Antitumour imidazotetrazines--IV. An investigation into the mechanism of antitumour activity of a novel and potent antitumour agent, mitozolomide (CCRG 81010, M & B 39565; NSC 353451)

8-Carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4-(3H )-one- mitozolomide (CCRG 81010, M & B 39565, NSC 353451) is a potent inhibitor of the growth of a number of experimental tumours and can potentially decompose to give either an isocyanate or the monochloroethyltriazene (MCTIC). In vitro CCRG 81010 is not cross-resistant with the bifunctional alkylating agents against the Walker carcinoma. To investigate the mechanism of the antitumour activity of CCRG 81010 a comparison has been made with BCNU and MCTIC on precursor incorporation into macromolecules in TLX5 mouse lymphoma cells. Whereas BCNU produces a rapid and extensive inhibition of both (methyl 3H) thymidine and [5-3H]uridine incorporation into acid-insoluble material, neither CCRG 81010 or MCTIC have an early effect on precursor incorporation. Inhibition of precursor uptake is also not produced by concentrations of 2-chloroethylisocyanate that inhibit intracellular glutathione reductase activity. The potential carbamoylating activity of CCRG 81010 has also been assessed by comparing its effect with that of BCNU and 2-chloroethyl isocyanate on enzymes known to be inhibited by carbamoylation. Such enzymes, glutathione reductase, chymotrypsin and gamma-glutamyltranspepidase are not inhibited by CCRG 81010 under conditions where BCNU and 2-chloroethyl isocyanate show complete inhibition of enzyme activity, suggesting an absence of carbamoylating species. The results suggest that the most likely antitumour metabonate produced from CCRG 81010 is the triazene MCTIC.     

Novel DNA groove binding alkylators: design, synthesis, and biological evaluation

A group of oligopeptides has been synthesized that are structurally related to the natural antiviral antitumor agents netropsin and distamycin but which bear alkylating functions. Cytostatic activity against both human and murine tumor cell lines as well as their in vitro activity against a range of viruses is reported. The biological activity of these agents is discussed both in terms of their alkylating reactivity and of their structural differences. The incorporation of alkylating moieties into these minor groove DNA binders results in up to 45-fold increase in cytostatic activity compared with netropsin and up to 18 times the activity of distamycin.     

The role of isocyanates in the toxicity of antitumour haloalkylnitrosoureas

The cytotoxicity of the antitumour nitrosoureas BCNU and CCNU and the isocyanates which they liberate (chloroethylisocyanate and cyclohexylisocyanate respectively) has been measured utilising an in vitro-in vivo bioassay. Lines of the TLX5 lymphoma and L1210 leukaemia were used which were either sensitive or resistant to nitrosoureas in vivo. An estimated logarithmic cell kill produced by each compound in vitro (before injecting the cells into animals) was calculated by reference to assays of the survival time of animals given from 2 × 10⁵ to 2 × 10⁰ cells of each line. Resistance to both BCNU and CCNU was observed in vitro in the cell lines of the TLX5 lymphoma made resistant to either BCNU or a dimethyltriazene in vivo. The latter tumour was cross-resistant in vivo to nitrosoureas. Resistance in vitro to nitrosoureas was also observed in a line of L1210 leukaemia which had had resistance to BCNU induced in vivo. The nitrosourea resistant TLX5 lymphomas were cross-resistant in vitro to both cyclohexylisocyanate and chloroethylisocyanate whereas the nitrosourea resistant L1210 line showed no cross-resistance to cyclohexylisocyanate and marginal cross-resistance to chloroethylisocyanate. The results suggest that the TLX5 lymphoma, which is naturally resistant to alkylating agents of the 2-chloroethylamine type, may be sensitive in vivo to nitrosoureas as a consequence of the intracellular release of isocyanates. This hypothesis was supported by the finding that the resistant TLX5 lymphoma showed no cross-resistance to other electrophilic agents, for example formaldehyde, monomethyltriazene or HN2. The transport of nitrosoureas into the sensitive and resistant cell lines was similar in profile and there was no difference in the concentration of non-protein thiols.     

