Stability and DNA alkylation rates of the simplest functional analogues of CC-1065, para-hydroxy and para-amino phenethyl bromides.

We have recently synthesised a series of compounds based on the simplest functional unit of CC-1065 containing a para substituted phenethyl halide moiety. These compounds alkylate N3 of adenines in a similar fashion to CC-1065, as well as N7 of guanines to a limited extent. In this work we compared the para amino substituted derivative (2) with the published hydroxyl compound (1) in terms of stability, DNA reactivity and pH dependence using gel electrophoresis techniques. The results show that 2 has a shorter lifetime and is at least 2.5 times more reactive with DNA than 1. It is completely hydrolysed between 30 and 60 min in buffer and its reaction with DNA is complete within 5 min. In contrast, only a fraction of 1 is hydrolysed after 60 min and retains reactivity towards DNA even after 3 h. The reactivities of both 1 and 2 with DNA are pH dependent and reaction rates rapidly decrease in the range pH 5.8-8.8. Preliminary molecular modelling studies suggest that the p-amino group on 2 enables the drug to bind to the AT-rich minor groove more effectively, thus stabilising the orientation of the substrate in the groove such that the reactive cyclopropyl ring is located close to the nucleophilic centre N3 of adenine. A possible mechanism of action of these drugs is presented based on these findings.

Lack of response to multiple genotoxic agents at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys.

We tested the ability of a series of known genotoxic agents to cause mutations at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys as measured by the ability to form clones in the presence of 6-thioguanine. Ethylmethane sulfonate (EMS, 300 mg/kg i.p.), chloroethylmethane sulfonate (CI-EMS, 35 or 50 mg/kg i.p.), and the Pharmacia & Upjohn antitumor agents adozelesin (1.6, 4, 6, or 8 microg/kg i.v.) and CC-1065 (6 microg/kg i.v.) were all negative in the hprt mutation test. Results with cyclophosphamide (CP, 75 mg/kg i.v.) were equivocal. Adozelesin, CC-1065, and CI-EMS treatments increased the percentage of T-lymphocytes with chromosome aberrations, as well as inducing types of aberrations not seen in control cells. EMS and CP were not tested for chromosome aberrations. We have previously shown that treatment of monkeys with 77 mg/kg ENU substantially increased the hprt mutant frequency, with a lag time of approximately 77 days between treatment and peak MF values. The results of the present study suggest a low sensitivity of the hprt mutation assay to certain classes of genotoxic agents in cynomolgus monkeys.

End points for biomonitoring: assay sensitivity/selectivity.

Estimation of population exposure and biological impact of potential hazards are central reasons for performing biomonitoring. The sensitivity of the biomonitoring methods and the linkage of the measured phenomenon to human disease are also important, but often overlooked, considerations. We are conducting experiments to evaluate the sensitivity of hprt mutation measurement in the nonhuman primate, the cynomolgus monkey. Our findings demonstrate in the monkey that hypoxanthine guanine phosphoribosyltransferase (hprt) mutations produced in vivo can be detected using technique originally worked out using human cells; cynomolgus monkeys were chosen to avoid many of the complications encountered in studying humans. Sequencing of mutants from the monkey using reverse transcriptase polymerase chain reaction methods has led us to conclude that there is similarity of the spectra observed between the spontaneous mutations detected in the two species. However, more recent data suggest that due to low sensitivity, the method is probably not appropriate for routine biomonitoring of randomly selected populations. For example, the inability of the hprt mutation assay to detect some very potent mutagens in the monkey and the effects of the time-dependent pattern of mutant occurrence serve to urge caution in interpretation of elevation or lack of elevation in mutant frequency. Mechanisms for splitting and archiving samples of human tissues/blood from populations at risk may prove valuable as methods improve.

Lung tumor induction in A/J mice and clastogenic effects in CD-1 mice of the sequence-selective DNA alkylating agents (+)-CC-1065 and (-)-CC-1065.

