INVITRO BINDING OF METABOLICALLY ACTIVATED [LEDAKRIN-C-14, OR 1-NITRO-9-C-14-(3'-DIMETHYLAMINO-N-PROPYLAMINO) ACRIDINE, A NEW ANTI-TUMOR AND DNA CROSS-LINKING AGENT, TO MACROMOLECULES OF SUBCELLULAR-FRACTIONS ISOLATED FROM RAT-LIVER AND HELA-CELLS

A new antitumor drug named Ledakrin or Nitracrine, 1-nitro-9-(3′-dimethylamino-n-ropylamino)acridine, which has been shown to be a latent DNA cross-linking agent in both mammalian and bacterial cells, was investigated to determine whether it irreversibly binds to cellular macromolecules in vitro. Incubation of [14C]-Ledakrin with subcellular fractions of either rat liver or HeLa cells in the presence of a NADPH-regenerating system led to an irreversible binding of as much as about 30 per cent of the drug radioactivity (up to 57 nmoles/mg protein) with subcellular macromolecules after exhaustive extraction with cold trichloroacetic acid, ethanol and ether. The binding seems to be covalent. The difference between irreversible binding in the presence of intact and heat-inactivated enzymatic subcellular fractions indicates that the metabolites of the drug, rather than Ledakrin itself, are responsible for the irreversible binding with macromolecules in vitro. The dependence of the macromolecule binding of Ledakrin radioactivity with subcellular macromolecules of post-mitochondrial or microsomes on microsomal enzyme, on substrate concentration, oxygen and NADPH, as well as induction of this reaction with phenobarbital or 3-methylcholanthrene rat pretreatment, indicates that the oxidative macromolecule binding of Ledakrin metabolites is catalyzed in vitro by mixed-function oxidases, probably by the unspecific drug metabolizing system involving cytochrome P-450 of liver microsomes. Irreversible binding in vitro was less pronounced under anaerobic conditions than in incubations under air. The reductive irreversible macromolecule binding of Ledakrin metabolites is catalyzed in vitro by unknown rat liver enzymes resistant to allopurinol or dicoumarol inhibition. To account for oxidative binding of Ledakrin through a metabolic activation in vitro, three pathways are considered likely: (1) C-hydroxylation; (2) N-alkylhydroxylamine formation and (3) aromatic N-hydroxylation. The elevated oxidative macromolecule binding of Ledakrin metabolites when an epoxide hydrase was inhibited is evidence for the formation of a reactive acridine epoxide intermediate during the drug binding reaction. The ineffectiveness of SKF 525-A, a specific inhibitor of microsomal C-oxidation, in decreasing the irreversible binding is indirect evidence that besides microsomal C-oxidation other oxidative activations are involved. Arylhydroxylamines formed under air in vitro with all subcellular fractions studied, as determined colorimetrically. An aliphatic N-hydroxylation of the amino group of Ledakrin side chain can be involved in the drug oxidative binding in vitro too. To account for irreversible macromolecule binding of Ledakrin metabolites under highly anaerobic conditions, a N-arylhydroxylamine arising from the nitro group reduction seems to be an intermediate determined colorimetrically. Moreover, the metabolism of the nitro group of Ledakrin to its parent 1-N-hydroxylamine was directly related to the irreversible binding of the drug metabolite(s) with subcellular macromolecules in vitro under nitrogen. Reduced glutathione trapped in vitro reactive electrophilic Ledakrin metabolite(s) formed most probably by establishing a chemically stable thioether bond and thereby protected macromolecules against irreversible binding. Finally, four reactive species are postulated in the irreversible macromolecule binding of Ledakrin metabolites in vitro. © 1979.