In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogues of halichondrin B.

Halichondrin B is a highly potent anticancer agent originally found in marine sponges. Although scarcity of the natural product has hampered efforts to develop halichondrin B as a new anticancer drug, the existence of a complete synthetic route has allowed synthesis of structurally simpler analogues that retain the remarkable potency of the parent compound. In this study, we show that two macrocyclic ketone analogues of halichondrir B, ER-076349 and ER-086526, have sub-nM growth inhibitory activities in vitro against numerous human cancer cell lines as well as marked in vivo activities at 0.1-1 mg/kg against four human xenografts: MDA-MB-435 breast cancer, COLO 205 colon cancer, LOX melanoma, and NIH: OVCAR-3 ovarian cancer. ER-076349 and ER-086526 induce G2-M cell cycle arrest and disruption of mitotic spindles, consistent with the tubulin-based antimitotic mechanism of halichondrin B. This is supported further by direct binding of the biotinylated analogue ER-040798 to tubulin and inhibition of tubulin polymerization in vitro by ER-076349 and ER-086526. Retention of the extraordinary in vitro and in vivo activity off halichondrin B in structurally simplified, fully synthetic analogues establishes the feasibility of developing halichondrin B-based agents as highly effective, novel anticancer drugs.

Mechanism of mouse skin tumor promotion by chrysarobin.

The skin tumor-promoting ability of 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) was compared with that of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,8-dihydroxy-9-anthrone (anthralin) in SENCAR mice. Although dose-response comparisons indicated that chrysarobin was several orders of magnitude less potent than TPA for promoting papilloma formation, this anthrone was 1.5 to 2 times more potent than anthralin. Maximal papilloma responses were achieved by 15 weeks of promotion with TPA whereas at least 25 weeks of promotion were necessary to achieve maximal papilloma responses with chrysarobin or anthralin indicating marked differences in tumor latency between the two classes of compounds. Interestingly, at optimal promoting doses, chrysarobin gave a carcinoma response (22% with 0.3 carcinomas per mouse at 45 weeks) similar to that of TPA suggesting that this compound may be more efficient at promoting carcinomas than papillomas. In two-stage promotion experiments, chrysarobin was incapable of functioning independently as a Stage I or II promoter despite its complete promoting activity. Chrysarobin and TPA were compared at optimal promoting doses for their ability to induce: (a) skin edema, (b) epidermal hyperplasia, and (c) epidermal ornithine decarboxylase. In each case, distinct differences were noted between the two compounds. When taken together, the data support the hypothesis that anthracene-derived skin tumor promoters work at least in part by a mechanism different from the phorbol esters.

Formation and disappearance of benzo[a]pyrene DNA-adducts in mouse epidermis.

The rates of formation and disappearance of benzo[a]pyrene (B[a]P) DNA-adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 200 nmol/mouse of [3H]B[a]P at various times prior to sacrifice. The formation and disappearance of total adducts as well as individual adducts was determined and in addition, the rate of DNA turnover was monitored concurrently so that adduct disappearance could be expressed as a function of epidermal cell turnover. Under these experimental conditions, covalent binding of B[a]P to epidermal DNA reached a peak 24 h after treatment. Interestingly, between 24-48 h after application of the hydrocarbon there was a very rapid drop in the level of bound B[a]P to value approximately 50% of the maximum level at 24 h. Thereafter, the level of bound B[a]P disappeared at a much slower rate. In dual-label experiments, where the epidermal DNA was pre-labeled with [14C]thymidine, [3H]B[a]P DNA-adduct disappearance between 24-48 h was clearly more rapid than could be explained on the basis of epidermal DNA turnover. By 72 h and beyond, however, [3H]B[a]P DNA-adduct disappearance approximately paralleled DNA turnover. Examination of the rate of formation and disappearance of individual B[a]P DNA-adducts (nine individual adducts) suggested that some deoxyadenosine adducts were removed more rapidly than deoxyguanosine adducts. The results indicate that at least some epidermal cells have the capacity to repair B[a]P DNA-adducts. The data are discussed in relation to the process of tumor initiation in mouse skin.

Covalent binding of 7,12-dimethylbenz(a)anthracene and 10-fluoro-7,12-dimethylbenz(a)anthracene to mouse epidermal DNA and its relationship to tumor-initiating activity.

10-Fluoro-7,12-dimethylbenz(a)anthracene (10-F-DMBA) is a more potent skin tumor initiator in SENCAR mice when compared with the parent hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA). To elucidate the mechanism for this difference, the covalent binding of these two hydrocarbons to the DNA of mouse epidermal cells in vivo and in vitro was compared. The quantity of 10-F-DMBA covalently bound to mouse epidermal DNA in vivo was greater than that of DMBA at all doses tested over the range of 4 to 200 nmol/mouse. The magnitude of this binding difference between 10-F-DMBA and DMBA was greater at the higher doses (e.g., 1.5-fold at 4 nmol/mouse versus 3.4-fold at 200 nmol/mouse). These results correlated closely with the dose-response relationships for tumor initiation by the two hydrocarbons. Analysis of the isolated DNA samples by Servacel DHB chromatography revealed the relative proportion of syn-diol-epoxide:DNA adducts derived from DMBA increased dramatically as a function of dose (approximately 30% at 4 nmol/mouse versus approximately 55% at 200 nmol/mouse). Conversely, the relative proportion of syn-diol-epoxide adducts derived from 10-F-DMBA was low and remained essentially constant over the same dose range. High-pressure liquid chromatographic analyses of the DNA adducts derived from DMBA- and 10-F-DMBA-treated mice revealed qualitatively similar profiles. However, as expected, there was a marked reduction in the relative proportion of syn-diol-epoxide:DNA adducts in the profiles of epidermal samples from 10-F-DMBA-treated mice. The major syn-diol-epoxide:deoxy-adenosine adduct was present at a level only 30% that found in high-pressure liquid chromatographic profiles of DMBA samples. Similar results were obtained when primary cultures of mouse epidermal cells were treated with the hydrocarbons. The results suggest that the increased total binding and possibly the decreased proportion of syn-diol-epoxide:DNA adducts confer greater tumor-initiating potency on 10-F-DMBA.