The in vivo genotoxic effects of the antitumor antibiotic, (+)-CC-1065, and its unnatural enantiomer, (-)-CC-1065, were investigated in two mouse models. These two compounds alkylate AT-rich regions of double stranded DNA with distinct sequence selectivities. (+)-CC-1065 dose-dependently increased the chromosomal aberration frequency in bone marrow cells of CD-1 mice from 1.2 +/- 0.8% in vehicle control animals to 5.0 +/- 1.2%, 11.4 +/- 3.9%, and 20.6 +/- 2.3% 24 hours following single intravenous doses of 2, 4, and 8 micrograms/kg, respectively. (-)-CC-1065 was significantly less potent with a maximal response at 8 micrograms/kg approximately one-third of that observed for (+)-CC-1065. (+)-CC-1065 induced a significant (P < or = 0.05), three-fold increase in the number of lung tumors/mouse in strain A/J mice from 0.27 +/- 0.15 for vehicle control animals to 0.83 +/- 0.15 24 weeks following a single intravenous dose of 8 micrograms/kg. This effect was paralleled by corresponding threefold increases in the percentage of mice with tumors and the percentage of mice with multiple tumors, compared to vehicle controls. (-)-CC-1065 at 8 micrograms/kg induced 0.67 +/- 0.15 tumors/mouse and resulted in slightly smaller increases in the tumor incidence and multiple tumor incidence, compared to (+)-CC-1065. The above results demonstrate that single intravenous doses of (+)- CC-1065 and (-)-CC-1065 which cause chromosomal damage in CD-1 mice also induce an increased incidence of lung tumors in A/J mice.(ABSTRACT TRUNCATED AT 250 WORDS)

CC-1065 bonding to intracellular and purified SV40 DNA: site specificity and functional effects.

CC-1065 is a minor-groove bonding agent capable of forming covalent adducts with the N-3 position of adenines within A-T-rich regions of duplex DNA. By examining the formation and location of CC-1065 adducts within the simian virus 40 (SV40) DNA molecule, the present study marks the first time that the precise sites of CC-1065 lesions have been identified at the level of eukaryotic genomic DNA. In naked DNA preparations, r values (moles of drug/mole of nucleotide base pair) > or = 0.0015 effected, after thermal treatment, a measurable decrease in intact supercoiled form I, as well as increases in forms II and III, indicating that both single-strand and apparent double-strand damage had occurred. A similar pattern of damage was observed in SV40-infected cells, albeit at higher CC-1065 levels. The amount of CC-1065 required to produce a 50% loss in form I was > 2-fold higher in infected cells (r = 0.029) than with purified DNA samples (r = 0.013). The appearance of double-strand damage at low drug levels suggested a high specificity of CC-1065 bonding to localized regions of the genome. The precise location of these CC-1065 adduction sites was examined by three methods: sequence analysis of the entire genome (GenBank), DNA polymerase termination assay of specific fragments of SV40, and restriction enzyme digestion analysis of the entire SV40 molecule. When sequence analysis of the entire genome was performed by examining both strands for the presence of the consensus CC-1065 binding sequence 5'-A/T-A/T-A/T-A/T-A*-3'[Reynolds et al. (1985) Biochemistry 24, 6228-6247], 294 single-strand adduction sites were predicted, compared to 20 sites where CC-1065 should bond to both strands within a 30-base-pair window and at which, when heated, a double-strand break should occur. DNA polymerase termination assay of actual adduction sites was performed on restriction fragments of SV40 DNA pretreated with CC-1065 in infected cells or in purified supercoiled DNA preparations and selected on the basis of the sequence analysis (i.e., regions 2510-2730, 3701-3920, 4400-4659, 4020-4320, and 5163-65). In general, double-strand lesions were detected in similar regions of the genome by the DNA termination assay and by sequence analysis. When restriction enzyme digestion and the DNA polymerase termination assay were compared throughout the genome, nearly identical patterns of adduct formation were observed. Interestingly, similar alkylation patterns were observed with either naked or infected cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation and repair of antitumor antibiotic CC-1065-induced DNA adducts in the adenine phosphoribosyltransferase and amplified dihydrofolate reductase genes of Chinese hamster ovary cells.

