BAY 12-9566, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity.

Matrix metalloproteinases (MMPs) have been implicated in tumor cell invasion, metastasis, and angiogenesis. BAY 12-9566, a novel, non-peptidic biphenyl MMP inhibitor, has shown preclinical activity on a broad range of tumor models and is currently in clinical development. The purpose of this study was to investigate the antiangiogenic activity of BAY 12-9566. In vitro, BAY 12-9566 prevented matrix invasion by endothelial cells in a concentration-dependent manner (IC50 = 8.4x10(-7) M), without affecting cell proliferation. In vivo, oral daily administration of BAY 12-9566 (50-200 mg/kg) inhibited angiogenesis induced by basic fibroblast growth factor in the Matrigel plug assay, reducing the hemoglobin content of the pellets. Histological analysis showed a reduction in the amount of functional vessels within the Matrigel. We conclude that the MMP inhibitor BAY 12-9566 inhibits angiogenesis, a property that further supports its clinical development as an antimetastatic agent.

Naamidine A is an antagonist of the epidermal growth factor receptor and an in vivo active antitumor agent.

The known 2-aminoimidazole alkaloid naamidine A (1) was isolated from a Fijian Leucetta sp. sponge as an inhibitor of the epidermal growth factor (EGF) receptor. The compound exhibited potent ability to inhibit the EGF signaling pathway and is more specific for the EGF-mediated mitogenic response than for the insulin-mediated mitogenic response. Evaluation in an A431 xenograft tumor model in athymic mice indicated that naamidine A exhibited at least 85% growth inhibition at the maximal tolerated dose of 25 mg/kg. Preliminary mechanism of action studies indicate that the alkaloid fails to inhibit the binding of EGF to the receptor and has no effect on the catalytic activity of purified c-src tyrosine kinase.

Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol).

Eleutherobin is a novel natural product isolated from a marine soft coral that is extremely potent for inducing tubulin polymerization in vitro and is cytotoxic for cancer cells with an IC50 similar to that of paclitaxel. This compound is cross-resistant along with other multidrug-resistant agents against P-glycoprotein-expressing cells and is cross-resistant with paclitaxel against a cell line that has altered tubulin. In mechanistic studies, eleutherobin shares with paclitaxel the ability to induce tubulin polymerization in vitro and is most likely cytotoxic by virtue of this mechanism. Human colon carcinoma cells exposed to eleutherobin contain multiple micronuclei and microtubule bundles, and they arrest in mitosis, depending on concentration, cell line, and length of exposure. These morphological abnormalities appearing in cultured cells are indistinguishable from those induced by paclitaxel. Electron microscopy reveals that eleutherobin induces homogeneous populations of long, rigid microtubules similar to those formed by paclitaxel. Thus, eleutherobin is a new chemotype with a mechanism of action similar to that of paclitaxel and, as such, has promising potential as a new anticancer agent.

Doxorubicin disaccharide analogue: apoptosis-related improvement of efficacy in vivo.

BACKGROUND: Although doxorubicin remains one of the most effective agents for the treatment of solid tumors, there is an intensive effort to synthesize doxorubicin analogues (compounds with similar chemical structures) that may have improved antitumor properties. We have synthesized a novel doxorubicin disaccharide analogue (MEN 10755) and have characterized some of its relevant biochemical, biologic, and pharmacologic properties. METHODS: The antitumor activity of this compound (MEN 10755) was studied in a panel of human tumor xenografts, including xenografts of A2780 ovarian tumor cells, MX-1 breast carcinoma cells, and POVD small-cell lung cancer cells. MEN 10755 was compared with doxorubicin according to the optimal dose and schedule for each drug. The drug's cytotoxic effects, induction of DNA damage, and intracellular accumulation were studied in A2780 cells. DNA cleavage mediated by the enzyme topoisomerase II was investigated in vitro by incubating fragments of simian virus 40 DNA with the purified enzyme at various drug concentrations and analyzing the DNA cleavage-intensity patterns. Drug-induced apoptosis (programmed cell death) in tumors was determined with the use of MX-1 and POVD tumor-bearing athymic Swiss nude mice. RESULTS: MEN 10755 was more effective than doxorubicin as a topoisomerase II poison and stimulated DNA fragmentation at lower intracellular concentrations. In addition, MEN 10755 exhibited striking antitumor activity in the treatment of human tumor xenografts, including those of the doxorubicin-resistant breast carcinoma cell line MX-1. CONCLUSIONS: The high antitumor activity of MEN 10755 in human tumor xenografts, including doxorubicin-resistant xenografts, and its unique pharmacologic and biologic properties make this disaccharide analogue a promising candidate for clinical evaluation.

