Preclinical Evaluation of DMA, a Bisbenzimidazole, as Radioprotector: Toxicity, Pharmacokinetics, and Biodistribution Studies in Balb/c Mice.

Radiotherapy, a therapeutic modality of cancer treatment, nonselectively damages normal tissues as well as tumor tissues. The search is ongoing for therapeutic agents that selectively reduce radiation-induced normal tissue injury without reducing tumoricidal effect, thereby increasing the therapeutic ratio of radiation therapy. Our laboratory established 5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'benzimidazolyl] benzimidazole (DMA) as noncytotoxic radioprotector in mammalian cells. DMA showed an excellent radioprotection in mice at single nontoxic oral dose by a dose-reduction factor of 1.28. An oxygen radical absorbing capacity assay confirmed its free-radical quenching ability. Single bolus dose and 28-days of repeated administration of DMA in mice for toxicity studies determined an LD50 of >2000 mg/kg body weight (bw) and 225 mg/kg bw, respectively, suggesting DMA is safe. Histopathology, biochemical parameters, and relative organ weight analysis revealed insignificant changes in the DMA-treated animals. The pharmacokinetic study of DMA at oral and intravenous doses showed its C(max) = 1 hour, bioavailability of 8.84%, elimination half-life of 4 hours, and an enterohepatic recirculation. Biodistribution study in mice with Ehrlich ascites tumors showed that (99m)Tc-DMA achieved its highest concentration in 1 hour and was retained up to 4 hours in the lungs, liver, kidneys, and spleen, and in a low concentration in the tumor, a solicited property of any radioprotector to protect normal cells over cancerous cells. We observed that the single-dose treatment of tumor-bearing mice with DMA 2 hours before 8 Gy total body irradiation showed an impressive rescue of radiation-induced morbidity in terms of weight loss and mortality without a change in tumor response.

Synthesis, DNA binding, topoisomerase inhibition and cytotoxic properties of 2-chloroethylnitrosourea derivatives of hoechst 33258.

A number of novel 2-chloroethylnitrosourea derivatives of Hoechst 33258 were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds as well as Hoechst 33258 well interact with AT base pair compared with GC pair. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than the parent compound Hoechst 33258. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase I (topo I) or topoisomerase II (topo II) inhibitors in plasmid relaxation assays.

Double-stranded DNA binding characteristics and subcellular distribution of a minor groove binding diphenyl ether bisbenzimidazole.

The interactions of Hoechst 33377 (H1) with 20 different oligomeric duplexes have been investigated via spectrofluorometric titrations and/or thermal denaturation experiments. H1 is shown to form 2:1 complexes with dsDNA binding sites of at least four contiguous A/T base pairs. H1 is also shown to possess the rare ability to meaningfully distinguish between different A.T rich sequences. For example, the combined equilibrium constants for complexation of the oligomeric duplex 5'-GCAATTGC-3' (15) by H1 are found to be 110-fold greater than for the duplex 5'-GCTTAAGC-3' (16). It is believed that the 5'-TpA-3' dinucleotide step in 16 disrupts the rigid "A-tract" conformation of 15 and discourages minor groove binding by agents capable of recognizing longer dsDNA sequences. Molecular models are presented which elucidate the structure of the (H1)(2)-dsDNA minor groove complex. The two H1 molecules bind to an A/T rich sequence of 6 bp in a slightly staggered, side-by-side, and antiparallel arrangement. Evidence suggests that the piperazine rings of the H1 side-by-side complex are capable of resting in the minor groove of G/C base pairs. Fluorescence microscopy studies using NIH3T3 cells indicate that H1 is capable of traversing the cytoplasmic membrane and selectively localizing to nuclear DNA. H1 also demonstrated the ability to inhibit endogenous transcription of the c-fos gene in NIH3T3 cells at micromolar concentrations. Cytotoxicity studies employing the same cell type show H1 to possess an LD(50) of 3.5 microM.

DNA-binding properties and cytotoxicity of extended aromatic bisamidines in breast cancer MCF-7 cells.

The DNA binding properties of three novel extended aromatic bisamidines (1-3) possessing different dicationic terminal side chains were studied. Data from the ethidium displacement assay showed that bisamidines 1-3 have significant affinity for DNA. We studied the cytotoxic activity of bisamidines 1-3 and Hoechst 33258 in the cultured breast cancer MCF-7 cells. These data show that in broad terms the cytotoxic potency of bisamidines 1-3 in the cultured breast cancer MCF-7 cells decreases with the size of the alkyl group substituent (cyclopropyl > isopropyl > cyclopentyl), in accord with their increases in DNA affinity, as shown by the binding constant values. The bisamidines 1-3 showed comparable antitumor activity to Hoechst 33258.

Cytotoxicity of an 125I-labelled DNA ligand.