CC-1065 is a potent antitumor antibiotic which bonds to duplex DNA specifically; the biological effects of the drug are presumably the consequences of its DNA interactions. In order to investigate the factors which may affect drug-DNA bonding in cells, a method using a thermal-alkaline treatment to induce phosphodiester bond breakage at the drug-DNA bonding sites and Southern DNA transfer-hybridization to quantify drug-DNA bonding at defined sequences in drug-treated cultured mammalian cells was developed. We have found that in vivo, in cultured Chinese hamster ovary (CHO) cells, CC-1065 bonds twice as efficiently in the highly amplified dihydrofolate reductase (DHFR) gene domains as in the nonamplified adenine phosphoribosyltransferase (APRT) gene domain. However, in vitro, in purified CHO cellular DNA, CC-1065 bonds equally to both the DHFR and APRT genes. We observed a significant degree of "gene-specific" preferential repair for drug-DNA adducts in the amplified DHFR gene domains, and it appears that this "gene-specific" repair reflects "transcribed-strand specific" repair. These results suggest that DNA amplification may affect drug-DNA adduct formation and transcription may affect its repair.

In vivo mutagenesis induced by CC-1065 and adozelesin DNA alkylation in a transgenic mouse model.

Although considerable work has focused on characterizing the bonding chemistry and sequence selective alkylation of DNA by cyclopropylpyrroloindole compounds, little is known about the molecular consequence of their N-3-adenine adducts in whole animal systems. We have utilized a transgenic mouse system, harboring a lambda phage shuttle vector, to assess the mutagenic potential of the antitumor compounds CC-1065 and adozelesin and, for the first time, to track the in vivo fate of their unique DNA modifications at the nucleotide level. Mice were inoculated with a single therapeutic dose of these agents and sacrificed at either 18 h, 3 days, or 15 days for extraction and analysis of liver DNA. Mutant frequencies obtained from drug treated and control animals were determined by in vitro packaging of the phage vector from genomic DNA followed by a colorimetric plaque assay to screen for phage in which the accompanying lacI repressor gene had mutated. Although undetectable at 18 h posttreatment, by 72 h a 3-fold increase in mutant frequency was observed in drug treated animals such that sequence analysis of drug induced mutations could be performed and a direct comparison made between in vitro and in vivo DNA alkylation. Base substitution involving guanine or cytosine accounted for 64% of the 41 mutations sequenced from drug treated animals. Only 7 of the mutations occurred at a cyclopropylpyrroloindole alkylation site while 23 occurred 1 to 4 nucleotides from a potentially alkylated adenine.

Evaluation of functional analogs of CC-1065 and the duocarmycins incorporating the cross-linking 9a-chloromethyl-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-on e (C2BI) alkylation subunit.

The DNA alkylation properties and in vitro cytotoxic activity of a series of analogs of CC-1065 and the duocarmycins incorporating the 9a-chloromethyl-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (C2BI) alkylation subunit are detailed. The C2BI-based agents have been shown to alkylate DNA within the minor groove in a fashion analogous to CC-1065 or duocarmycin. The stereoelectronically-controlled adenine N3 addition to the least substituted cyclopropane carbon occurs with a selectivity that represents a composite of the two enantiomers of the corresponding CBI-based agents. Additional high affinity alkylation sites were detected which were not prominent alkylation sites for either enantiomer of the CBI-based agents. Such sites may represent induced high affinity alkylation sites resulting from DNA cross-linking following complementary strand alkylation at a high affinity alkylation site and each such site detected proved consistent with predicted models of an adenine-adenine cross-linking event. Further, consistent with this interpretation, the C2BI agents were shown to constitute efficient cross-linking agents with DNA cross-linking being observed at the same concentrations as DNA alkylation. In comparison to the parent CBI-based agents, the C2BI-based agents proved to be approximately 100-10,000x less effective at DNA alkylation and 100-10,000x less potent in cytotoxic assays. This is suggested to be the consequence of a significant steric deceleration of the adenine N3 alkylation reaction attributable to the additional 9a-chloromethyl substituent. Consistent with this interpretation, the noncovalent binding constant of C2BI-CDPI2 for poly[dA]-poly[dA]-poly[dT] proved nearly identical to that of CDPI3 under kinetic binding conditions, and prolonged incubation of C2BI-CDPI2 with poly[dA]-poly[dT] (72 h, 25 degrees C) provided covalent complexes with a helix stabilization comparable to that observed with (+)- or (-)-CPI-CDPI2 indicating that the size of the C2BI subunit inhibits but does not preclude productive DNA alkylation.