Preclinical antitumor activity of water-soluble paclitaxel derivatives.

PURPOSE: Five water-soluble paclitaxel derivatives were extensively evaluated for their antitumor activities relative to the parent drug. METHODS: Both subcutaneous (s.c.) murine (M109 lung) and human (A2780 ovarian, L2987 lung) tumor models were used for this purpose. RESULTS: Consecutive daily intravenous (i.v.) paclitaxel therapy of mice bearing s.c. M109, beginning on day 4 or 5 posttumor implant and continuing for 5 days, resulted in a range of maximum gross log cell kill (LCK) values (reflective of delays in tumor growth) and maximum relative median survival time (% T/C) values (reflective of increases in lifespan) of 1.0-2.1 and 132-162% (and one outlying result of 235%), respectively. Against the same tumor model, using the same treatment schedule, each of the water-soluble derivatives was active, with maximum LCK of 1.3-2.5 and T/C of 124-254%. These LCK and %T/C values were always within 0.5 LCK and 15%, respectively, of the concomitantly obtained maximum effects of paclitaxel. When tested in several experiments against staged (50-100 mg) s.c. A2780 tumors, using various i.v. treatment schedules, the water-soluble derivatives achieved a maximum LCK of 1.4-3.8. Evaluated in parallel, paclitaxel achieved a maximum LCK of 2.1-4.5 following every other day x 5 i.v. therapy. When paclitaxel was assayed in several experiments using the staged (50-100 mg) s.c. L2987 tumor model, maximum LCK of 0.9->4.1 were produced following every other day x 5 i.v. therapy. Concomitant testing of the water-soluble derivatives, using the same i.v. treatment schedule, resulted in maximum LCK of 0.2->4.1. In each of the tumor models used, the consistently active, and usually the most active, water-soluble derivative was BMS-185660. The levels of activity observed were comparable (within 1 LCK) to those achieved concomitantly using paclitaxel, and its potency was only slightly inferior to the parent drug. CONCLUSIONS: Based on the evaluations performed in three distal site tumor models, we conclude that BMS-185660 is a water-soluble paclitaxel derivative with preclinical antitumor activity comparable to that of the parent drug.

Effect of linker variation on the stability, potency, and efficacy of carcinoma-reactive BR64-doxorubicin immunoconjugates.

The internalizing anti-Le(y) monoclonal antibody (MAb) BR64 was conjugated to the anticancer drug doxorubicin (DOX) using an acid-labile hydrazone bond to the DOX and either a disulfide or thioether bond to the MAb. The resulting disulfide (BR64-SS-DOX) and thioether (BR64-S-DOX) conjugates were evaluated for stability, potency, and antigen-specific activity in both in vitro and in vivo model systems. The BR64-SS-DOX conjugates demonstrated antigen-specific activity both in vitro and when evaluated against antigen-expressing, DOX-sensitive human carcinoma xenografts. However, the stability and potency of disulfide conjugates were poor, and in vivo activity superior to unconjugated DOX was seen only at doses approaching the maximum tolerated dose. Furthermore, BR64-SS-DOX conjugates were not active against antigen-expressing, DOX-insensitive colon tumor xenografts. In contrast, the BR64-S-DOX conjugates demonstrated good stability both in vitro and in vivo. The increased stability of the BR64-S-DOX conjugates resulted in the delivery of more biologically active DOX to tumors with a concomitant increase in potency and efficacy over that which could be achieved with either unconjugated DOX or BR64-SS-DOX conjugates. Delivery of DOX by BR64-SS-DOX conjugates resulted in complete regressions and cures of both DOX-sensitive lung xenografts and DOX-intensitive colon tumor xenografts. These results demonstrate the importance of linker stability when delivering drugs such as DOX to carcinomas via internalizing antibodies and are likely to have direct relevance to the clinical utility of MAb-directed delivery.

Paclitaxel (Taxol(R)) inhibits motility of paclitaxel-resistant human ovarian carcinoma cells.

