In vitro activity of the novel indoloquinone EO-9 and the influence of pH on cytotoxicity.

The novel indoloquinone compound EO-9 is shortly to undergo phase I clinical evaluation as a potential bioreductive drug. Preclinical studies have shown that EO-9 has greater activity against cells derived from human solid tumours than leukaemias in vitro. The results of this study extend the preclinical data available on EO-9 by demonstrating that EO-9 induces a broad spectrum of activity (IC50 values range from 8 to 590 ng ml-1) against a panel of human and murine tumour cell lines. Some evidence exists of selectivity towards leukaemia and human colon cell lines as opposed to murine colon cells. The response of cells to Mitomycin C were not comparable to EO-9 suggesting that the mechanism of action of these compounds is different. The cytotoxic properties of EO-9 under aerobic conditions are significantly influenced by extracellular pH. Reduction of pH from 7.4 to 5.8 increases cell kill from 40% to 95% in DLD-1 cells. In addition, EO-9 is unstable at acidic pH (T1/2 = 37 min at pH 5.5) compared to neutral pH T1/2 = 6.3 h). The major breakdown product in vitro was identified as EO-5A which proved relatively inactive compared to EO-9 (IC50 = 50 and 0.6 ug ml-1 respectively). These studies suggest that if EO-9 can be delivered to regions of low pH within solid tumours, a therapeutic advantage may be obtained.

Conjugation of model substrates or microsomally-activated aflatoxin B1 with reduced glutathione, catalysed by cytosolic glutathione-S-transferases in livers of rats, mice and guinea pigs.

Glutathione-S-transferase (GST) activity has been examined in liver cytosol fractions from guinea pigs, mice, control fed rats or rats with pre-neoplastic nodular liver lesions. The levels of activity in unfractionated cytosols have been assayed using the model substrates 1-chloro-2,4-dinitrobenzene (CDNB), 3,4-dichloronitrobenzene (DCNB) and monobromobimane (mBrB) with reduced glutathione (GSH). The order of activities in the various liver fractions using CDNB as substrate were: mouse greater than pre-neoplastic nodular rat greater than guinea pig greater than control rat and paralleled the capacities of the cytosols to catalyse the formation of aflatoxin B1-GSH from microsomally-activated aflatoxin B1 (AFB1) and GSH. Quantitative differences between the activities of the cytosols using the three model substrates were observed. In the mouse fractionation of GST activity by isoelectric focusing (I.E.F.) on preparative granular gels showed that the most basic component (isoelectric point pH 9.4) with the highest conjugating activity with respect to microsomally-activated AFB1 did not correspond with the peak of most activity for conjugating CDNB. In the pre-neoplastic nodular rat liver the CDNB conjugating activities of all fractions separated on granular I.E.F. gels, were higher than the corresponding fractions isolated from control rat liver, with particular enhancement of the peak containing the 3:3 isoenzyme. In contrast to control rat liver the 7:7 isoenzyme was detected in pre-neoplastic nodular liver preparations. These isoenzymes (3:3 and 7:7) did not contribute significantly to the enhanced level of AFB1-GSH formation catalysed by cytosol fractions prepared from pre-neoplastic nodular rat liver. The microsomally-activated AFB1-conjugating activity of unfractionated rat liver cytosols was increased to a relatively greater extent than CDNB conjugating activity during the induction of pre-neoplastic nodular liver lesions, and the elevated level of the activated AFB1-conjugating activity was found to be associated with the most basic fraction (isoelectric point pH 9.0). Analytical isoelectric focusing gels using mBrB as substrate demonstrated the presence of a basic GST isoenzyme in the pre-neoplastic nodular rat liver, not detected in preparations from the livers of control rats. The low level of activated AFB1-conjugating activity present in unfractionated guinea-pig cytosol was found to correspond with the fraction containing the peak of CDNB conjugating activity on preparative isoelectric focusing (isoelectric point pH 7.5). The lack of correlation between the conjugation of model substrates and the conjugation of xenobiotics could be of import

Chemical rearrangement of phenol-epoxide metabolites of polycyclic aromatic hydrocarbons to quinone-methides.

