions associated with AQ.