Akira Okura

In vivo Anti‐tumor Activity of a Novel Indolocarbazole Compound, J‐107088, on Murine and Human Tumors Transplanted into Mice

J‐107088 (6‐N‐(1‐hydroxymethyl‐2‐hydroxy)ethylamino‐12,13‐dihydro‐2,10‐dihydroxy‐13‐(β‐D‐glu‐copyranosyl)‐5H‐indolo[2,3‐a]‐pyrrolo [3,4‐c]carbazole‐5,7(6H)‐dione) is a derivative of NB‐506, an indolocarbazole compound previously reported as an anti‐tumor agent targeting topoisomerase L The optimal administration schedule of J‐107088 was found to be intermittent injections. The GID75 (75% growth inhibiting total dose) values of J‐107088 against LX‐1 lung cancer and PC‐3 prostate cancer when given by intermittent injection (twice a week for 2 consecutive weeks) were 200 and 15 mg/m2, respectively, whereas the 10% lethal dose (LD10) values of J‐107088 against LX‐1‐ and PC‐3‐bearing mice were 578 and 1200 mg/m2. The ratio of LD10/GID75 indicates the therapeutic window of an anti‐tumor agent. Although the ratios of doxorubicin, paclitaxel and cisplatin against PC‐3 were <0.3, <0.5 and <0.2, J‐107showed the widest therapeutic window among the anti‐tumor drugs tested. J‐107088 was also effective on cells that had acquired resistance related to P‐glycoprotein. Furthermore, J‐107088 was found to be highly effective in inhibiting proliferation of micro‐metastases of tumors to the liver in mice. Therefore, J‐107088 is considered to be a promising candidate as an anti‐tumor drug for treatment of solid tumors in humans.

Sequence-selective DNA cleavage by a topoisomerase I poison, NB-506

An indolocarbazole compound, NB‐506, inhibits the activity of topoisomerase I by stabilizing the DNA‐topoisomerase I complex (cleavable complex). NB‐506 inhibited the re‐ligation step of topoisomerase I activity more potently than camptothecin or its derivative, topotecan. A cleavage assay using an end‐labeled fragment of DNA revealed that the pattern of cleavage induced by NB‐506 was different from that induced by camptothecin. The preferred cleavage sites of NB‐506 were found to be not only T but also A or C at the 3′‐terminus of the cleaved DNA (position −1), while the DNA cleavage sites of camptothecin always had T at position −1. At the 5′‐terminus of the cleaved DNA (position +1), NB‐506 showed a preference for G, which is a feature shared in common with camptothecin. Therefore, the difference in cleavage patterns was most likely due mainly to the preferred base at position −1. Moreover, the re‐ligation rate was significantly slower at NB‐506‐selective sites, which had C at position‐1, than at camptothecin‐selective sites or at sites cleaved by both NB‐506 and camptothecin. Our data suggest that NB‐506 is an unique topoisomerase I poison and that its potent inhibition of topoisomerase I is partly dependent on retardation of re‐ligation at sites selectively induced by NB‐506. Int. J. Cancer 75:145–150, 1998.© 1998 Wiley‐Liss, Inc.

Inhibition of Topoisomerase II by a Novel Antitumor Cyclic Depsipeptide, BE‐22179

BE‐22179, a novel cyclic depsipeptide antibiotic having two 3‐hydroxyquinoline moieties, inhibited the DNA‐relaxing activity of L1210 topoisomerase II completely at 0.08 μM. This effect was far stronger than that of VP‐16. However, it did not show any marked effect on topoisomerase II‐mediated DNA cleavage. BE‐22179 was ineffective in inhibiting the DNA relaxation by topoisomerase I at concentrations up to 10 μM, but showed DNA‐intercalating ability (DNA unwinding) at 30 μM. The structure of BE‐22179 is quite novel for a topoisomerase II inhibitor. Echinomycin, a quinoxaline antibiotic structurally related to BE‐22179, interfered with DNA relaxation by topoisomerase II, though the effect was not due to inhibition of the catalytic activity of topoisomerase II but to conformational change of DNA based on its intercalation into DNA. Therefore, the potent inhibitory activity on topoisomerase II might not be a common activity of quinoxaline antibiotics, but might rather be specific to BE‐22179. BE‐22179 prevented DNA synthesis as well as RNA synthesis in L1210 cells and inhibited the growth of the cells. However, it remains unclear to what extent the topoisomerase II inhibition was responsible for the cytotoxicity of BE‐22179.