Evidence of the involvement of triol-epoxide and phenol-epoxide metabolites in the metabolic activation of polycyclic hydrocarbons is accumulating. It is proposed that the phenolic OH-groups present in such epoxides will activate the epoxide moieties and permit their rearrangement to quinone-methides. These quinone-methides are highly reactive, potentially-isolable chemical entities with strong alkylating activity. In one resonance form they are resonance-stabilized carbonium ions. Only epoxides that also possess phenolic OH-groups in certain positions will form quinone-methides: these appear to include 9-hydroxybenzo [a] pyrene 4,5-oxide and the triol-epoxides 9-hydroxy-trans-1,2-dihydro-1,2-dihydroxychrysene 3,4-oxide and 2-hydroxy-trans-9,10-dihydro-9,10-dihydroxybenzo[a]pyrene 7,8-oxide.

Genetic activity spectra of some antischistosomal compounds, with particular emphasis on thioxanthenones and benzothiopyranoindazoles.

In this review we note that hycanthone (Etrenol) is mutagenic for bacteriophage, bacteria, yeast, Neurospora, Drosophila, and for mammalian tissue culture cells, and we point out other genetic activities of this thioxanthenone and of related compounds. One alarming genetic activity is the ability of hycanthone to cause transformation of tissue culture cells in vitro in a test designed to detect carcinogens, results that parallel the direct demonstration of carcinogenic activity of hycanthone in the mouse in vivo. These and other results are compatible with the somatic mutation theory of cancer induction. Factors likely to affect the quantitative genetic activity of hycanthone and its congeners are summarized. Attempts are made to weave the more critical experimental evidence into a molecular model that accounts for the genetic activities of this series of compounds. We conclude that hycanthone is a directly acting mutagen that intercalates into DNA and preferentially alkylates deoxyguanosine residues via formation of a strongly electrophilic molecular species, the carbonium ion. Finally, we show that genetic activity can be dissociated from schistosomicidal activity by appropriate modifications in the thioxanthenone molecule. Preliminary experiments on a newly synthesized piperazinyl N-oxide derivative demonstrate no detectable mutagenic activity; yet considerable schistosomicidal activity is retained.

Microsomal activation to mutagens of antischistosomal methyl thioxanthenones and initial tests on a possibly non-mutagenic analogue.

Five methylthioxanthenone and methylbenzothiopyranoindazole analogues, including lucanthone (Miracil D), are non-mutagenic for Salmonella typhimurium but are activated to mutagens by a rat liver microsome preparation. Hydroxymethyl analogues, including hycathone (Etrenol), are mutagenic in the absence of microsomes. It seems reasonable to assume that the hydroxymethyl derivatives are the more proximal mutagens and that Salmonella is unable to carry out the hydroxylation necessary for mutagen activation. During the pase 24 years, several million patients with schistosomiasis have been treated with lucanthone, and in recent years about 700 000 persons with hycanthone. The possible long-term deleterious effects of these agents for man even now remain to be determined. Our studies indicate that particular modifications in the structure of thioxanthenones drastically alter their mutagenicity. One apparently non-mutagenic thioxanthenone has been found. A number of the less mutagenic compounds also exhibit decreased acute toxicity in the mouse while retaining appreciable antischistosomal activity, suggesting that genetic and schistosomicidal activities may be dissociated from each other.

Hycanthone analogs: dissociation of mutagenic effects from antischistosomal effects.

N-Oxidation at the diethylamino group of hycanthone, of lucanthone, and of two chlorobenzothiopyranoindazoles resulted in a marked reduction in mutagenic activity, while antischistosomal activity was retained or even enhanced. Introduction of chlorine into the 8-position of benzothiopyranoindazoles reduced acute toxicity but had no effect on chemnotherapeutic potency. These dissociations of biological activities indicate that safer antischistosomal compounds of this class can be developed.