Protection by Salvia extracts against oxidative and alkylation damage to DNA in human HCT15 and CO115 cells

DNA damage induced by oxidative and alkylating agents contributes to carcinogenesis, leading to possible mutations if replication proceeds without proper repair. However, some alkylating agents are used in cancer therapy due to their ability to induce DNA damage and subsequently apoptosis of tumor cells. In this study, the genotoxic effects of oxidative hydrogen peroxide (H?O?) and alkylating agents N-methyl-N-nitrosourea (MNU) and 1,3-bis-(2-chloroethyl)-1-nitosourea (BCNU) agents were examined in two colon cell lines (HCT15 and CO115). DNA damage was assessed by the comet assay with and without lesion-specific repair enzymes. Genotoxic agents were used for induction of DNA damage in both cell lines. Protective effects of extracts of three Salvia species, Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), against DNA damage induced by oxidative and alkylating agents were also determined. SO and SF protected against oxidative DNA damage in HCT15 cells. SO and SL decreased DNA damage induced by MNU in CO115 cells. In addition to chemopreventive effects of sage plant extracts, it was also important to know whether these plant extracts may interfere with alkylating agents such as BCNU used in cancer therapy, decreasing their efficacy. Our results showed that sage extracts tested and rosmarinic acid (RA), the main constituent, protected CO115 cells from DNA damage induced by BCNU. In HCT15 cells, only SF induced a reduction in BCNU-induced DNA damage. Sage water extracts and RA did not markedly change DNA repair protein expression in either cell line. Data showed that sage tea protected colon cells against oxidative and alkylating DNA damage and may also interfere with efficacy of alkylating agents used in cancer therapy.     

The effects of chlorambucil on the utilization of exogenous thymidine by tumour cells.

Treatment of an alkylating agent-sensitive strain of the Yoshida ascites sarcoma with chlorambucil resulted in an inhibition of the incorporation of [³H]thymidine into DNA, which could be overcome by incubating cells in high extracellular concentrations of thymidine. Increase in cellular DNA content and the dilution of specific radioactivity in pre-labelled DNA indicated that DNA synthesis was continuing at times when [³H]thymidine incorporation was inhibited. Uptake and phosphorylation of thymidine were not impaired by the treatment and the reduced incorporation of [³H]thymidine into DNA is attributable to a block in the utilization of TTP derived from exogenous nucleoside.     

Modulation of nitrosourea resistance in human colon cancer by O6-methylguanine.

Human colon cancer is resistant to a variety of alkylating agents including the nitrosoureas. To specifically evaluate nitrosourea resistance, we studied the role of O6-alkylguanine-DNA alkyltransferase (alkyltransferase) which is known to repair nitrosourea-induced cytotoxic DNA damage. Alkyltransferase activity varied over a similar wide range in 25 colon cancer biopsies and 14 colon cancer cell lines but the activity was not correlated with differentiation status, Dukes' classification or in vitro growth characteristics. 1,3-Bis-(2-chloroethyl)-1-nitrosourea (BCNU) resistance and alkyltransferase activity were highly correlated (R2 = 0.929, P less than 0.001) in 7 different colon cancer cell lines, suggesting that the alkyltransferase is an important component of nitrosourea resistance in colon cancer cells. In the BCNU-resistant, high alkyltransferase VACO 6 cell line, inactivation of the alkyltransferase by O6-methylguanine caused a proportional decrease in the BCNU IC50, consistent with that predicted by the regression line. Enzyme inactivation was also associated with a marked increase in DNA cross-link formation. Because alkyltransferase correlates with BCNU resistance in colon cancer, and resistance can be reversed by inactivating the protein, the alkyltransferase may have an important role in nitrosourea resistance in human colon cancer cells. These data provide the rationale for clinical trials in colon cancer with biochemical modulators of the alkyltransferase to increase the therapeutic response to nitrosoureas.     