Synthesis and biochemical evaluation of the CBI-PDE-I-dimer, a benzannelated analog of (+)-CC-1065 that also produces delayed toxicity in mice.

A practical synthesis of CBI (2) was developed and applied to the synthesis of benzannelated analogs of CC-1065, including CBI-PDE-I-dimer (13) and CBI-bis-indole [(+)-A'BC]. The CBI-PDE-I-dimer was shown to have similar DNA sequence selectivity and structural effects on DNA as (+)-CC-1065. Of particular importance was the observed duplex winding effect that has been associated with the pyrrolidine ring of the nonalkylated subunits of (+)-CC-1065 and possibly correlated with its delayed toxicity effects. The effect of CBI-PDE-I-dimer was also compared to (+)-CC-1065 in the inhibition of duplex unwinding by helicase II and nick sealing by T4 ligase and found to be quantitatively similar. The in vitro and in vivo potencies of the CBI compounds corresponded very closely to the corresponding CPI derivatives. Finally, CBI-PDE-I-dimer was like (+)-CC-1065 in causing delayed toxicity in mice.

Calf thymus DNA binding/bonding properties of CC-1065 and analogs as related to their biological activities and toxicities.

CC-1065 is a potent natural antitumor antibiotic that binds non-covalently and covalently (N-3 adenine adduct) in the minor groove of B-form DNA. Synthetic analogs of CC-1065 do not exhibit the delayed death toxicity of CC-1065 and are efficacious anticancer agents, some of them curative in murine tumor models. In an attempt to understand the different biological properties of CC-1065 and analogs, we have determined the following quantities for CC-1065, enantiomeric CC-1065, and three biologically active analogs and their enantiomers: the calf thymus DNA (CT-DNA) induced molar ellipticity of the adduct (or how rigidly the adduct is held in the right-hand conformation of the minor groove); the stability of the adduct with respect to long incubation times and to digestion by snake venom phosphodiesterase I (SVPD); the stabilizing effect on the CT-DNA helix of the covalently and non-covalently bound species with respect to thermal melting; and the CT-DNA binding/bonding (non-covalent/covalent) profiles at a low molar ratio of nucleotide to drug. The major observations from these studies are as follows: (i) molecules which show large DNA interaction parameters, stable adducts, and significant non-covalent binding exhibit delayed death toxicity; (ii) molecules which show intermediate DNA interaction parameters and stable adducts, but do not show significant non-covalent binding, do not exhibit delayed death toxicity and are biologically active; (iii) molecules which show small DNA interaction parameters and unstable DNA adducts are biologically inactive. The results suggest that a window exists in the affinity for the minor groove of DNA wherein an analog may possess the correct balance of toxicity and activity to make a useful anticancer agent. Outside of this window, the analog causes delayed deaths or has no significant biological activity.

19-Deformyl-4'-deoxydesmycosin (TMC-016): synthesis and biological properties of a unique 16-membered macrolide antibiotic.

19-Deformyl-4'-deoxydesmycosin was synthesized by the following synthetic route: 19-Deformylation of desmycosin, 3,2',4''-tri-O-trimethylsilylation, 4'-O-sulfonylation, 4'-iodination, reductive deiodination and 3,2',4''-tri-O-detrimethylsilylation. Deformylation of the aldehyde group at the C-19 position was achieved by two different methods: A) A simple one-step deformylation using Wilkinson's catalyst ((Ph3P)3RhCl). B) Reductive decarboxylation of the 19-carboxyl derivative following NaClO2 oxidation of the aldehyde. 19-Deformyl-4'-deoxydesmycosin showed very strong antimicrobial activity in vitro and in vivo.