Antimetastatic Effect of a Novel Indolocarbazole (NB‐506) on IMC‐HM Murine Tumor Cells Metastasized to the Liver

IMC‐HM cells were isolated from spontaneously induced ascitic IMC carcinoma cells that had been maintained intraperitoneally in CDF1 mice. Metastasis to the liver of subcutaneously implanted IMC‐HM cells was detected 10 days after implantation into the flanks of mice (day 10), but metastasis to other organs was limited. Thereafter, however, tumor cells spread rapidly to lymph nodes, lung, spleen, ovary and other organs, and the mice died on day 13 to 18. We report here, together with the properties of IMC‐HM cells, the effects of adriamycin, cisplatin, etoposide and a new indolocarbazole antitumor compound (NB‐506) on this model of metastasis. Although these anticancer agents all inhibited the growth of the subcutaneous tumors, their effects on the life span of the tumor‐bearing mice varied. Treatment with NB‐506, started on day 1, more than doubled the survival period at doses 30 mg/m2 to 900 mg/m2. Further, treatment with NB‐506, started on day 4 after resection of the primary tumor, inhibited growth of the metastasized tumor in the liver and other organs. Etoposide also increased the life span at a limited range of doses. However, the life‐prolonging effects of adriamycin and cisplatin were marginal. These results demonstrate that IMC‐HM carcinoma is a good model for spontaneous metastasis to the liver followed by lethal spread to many organs. Moreover, NB‐506 was found to be highly effective against the growth not only of subcutaneous tumors, but also of tumors metastasized to the liver.

ED-110, a Novel Indolocarbazole, Prevents the Growth of Experimental Tumors in Mice

A new indolocarbazole compound, ED‐110, which was obtained by glucosylating a microbial product (BE‐13793C) and is a potent topoisomerase I inhibitor, showed characteristic inhibitory effects on the growth of 12 human tumor cell lines tested. The IC50 values of ED‐110 against 9 of the 12 lines ranged from 11.5 μg/ml to 0.07 μg/ml, while the remaining 3 lines were quite resistant (IC50, >100μg/ml). In in vivo experiments, i.p. treatment with ED‐110 increased the survival period by more than two‐fold in mice implanted i.p. with P388, L1210, L5178Y or EL4 murine leukemic cells. The minimum effective dose increasing the life‐span of mice bearing P388 leukemia by 25% was <2.5 mg/kg/day × 10 and the maximum tolerated dose was > 160 mg/kg/day × 10. ED‐110 was also effective against the spontaneous metastasis of mouse Meth A fibrosarcoma cells and the growth of xenografted MKN‐ 45 human stomach cancer cells as well as s.c. implanted mouse colon 26 and IMC carcinoma cells. These results indicated that ED‐110 may have potential as a new antineoplastic agent with a large chemotherapeutic index and a wide range of effective doses.

BE‐23372M, a Novel and Specific Inhibitor for Epidermal Growth Factor Receptor Kinase

The fungal metabolite BE‐23372M is a structurally novel protein kinase inhibitor. Its IC50 for epidermal growth factor (EGF) receptor kinase was 0.03 μM. IC50 values of BE‐23372M for other protein tyrosine kinases, erbB‐2, p43v‐abl, insulin receptor kinase, and p60c‐src were 0.42, 1.0, 3.3, and 4.5 μM, respectively, and the IC50 for protein kinase C, a serine/threonine kinase, was 4.1 μM. Cdc2 kinase, casein kinases I and II and cAMP‐dependent protein kinase were not inhibited by 20 μM BE23372M. A kinetic study showed that BE‐23372M was competitive with respect to the substrate peptide and to ATP. Autophosphorylation of solubilized EGF receptor kinase was clearly inhibited by 0.1 μM BE‐23372M. Autophosphorylation of EGP receptor in A431 cells was also inhibited. These results show that BE‐23372M is a potent and specific EGF receptor kinase inhibitor. It should be a valuable tool for EGF receptor kinase research.

Stereo(C7)‐dependent Topoisomerase II Inhibition and Tumor Growth Suppression by a New Quinolone, BO‐2367

A new antimicrobial quinolone (—)BO‐2367, (‐)‐7‐[(1R*,2R*,6R*)‐2‐amino‐8‐azabicyclo[4.3.0.]‐non‐3‐en‐8‐yl]‐l‐cyclopropyl‐6,8‐difluoro‐l,4‐dihydro‐4‐oxo‐3‐quinorinecarboxyric acid, strongly inhibited both mammalian and bacterial topoisomerase II. The IC50 values of (—)BO‐2367 against the DNA relaxation activity of L1210 topoisomerase II and the supercoiling activities of Escherichia coli gyrase and Micrococcus luteus gyrase were 3.8, 0.5, and 1 μM, respectively. This compound enhanced double‐stranded DNA cleavage mediated by topoisomerase II not only with purified enzyme, but also with intact L1210 cells. All these activities of (—)BO‐2367 were more than 2‐fold stronger than those of VP‐16. Intriguingly, (+)BO‐2367, which has an enantiomeric substitnent at the C7 position of (‐)BO‐2367, did not affect the activity of the mammalian topoisomerase II, while it inhibited E. coll gyrase. Intraperitoneal injection of (‐)BO‐2367 at 0.08 mg/kg increased the lifespan of CDF1female mice bearing ascitic L1210 leukemia by 2.4 times, and subcutaneous injection at 1.25 mg/kg completely inhibited the growth of colon 26 carcinoma implanted subcutaneously. These results suggest that (—)BO‐2367 is a potent antitumor agent which targets topoisomerase II. These enantiomers should be a useful tool for studying drug‐topoisomerase II interactions.