Sequential inhibitory effects of antitumor agents related to levodopa and dopamine upon DNA synthetic enzymes

Novel antitumor agents related to levodopa and dopamine exhibit a selective and rapid inhibition of DNA synthesis as measured by thymidine incorporation. Our investigations have attempted to determine the biochemical basis of the selective inhibition of tumor cells and in this present study we examined the effects of these agents on thymidylate synthase. The dihydroxybenzene derivatives were found to inhibit thymidylate synthase in situ at concentrations ranging between 100 and 800 microM. The quinols did not inhibit partially purified thymidylate synthase, although the oxidized quinones did cause inhibition at concentrations between 10 and 100 microM. Time course experiments suggested that the inhibition of thymidylate synthase in situ by the dihydroxybenzene derivatives occurs after the inhibition of thymidine incorporation, indicating that an earlier event was critical to the inhibition of DNA synthesis. With the use of a novel in situ assay which measured the release of [3H]water from [5-3H] uridine in intact cells, we were able to show that one of the earliest biochemical events is the inhibition of ribonucleotide reductase and that the inhibition of thymidylate synthase, which is delayed by approximately 30 min, was indirectly mediated possibly through effects on ribonucleotide reductase.     

Studies on the protection by imidazoles against the cytotoxicity of the antitumour alkylating agents melphalan and CB 1954

The effect of two imidazoles, the purine precursor 4-amino-imidazole-5-carboxamide (AIC) and an aryl derivative 4-amino-2-phenylimidazole-5-carboxamide (2-phenyl AIC), which protect against the cytotoxicity of the monofunctional alkylating agent CB 1954, have been investigated as protectors against the cytotoxicity of melphalan, a difunctional alkylating agent. The effects of the imidazoles on the melphalan-induced depression of thymidine incorporation into Walker cells has been compared with the effect on the depression produced by CB 1954. In addition, the effects of 2-phenyl AIC alone on precursor incorporation have been studied. It has a rapid and potent depressant effect on adenine incorporation, a moderate depressant effect upon pyrimidine incorporation but no effect on leucine incorporation or upon the uptake of glucose into the Walker cell.     

The effect of the nucleoside transport inhibitor dipyridamole on the incorporation of [3H]thymidine in the rat

Dipyridamole is a non-specific inhibitor of nucleoside transport into mammalian cells. It is currently undergoing clinical evaluation in combination with various antimetabolites in an attempt to enhance the activity of these anticancer drugs by blocking the salvage of extracellular nucleosides, an important determinant of their cytotoxicity. In the present study, the effect of i.v. infusions of dipyridamole on [3H]thymidine incorporation into DNA has been examined in the anaesthetized rat. The tissues studied were bone marrow, gastrointestinal tract epithelium and the ascitic form of the Walker carcinosarcoma. Dipyridamole at 10 mg/kg, given over 3 hr, led to plasma levels of less than 5 microM and did not reduce [3H]thymidine incorporation into any of the tissues studied. At 40 mg/kg dipyridamole (plasma levels 10-15 microM) [3H]thymidine incorporation into the DNA of bone marrow and gastrointestinal tract epithelium was reduced to 20-30% of control values. Increasing the dose to 100 mg/kg did not lead to a further suppression of incorporation. Measurement of [3H]thymidine plasma pharmacokinetics and the intracellular distribution of tritium suggested that the inhibition of [3H]thymidine incorporation was due to reduced cellular uptake. In contrast to the effects on normal tissues, even at a lethal dose (200 mg/kg) dipyridamole did not significantly inhibit [3H]thymidine incorporation into Walker tumour cells. The levels of dipyridamole found in the ascitic fluid, at 100 mg/kg approximately half those in plasma, argue against a pharmacokinetic basis for this difference. Dipyridamole was found to bind extensively (97%) to rat plasma proteins, which may explain the discrepancy between the concentrations of dipyridamole required to inhibit nucleoside incorporation in vitro, in serum-free media, and those needed in vivo. From a comparison of the plasma levels of dipyridamole which cause an inhibition of [3H]thymidine incorporation in the rat with those which can be achieved safely in patients, it is concluded that dipyridamole is unlikely to markedly reduce nucleoside salvage in man.     

Effect of frentizole on mitogen-induced blastogenesis in human lymphocytes.

Frentizole is a new immunoregulatory agent developed as an alternative to cytotoxic agents. In human lymphocytes, Frentizole inhibits the response to various phytomitogens without decreasing cell viability. Frentizole (500 ng/ml) inhibited thymidine incorporation into DNA most effectively when added to lymphocyte cultures at the same time as the addition of the mitogen. Frentizole (500 ng/ml) markedly inhibited the response to Con A (% inhibition, corporation was dose dependent with 125 ng/ml of Frentizole sufficient to inhibit significantly the response of all three mitogens. Frentizole (62.5 ng/ml) maximally inhibited uridine incorporation, and inhibition of uridine incorporation was independent of the phytomitogen employed. Increasing concentrations of Frentizole were found to have no effect on thymidine or uridine incorporation into unstimulated normal lymphocytes.     