The subcellular distribution and cytotoxicity of a DNA-binding ligand [125I]-Hoechst 33258 following incubation of K562 cells with the drug was investigated. The ability of a radical scavenger, dimethyl sulphoxide, to protect cells from the 125I-decay induced cell death was also studied. Three different concentrations and specific activities of the drug were used to provide different ligand : DNA binding ratios. The results demonstrated a trend toward improved delivery of the ligand to the nucleus and to chromatin at higher ligand concentrations, with concomitant increased sensitivity to 125I-decay induced cytotoxicity and decreased protection by dimethyl sulphoxide. This correlation of radiobiological parameters with subcellular drug distribution is consistent with the classical dogma that attributes cytotoxicity to DNA double-stranded breakage in the vicinity of the site of decay, where the high LET nature of the damage confers minimal sensitivity to radical scavenging.

Microbial mutagenic effects of the DNA minor groove binder pibenzimol (Hoechst 33258) and a series of mustard analogues.

A series of aniline mustards and half-mustards targeted to DNA by linkage (through a polymethylene chain) to the bisbenzimidazole chromophore of pibenzimol (Hoechst 33258) have been evaluated for their mutagenic properties, as estimated in three strains of Salmonella typhimurium, and for their mitotic crossing-over and petite mutagenesis activities in Saccharomyces cerevisiae strain D5. Agarose gel electrophoresis studies showed that only the derivative with the longest linker chain cross-linked DNA, with the remaining compounds being monoalkylators. The parent (non-alkylator) minor groove binding ligand (Hoechst 33258) was inactive in the bacterial strains TA98 or TA100 but weakly mutagenic in TA102, and caused neither mitotic crossing-over nor 'petite' mutagenesis in yeast. Aniline half-mustard itself (monoalkylator) was an effective base-pair substitution mutagen (events in S. typhimurium strain TA100) with some frameshift mutagenesis activity in TA98, but showed only weak effects in the yeast assays, whereas aniline mustard (cross-linker) was inactive in these bacterial systems but caused substantial amounts of mitotic crossing-over in yeast. The composite molecules studied here showed effects more characteristic of the minor groove binding chromophore than of alkylating moieties. All showed weak mutagenic activity in TA102 and none in TA98. The only compound to show significant mitotic crossing-over ability was the long-chain derivative which cross-linked DNA. For most of the compounds, the mutagenicity data provided no supportive evidence for DNA alkylation. Since other evidence suggests this does occur readily, it is likely to have a different target to that seen with untargeted aniline mustards. The significant antitumor activity and low mutagenic potential shown by these compounds make them worthy of further study.

Transcriptional regulation of differentiation, selective toxicity and ATGCAAAT binding of bisbenzimidazole derivatives in human melanoma cells.

To study the relationship between the structure of minor groove ligands and their affinity for specific DNA sequences that regulate gene transcription, three analogues of the A-T-specific DNA minor groove ligands Hoechst 33258 and Hoechst 33342 were synthesized with 5, 8 or 12 carbons in an aliphatic chain attached to the phenolic oxygen of the molecule. There was a striking bimodal relationship between toxicity to HeLa cells and the lipophilicity of the five analogues, toxicity being low for the compounds with a free hydroxyl (Hoechst 33258) or a 12-carbon substituent, yet high for the 5-carbon analogue. Selective killing of human melanoma cells compared with normal fibroblasts was observed for the Hoechst analogue with a 12-carbon chain attached. Hoechst 33258 itself was selectively toxic for the MM96E melanoma cell line compared with other cell lines, induced a highly dendritic morphology, increased tyrosinase activity and tyrosinase mRNA but decreased the level of gp75 (TRP-1) mRNA; message for a third pigment gene, Pmel-17, was unchanged. Tyrosinase activity was decreased in the resistant A2058 melanoma cell line and transcription was affected to a lesser extent than in MM96E. Expression of gp75 protein and two intermediate filament proteins was inhibited by Hoechst 33258 in MM96E cells. There was no major difference in the amount of 125I-Hoechst 33258 taken up by sensitive and resistant cells. Of the five derivatives studied, the parent drug Hoechst 33258 and the 2-carbon analogue (Hoechst 33342) were found to have the most inhibitory effect on affinity of octamer binding proteins for the ATGCAAAT consensus sequence found in the promoter region of certain genes associated with proliferation and differentiation. In contrast to Distamycin A (also an A-T-specific minor groove ligand), Hoechst 33258 displaced proteins already bound to the octamer motif. The G-C ligand chromomycin A3 exhibited a different spectrum of cell toxicity and tyrosinase stimulation compared with Hoechst 33258. Chromomycin A3 but not Hoechst 33258, strongly inhibited the zinc-dependent transcriptional activity of the sheep metallothionein-Ia promoter in reporter gene assays of transfected cells. Since the six metal-responsive elements of the promoter are GC-rich, this provides independent evidence for the sequence-specificity of transcriptional inactivation by one of these drugs in melanoma cells. Overall, the results suggest that Hoechst 33258 acts by inhibiting the transcription of specific genes, cell lines evidently differing in the accessibility to drugs of certain A-T-rich sequences.