Studies on the mode of action of the antitumour acridine 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA)

Abstract

1. The mode of action of a novel antitumor acridine 4’-(9-acridinylamino)-methansulphon-m-anisidide (m-AMSA) has been investigated. Two congeners of m-AMSA, 4’-(9-acridinylamino)methanesulphonanilide (AMSA) and 4’-(9-acridinylamino)methanesulphon-o-anisidide (o-AMSA) were also studied for comparative purposes. M-AMSA is being evaluated clinically at present under the auspices of the National Cancer Institute, U.S.A. 2. Treatment of mice bearing the mast cell tumour, mastocytoma P815, with m-AMSA provided some complete cures. AMSA was almost as effective as m-AMSA but its dose potency was 6-fold lower. o-AMSA was inactive. 3. m-AMSA was found to have a short half-life in mice. Loss of m-AMSA from plasma was due, in part, to the formation of highly fluorescent covalent protein adducts. The rate of this reaction was similar for all three AMSA congeners, and is probably not required for antitumour activity. 4. The reaction of AMSA drugs with proteins was shown to occur by nucleophilic displacement of the methanesulphonanilide moiety. Evidence is presented that the thiols are the predominant reaction centres in proteins. 5. A tissue culture model for the antitumour action of m-AMSA was developed using mastocytoma P815 cells. Profound growth inhibition and eventual killing was demonstrated using drug concentrations and durations of exposure attainable in mice. The potencies of the three AMSA congeners paralleled their antitumour potencies in vivo, except at very high drug concentrations. 6. The rates of biotransformation of AMSA drugs, and their extent of uptake by cells in culture, could not account for the differing potencies of the three AMSA congeners. 7. M-AMSA prevented the progression of mastocytoma cells through the cell division cycle under conditions where net cell growth was unaffected. Physiologically attainable drug concentrations inhibited chromosome condensation in cells which were less than 10 minutes from the G2-phase/mitosis boundary at the time of drug addition. The sedimentation rate on alkaline sucrose gradients of DNA from cells treated with m-AMSA was lower than that from untreated cells, suggesting that this agent may cause fragmentation of DNA. 8. A new method for the investigation of the cell cycle stage selectivity of antitumour drugs was developed. This technique demonstrated that m-AMSA and AMSA have significant cycle stage selectivity, the growth of cells late in cycle being most affected. 9. Spectrophotometric determinations of binding parameters for the interaction of AMSA derivatives with native DNA indicated that m-AMSA bound with lower affinity that did o-AMSA or AMSA. This conclusion was supported by the helix stabilization caused by these drugs. However, the association constants were sufficiently high for each derivative to ensure that essentially all intracellular drug available for binding to DNA would be bound. 10. The ration of the association constant for native DNA to that for a single-stranded viral RNA was higher for m-AMSA than for AMSA. This selectivity, if operative in vivo, could account for the high dose potency of m-AMSA.