Studies on the mechanism of action of the tumour inhibitory triazenes

A series of imidazole and phenyl dialkyi triazenes were synthesized and investigated for their anti-cancer activity in experimental animals. Active triazenes had a broad spectrum of anti-tumour action and like bischloroethylnitrosourea (BCNU) were active against tumours not sensitive to conventional alkylating agents. It was confirmed that at least one N-methyl group was necessary for anti-cancer activity but there was no correlation between dealkylation by liver microsomes and activity since a diethyl triazene was readily dealkylated but not active. A factor appears to be present in normal cell cytoplasm which can detoxify triazenes but which is absent from tumours sensitive to these agents.     

Comparative chemical and biological studies of four prototype phosphoraziridine antineoplastic agents

The chemical alkylating activities of four prototype phosphoraziridine antineoplastic agents were compared with their biological effects on V-79 Chinese hamster lung fibroblasts. It was found that the chemical reactivity patterns correlate well with all of the biological parameters examined in this study, i.e. cytotoxicity, DNA synthesis, and production of alkali labile strand breaks. Specifically, the 2,2-dimethylaziridine derivatives (AB-132 and AB-163) showed higher initial activities reaching a plateau after a short reaction time in all of the systems used in this study while the unsubstituted aziridine derivatives (AB-100 and D-63) reacted more slowly but continued to exert their action in a linear fashion to produce greater overall effects. These findings are consistent with the conclusion that the difference between the time-dependent biological activities of these drugs closely follows the different chemical mechanisms of their alkylating reactions (SN1 vs SN2). The more rapid action and subsequent hydrolytic inactivation of the 2,2-dimethylphosphoraziridines as effective alkylators could be the basis of their lower hemopoietic toxicity compared to conventional alkylating agents including their own C-unsubstituted aziridine analogs. The much more rapid action of the 2,2-dimethylphosphoraziridines on DNA inside the cell may have some bearing on their radiation potentiating activity, but this aspect and the cholinesterase inhibitory activity of these agents (which may depend on phosphorylation) were not investigated in the present study.     

Inhibition of intracellular esterases by antitumour chloroethylnitrosoureas. Measurement by flow cytometry and correlation with molecular carbamoylation activity

Antitumour chloroethylnitrosoureas (Cnus) decompose in physiological conditions yielding alkylating species and organic isocyanates. While antitumour activity is mainly attributed to the alkylation of DNA, carbamoylation of intracellular proteins by isocyanates may also have pharmacological and toxicological relevance. We previously reported a novel dynamic flow cytoenzymological assay for esterase inhibition in intact murine cells by BCNU and related isocyanates, and proposed that this might form the basis of an assay for intracellular carbamoylation. We have now examined a wide range of Cnus, isocyanates, and alkylating agents for their ability to inhibit cellular esterases. BCNU, CCNU, their derived isocyanates, and the 4-OH metabolites of CCNU exhibited potent inhibitory activity (I50 values 5.5 x 10(-5)-7.3 x 10(-4) M). Chlorozotocin and GANU were relatively inactive (I50 much greater than 10(-2) M). ACNU, TCNU and the 2-OH metabolites of CCNU exhibited intermediate activity (I50 values 1.1 x 10(-3)-2.3 x 10(-2) M). Compounds not able to form isocyanates were essentially inactive. Poor membrane permeability was also implicated in the weak activity of chlorozotocin and GANU. There was overall a good correlation between esterase inhibition and chemical carbamoylating activity, but some particular differences were identified. We concluded that flow cytoenzymological assay appears to have the potential to provide useful measurement of intracellular protein carbamoylation by existing Cnus and novel derivatives, and also offers the advantage of cell subpopulation identification for in vivo evaluation of these agents.     