The effect of paclitaxel on the adhesive and motility properties of human ovarian carcinoma cell lines was investigated. Paclitaxel significantly inhibited the motility of OVCAR 5, SK-OV-3, and HOC-1OTC ovarian carcinoma cell lines (IC50 = 2.1 x 10(-8), 2 x 10(-9), and 1.9 x 10(-8) m, respectively) but did not affect the adhesion of these cells to the subendothelial matrix. The association between inhibition of motility and cytotoxic activity was investigated using an A2780 subclone (1A9) and three paclitaxel-resistant variants (designated 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18). Although paclitaxel did not significantly affect the adhesion to subendothelial matrix of the sublines, it completely inhibited their migration. Inhibition of migration was similar in 1A9 cells and the resistant sublines, with an IC50 of 1 x 10(-8) for 1A9 cells and 5.4 x 10(-9), 1.1 x 10(-8), and 5.2 x 10(-9) m for 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively. Paclitaxel inhibited motility induced by soluble attractant (chemotaxis) and immobilized attractant (haptotaxis). Inhibition of cell motility occurred in the absence of an antiproliferative effect, because higher concentrations of paclitaxel were required to inhibit tumor cell proliferation (IC50 = 1.9 x 10(-7) and 4.6 x 10(-6), 1 x 10(-5), and 3.1 x 10(-6) m for 1A9 and 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively). These data show that paclitaxel is a potent inhibitor of ovarian carcinoma cell motility and that this activity is independent of its cytotoxic activity.

Structure-activity relationships of VP-16 analogues.

A total of 27 selected analogues of VP-16 and VM-26 were compared with VP-16 and VM-26 for their relative abilities to stabilize the enzyme-substrate intermediate normally formed between eukaryote topoisomerase II and DNA. This activity was compared with cytotoxicity results obtained using the human colon HCT116 cell line and antitumor results obtained after intraperitoneal injection of mice with murine leukemia P388. The most potent analogues were those containing OH groups (demethyl) in either the 3' and 4' or the 3', 4', and 5' positions, the latter being twice as potent as VP-16. VM-26 was only 40% more potent than VP-16 in this assay. It was generally found that the 4'-esters had little activity in vitro, yet were cytotoxic and had antitumor activities. All other analogues with little in vitro activity were not very cytotoxic and had little if any antitumor activity. A very good correlation exists between stabilization of topoisomerase II-DNA intermediates, cytotoxicity, and antitumor activity.

Reversal of the human and murine multidrug-resistance phenotype with megestrol acetate.

MA is an orally active PG derivative with an excellent safety profile that is used primarily for the treatment of carcinomas of the breast and endometrium. We investigated the potential application of MA as an MDR-reversal agent using cell culture and human tumor xenograft models. The reversing activity of MA in vitro was compared with that of PG and VER in two human MDR cell lines, the colon carcinoma HCT-116/VM46 and the breast carcinoma MCF-7/ADR, and in a murine cell line, J774.2. At concentrations as low as 3 microM, MA was capable of partially restoring sensitivity to Act D in the HCT-116/VM46 cells and sensitivity to DOX in the MCF-7/ADR cells. Although less effective than VER, MA was about 2.5 times more potent than PG in reversing MDR at equimolar concentrations. Increased accumulation of DOX in drug-resistant cells that were treated simultaneously with MA was observed by flow cytometry. In vivo, using established human colon and breast carcinoma xenografts implanted s.c. in athymic mice, the combined therapy with MA and DOX resulted in enhanced antitumor activity relative to that of DOX alone in the MDR sublines. These results suggest that MA may be a promising clinical MDR-reversing agent.

Selective proteolytic activity of the antitumor agent kedarcidin.

Kedarcidin is a potent antitumor antibiotic chromoprotein, composed of an enediyne-containing chromophore embedded in a highly acidic single chain polypeptide. The chromophore was shown to cleave duplex DNA site-specifically in a single-stranded manner. Herein, we report that in vitro, the kedarcidin apoprotein, which lacks any detectable chromophore, cleaves proteins selectively. Histones that are the most opposite in net charge to the apoprotein are cleaved most readily. Our findings imply that the potency of kedarcidin results from the combination of a DNA damaging-chromophore and a protease-like apoprotein.

Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates.

Immunoconjugates (BR96-DOX) were prepared between chimeric monoclonal antibody BR96 and the anticancer drug doxorubicin. The monoclonal antibody binds an antigen related to Lewis Y that is abundantly expressed at the surface of cells from many human carcinomas; it has a high degree of tumor selectivity and is internalized after binding. BR96-DOX induced complete regressions and cures of xenografted human lung, breast, and colon carcinomas growing subcutaneously in athymic mice and cured 70 percent of mice bearing extensive metastases of a human lung carcinoma. Also, BR96-DOX cured 94 percent of athymic rats with subcutaneous human lung carcinoma, even though the rats, like humans and in contrast to mice, expressed the BR96 target antigen in normal tissues.