Toxicity, uptake, and subcellular distribution in rat hepatocytes of roxithromycin, a new semisynthetic macrolide, and erythromycin base.

Rat hepatocytes were used to study the toxicity of a new semisynthetic macrolide, roxithromycin, in comparison with erythromycin base and erythromycin estolate. Roxithromycin caused lactate dehydrogenase leakage close to that of erythromycin estolate and higher than erythromycin base after 21 h of exposure to the drugs. This effect was, at least in part, explained by the higher uptake: roxithromycin was two to three times more concentrated by liver cells than erythromycin base. For both roxithromycin and erythromycin base, the uptake depended on time, temperature, and extracellular antibiotic concentration. The accumulated macrolides egressed rapidly when cells were incubated in antibiotic-free medium. No uptake and no loss of accumulated drugs were observed at 4 degrees C. After accumulation by hepatocytes, roxithromycin and erythromycin base underwent similar subcellular distribution, mostly concentrating in cytosol and lysosomes. The small amount accumulated in the other particulate fractions followed the order mitochondria much greater than nuclei greater than microsomes. Roxithromycin, however, was less concentrated than erythromycin base in the microsomes.

Evaluation of DNA binding characteristics of some CC-1065 analogs.

The factors influencing the binding of CC-1065 to DNA were examined using racemic analogs with varying chain lengths. The ability of these agents to bind DNA appeared to be related to cytotoxic potency, however this did not appear to be a direct quantitative correlation. Two enantiomers of a bis-indole analog of CC-1065 were studied for DNA binding and cytotoxic activity. The agent with the same stereochemical configuration as CC-1065 was a potent cytotoxin, but its enantiomer was essentially inactive. Both enantiomers showed significant binding to DNA, but the biologically less active isomer showed less overall binding. In all cases, the agents preferred AT-rich DNA, and all bound to similar regions in DNA as evidenced by positions of drug-initiated thermal breaks in single end-labelled fragments of phi X 174RF DNA. The overall similarity in site specificity for binding of the structurally diverse agents suggests that much of the specificity observed in binding of the agent to DNA lies in the DNA itself. Thus, it may be difficult to change minor groove specificity for agents of this type simply by designing structures that can encompass guanine or cytosine residues. Other modifications, such as changing the specificity of the alkylating moiety, may be required to achieve this goal.

Mutagenicity of the antitumor antibiotic CC-1065 and its analogues in mammalian (V79) cells and bacteria.

CC-1065 is a very potent antitumor antibiotic which binds in the minor groove of DNA with alkylation at N-3 of adenine. Since CC-1065 caused delayed deaths in mice at therapeutic doses, analogues were prepared whose antitumor and biochemical activities have been reported. In this study, the mutagenicity for V79 cells (6-thioguanine resistance) and Salmonella (histidine auxotrophy or azaguanine resistance) of selected analogues was compared to DNA-binding activity and the structure-activity relationship was determined. CC-1065, U-62,736, U-66,866, U-66,694, U-67,786, and U-68,415 all have an A segment with an intact cyclopropyl group and different B segments. The cyclopropyl group is absent from U-66,226 and U-63,360. Elimination of the cyclopropyl ring diminished the cytotoxic and mutagenic potency of the compounds such that U-63,360 was nearly three orders of magnitude less potent than CC-1065 in V79 cells. For the compounds with an intact cyclopropyl group, the order of cytotoxic and mutagenic potency (molar basis) in V79 cells generally correlated with binding to calf thymus DNA, and increased with the length of the B segment. Thus, the order of cytotoxicity was CC-1065 greater than U-68,415 greater than U-66,694 greater than U-66,866 greater than U-62,736. U-67,786 fell outside this pattern since it was more cytotoxic and mutagenic than U-66,694, although it was of a similar size and had similar DNA-binding activity. These results show that an electrophilic carbon afforded by an intact cyclopropyl group of this type is necessary but not sufficient to account for the high cytotoxic and mutagenic potency of CC-1065 and U-68,415. The size and characteristics of the B segment also affect the potency. At an equitoxic (10 or 50% lethal dose) dose, an inverse relationship exists between cytotoxic and mutagenic potency such that at the 50% lethal dose, the least cytotoxic compound (U-62,736) was more mutagenic than the most cytotoxic compound (CC-1065). We speculate that the more cytotoxic analogues are less mutagenic (at an equitoxic dose) because they may have greater structure-directed binding to less mutable DNA sites in the minor groove.