Lack of genotoxic and cytotoxic effects of the herbicide lenacil on mouse tumor cells and on some Salmonella typhimurium strains

The effects of 3-cyclohexyl-6,7-dihydro-1H-cyclopentapyrimidine-2,4(3H,5H)-dione (lenacil) on macromolecular synthesis, thymidilate synthetase activity, viability and cell cycle progression were studied using Friend leukemia (FL). P388 and Ehrlich ascites tumor cells in suspension, and its cytogenetic effects were studied in a Salmonella/mammalian microsome assay using both frameshift and base-substitution tester strains. At a concentration of 0.5 mmol/l lenacil inhibited 45 to 70% thymidine incorporation into DNA fraction, while incorporations of uridine into RNA and leucine into protein were less affected. Thymidilate synthetase activity in P388 cells as assayed by the release of tritiated water from 5-3H-deoxyuridine was inhibited by the compound to about 20%. Lenacil neither showed an in vivo inhibitory action on thymidine incorporation into acid-insoluble material in P388 cells, nor on thymidilate synthetase activity after a 24 or 48 h treatment. The compound did not change the melting temperature of isolated DNA. Studies of lenacil's effect on cell cycle kinetics of FL cells demonstrated that 48 h treatment increased the percentage of S-phase cells. Lenacil exerted a weak cytotoxic effect on FL cells. At concentrations above 0.1 mmol/l it inhibited cell growth the effect being nonlethal. Cytogenetic studies of lenacil revealed no indication of its mutagenicity against Salmonella typhimurium TA97, TA98, TA100 and TA102.     

The effects of nitrogen mustards on the proliferative and Ig synthetic response of human peripheral blood lymphocytes

Maximal inhibition of pokeweed mitogen-stimulated Ig production and [3H]thymidine incorporation was shown to occur when unfractionated human peripheral blood mononuclear cells were cultured with concentrations of the nitrogen mustards melphalan, mechlorethamine or chlorambucil in the 20-100-microM range, whereas concentrations of microsome-activated cyclophosphamide (A-Cy) in the 2-mM range were required for equivalent inhibition. Around 400 microM A-Cy, IgM secretion was not inhibited, but secretion of IgA and IgG was. The [3H]thymidine incorporation of enriched populations of both large and small B and T cells all showed about 20-50-fold greater sensitivity to melphalan than to A-Cy, despite a difference of only 6-fold in alkylating activity between these drugs. Large (250 micron 3) B and T cells were only marginally more sensitive to melphalan and A-Cy than small (210 micron 3) T and B cells. Kinetic studies showed that IgG and IgA secreted by day 7 could be maximally inhibited by melphalan added as late as day 3, and IgM synthesis as late as day 2. In contrast, inhibition of Ig production by A-Cy steadily declined after the first day, especially IgM, which was no longer inhibitable by A-Cy on day 3. Inhibition of cumulative Ig production did not occur when A-Cy or melphalan was added on day 5 or later. Cell recombination experiments performed with drug pulsed and untreated monocytes plus B cells and irradiated T cells showed that inhibition of [3H]thymidine or Ig production was most striking when monocytes + B cells (rather than T cells) were exposed to melphalan in the first 16 h. When A-Cy was used in the first 16 h, inhibition of Ig production was partial and inconsistent, and inhibition of monocytes + B cell or T cell [3H]thymidine incorporation was not evident. We conclude that the nitrogen mustards melphalan and A-Cy can inhibit pokeweed mitogen-stimulated DNA synthesis by human T or B cells and Ig production in vitro, but that their mechanisms of action differ.     

Molecular basis of the antineoplastic activity of 3'-amino-3'-deoxythymidine

3'-Amino-3'-deoxythymidine decreased the incorporation of [2-14C]thymidine into DNA of L1210 cells in vitro, and produced an accumulation of [2-14C]thymidine di- and triphosphate. The extent of these effects varied with the amount of recovery time after removal of 3'-amino-3'-deoxythymidine prior to addition of labeled thymidine. The distribution of radioactivity in the acid-soluble fraction derived from [3H]3'-amino-3'-deoxythymidine was as follows: 50% as 3'-amino-3'-deoxythymidine, 20% as the monophosphate, 10% as the diphosphate, and 20% as the triphosphate derivatives. No incorporation of [3H]3'-amino-3'-deoxythymidine into L1210 DNA could be detected. 3'-Amino-3'-deoxythymidine-5'-triphosphate is a competitive inhibitor against dTTP with a Ki of 3.3 microM, whereas the Km for dTTP was 8 microM using activated calf thymus DNA as the template and DNA polymerase-alpha. These data indicate that a major site of inhibition by 3'-amino-3'-deoxythymidine is inhibition of the DNA polymerase reaction.     