[Chronic toxicity and recovery tests of rokitamycin in beagle dogs].

Six-month oral chronic toxicity study and two-month recovery tests on rokitamycin (TMS-19-Q) were performed in Beagle dogs at daily doses of 90, 180 and 360 mg/kg. The results were summarized as follows: No death occurred to any animals in these experiments. In higher dosage groups (180 and 360 mg/kg), vomiting, salivation and reduction of body weight gain with decrease in food intake were observed. Body temperature and pulse rate were normal, and ECG, sperm and vaginal smear tests, urinalysis, blood and biochemical examinations also revealed normal values. No marked changes in organ weights, necropsy, histopathological and electron microscopic observations were detected.

[Comparative toxicity study of rokitamycin and josamycin in rats].

A comparative toxicity study on macrolide antibiotics, rokitamycin (TMS-19-Q) and josamycin, was performed in male rats. These drugs were given orally for 1 month at daily doses of 250, 500, 1,000, and 2,000 mg/kg. No animals died in the study. No abnormal symptoms or changes in body weights and food consumption were observed. Results of urinalysis, hematological analysis and biochemical analysis of serum and organ weights were normal except that a dose-dependent hypertrophy of caecum was observed in all dosage groups. Necropsy revealed no notable drug related abnormalities except in caecum of all test groups. Pathological examination found no significant drug-related abnormalities in the test groups. No toxicological signs were detected in the group administered with 2,000 mg/kg of either drug and no difference was found between the 2 drugs.

[Reproduction study of rokitamycin. Teratological study in rabbits].

A teratological study was performed on rokitamycin (TMS-19-Q), a new macrolide antibiotic, using rabbits. TMS-19-Q, at dose levels of 100, 300 and 600 mg/kg, was orally administered from the day 6 to the day 18 of gestation to dams. Diarrhea and decrease in body weight gain and food consumption were observed in dams at or above 300 mg/kg. Decrease in numbers of live fetuses was observed at dose levels of 300 and 600 mg/kg but there was no teratogenicity at any dose levels. The maximum non-toxic dose level for TMS-19-Q in this study was 100 mg/kg.

Genotoxicity of the antitumor antibiotic CC-1065.

CC-1065, a very potent antitumor antibiotic, is active against several animal tumors, and against human tumors in the cloning assay at doses 50-1000 times lower than other agents such as adriamycin. It binds and alkylates DNA, and inhibits DNA synthesis, suggesting a potential for genotoxicity. Therefore, the genotoxic effects of CC-1065 were tested in several assay systems. CC-1065 was weakly mutagenic in the Ames Salmonella mutation assay (strain TA100) without S9 activation, but lacked mutagenic activity in TA98 with or without activation. CC-1065 was a very potent mutagen in the Salmonella forward mutation assay (induction of 8-azaguanine resistance), increasing the mutation frequency 19-fold over background at 0.1 ng/ml without activation. In mammalian (V79) cells it was a very potent mutagen without activation, increasing the mutation frequency 20-fold over background a 0.5 ng/ml. CC-1065 induced chromosome aberrations in V79 cells at very low (less than 0.1 ng/ml) doses, making this assay the most sensitive. CC-1065 increased the induction of micronuclei in rats 10- to 20-fold over the background at 200 and 400 micrograms/kg, but not at 100 micrograms/kg. CC-1065 failed to cause DNA breaks or DNA--protein cross-links as measured by the DNA damage/alkaline elution assay.