Mechanism of action of the nitrosoureas -- III. Reactions of bis-chloroethyl nitrosourea and bis-fluoroethyl nitrosourea with adenosine.

Previous papers in this series have provided evidence for the formation of haloethyl nucleoside derivatives from the interaction of the therapeutic nitrosoureas with cytidine and guanosine. Such derivatives could be important in explaining the cytotoxic action of bis-chloroethyl nitrosourea (BCNU), bis-fluoroethyl nitrosourea (BFNU), and related therapeutic agents. We now report the formation of 1-haloethyl adenosines from the reaction of BCNU and BFNU with adenosine. These 1-substituted haloethyl adenosines cyclize to form 1,N⁶-ethanoadenosine: 1-chloroethyladenosine with a half-life of 20 min in neutral aqueous solution at 37°, and 1-fluoroethyladenosine with a half-life of 20 hr under the same conditions. 1-Hydroxyethyladenosine is also a major product of the reaction of either BCNU or BFNU with adenosine, but it is not formed from the hydrolysis of either 1-haloethyladenosine. Accordingly, a reaction mechanism involving a cyclized nitrosourea derivative is proposed to explain the formation of this and other hydroxyethyl nucleosides.     

In vitro and in vivo studies of a promising antileukemic thymidine analogue, 5-hydroxymethyl-2' deoxyuridine

The toxicity and metabolism of a thymidine analogue, 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) were studied with human leukemia cells (HL-60) and with human platelets. 3 X 10(-5) M 5HmdUrd caused a 50% inhibition in the proliferation of HL-60 cells. The compound was hydrolyzed to 5-hydroxymethyluracil (5HmUra) by the enzyme thymidine phosphorylase (EC 2.4.2.4) present in leukemia cells; this catabolic product was non-toxic. The catabolism of 5HmdUrd by human platelet thymidine phosphorylase could be inhibited by 6-aminothymine. The toxicity of 5HmdUrd was effectively reversed by deoxycytidine and 5HmdUrd increased the incorporation of deoxycytidine into dCTP and DNA several fold. The two latter phenomena are explicable in terms of a feedback action to ribonucleotide reductase, resulting in deoxycytidylate starvation, which is a known effect of excess thymidine. We report here also our preliminary observations that 5HmdUrd is active against mouse leukemia in vivo.     

Antineoplastic agents. 1. Synthesis and antineoplastic activities of chloroethyl- and methylnitrosourea analogues of thymidine

A new class of chloroethyl- and methylnitrosourea analogues of thymidine, 5a,b, 6, 10, and 11, has been synthesized from the corresponding amino nucleosides, 2 and 7. The 3'-chloroethyl and 3'-methyl derivatives, 10 and 11, inhibited L1210 cell growth in culture (ED50 = 1.5 and 1.0 micrometer, respectively) more effectively than 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (ED50 = 4 micrometer) and the 5'-nitrosourea analogues. Neither the alkylating nor the carbamoylating activities of these compounds correlated with their biological activity.     

The mechanisms of the inhibitory effects of liver extract on lymphocyte proliferation. III. The effects of arginase on DNA polymerase activities

The incorporation of labeled precursors into DNA, RNA and protein in phytohaemagglutinin (PHA)-prestimulated human lymphocytes was maximally inhibited by liver extract (LEx) or arginase at 24 h. The activities of DNA polymerase alpha, beta and gamma were less inhibitable by these agents than was [3H]thymidine incorporation. The inhibition of DNA, RNA and protein syntheses by either LEx or arginase is probably due to arginine depletion by arginase activity, since their syntheses were similarly inhibited when cultured in an arginine-free medium in the absence of arginase. These results indicate that arginase nonspecifically inhibits the activities of DNA polymerase. The inhibition is probably due to arginine depletion.