Kedarcidin chromophore: an enediyne that cleaves DNA in a sequence-specific manner.

Kedarcidin chromophore is a 9-membered enediyne, recently isolated from an actinomycete strain. In vivo studies show this molecule to be extremely active against P388 leukemia and B16 melanoma. Cytotoxicity assays on the HCT116 colon carcinoma cell line result in an IC50 value of 1 nM. In vitro experiments with phi X174, pM2 DNA, and 32P-end-labeled restriction fragments demonstrate that this chromophore binds and cleaves duplex DNA with a remarkable sequence selectivity producing single-strand breaks. The cleavage chemistry requires reducing agents and oxygen similar to the other naturally occurring enediynes. Certain cations (Ca2+ and Mg2+) prevent strand cleavage. High-resolution 1H NMR studies on the chromophore in the presence of calcium chloride implicate the 2-hydroxynaphthoyl moiety in DNA binding. Interestingly, the kedarcidin chromophore appears structurally related to neocarzinostatin yet recognizes specific DNA sequences in a manner similar to calicheamicin gamma 1I, an enediyne with a significantly different structure. Moreover, kedarcidin and calicheamicin share a DNA preferred site, the TCCTN-mer. These observations indicate that the individual structural features of these agents are not solely responsible for their DNA selectivity. Rather, a complementarity between their overall tertiary structure and the local conformation of the DNA at the binding sites must play a significant role in the recognition process.

Preclinical antitumor activity of orally administered platinum (IV) complexes.

Several novel platinum (IV) mixed ammine/amine dicarboxylate dichlorides of general structure [Pt(IV)Cl2(OCOY)2NH3(XNH2)], where Y is aliphatic or aromatic and X is alicyclic or aliphatic, known to be particularly well absorbed following oral administration, were evaluated by that route for their antitumor activity. Testing of the Pt(IV) derivatives took place concomitantly with i.v. administered cisplatin and carboplatin in two s.c. staged tumor models, the murine M5076 sarcoma and human A2780 ovarian carcinoma. Based upon repetitive experiments which included an evaluation of different vehicles and treatment schedules, each of the orally administered Pt(IV) dicarboxylates was reproducibly active in the M5076 tumor, producing mean maximum gross log cell kill (LCK) values of between 1.5 and 2.0, and lifespan increases, reflected by mean maximum treated/control median survival (T/C) values, of 139-151%. Cisplatin and carboplatin given i.v. yielded mean maximum LCK of 3.5 and 2.5, respectively, as well as mean maximum T/C values of 166% and 164%, respectively, in the same tumor model. The best of the derivatives in the M5076 experiments, JM-216 [ammine/cyclohexylamine diacetato dichloride Pt(IV)], produced LCK values that averaged only 0.5 lower than that of carboplatin, and increases in lifespan not significantly different than that of carboplatin. Against the A2780 tumor, the Pt(IV) dicarboxylates produced individual best effects of between 0.8-1.1 LCK, based on data from two or three experiments. The mean maximum LCK values for cisplatin and carboplatin were 1.8 and 2.2 LCK, respectively. JM-225, ammine/cyclopentylamine diacetato dichloride Pt(IV), was active in two of three experiments, including one result comparable to that of carboplatin. The Pt(IV) mixed ammine/amine dicarboxylate dichlorides represent a novel class of Pt derivative capable of expressing oral antitumor activity in both murine and human tumor models.

Mechanisms of resistance to etoposide and teniposide in acquired resistant human colon and lung carcinoma cell lines.

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.

Preclinical antitumor activity of a soluble etoposide analog, BMY-40481-30.

BMY-40481-30 is a new, water-soluble derivative and probable prodrug of etoposide characterized by the presence of a phosphate group in position 4' of the E ring of the etoposide molecule. The compound was only weakly cytotoxic in vitro and, consequently, an investigation of its antitumor activity was conducted in several murine and human tumor (xenograft) models. Etoposide was administered ip or po whereas BMY-40481-30 was given ip, po or iv. The potency of the derivative, when administered parenterally, as defined on the basis of maximum tolerated dose (MTD), was less than the parent compound on a weight (mg/kg) basis in some experiments but comparable to etoposide in other instances. Comparison at the MTD of the two compounds showed that BMY-40481-30 administered ip was as active as etoposide against ip P388 leukemia. BMY-40481-30 given iv was more active than etoposide given ip in two of five experiments versus iv P388 leukemia, but the two compounds were comparably active in the other three studies. Of particular interest was the finding that the derivative was more active than the parent compound at many of the comparable (on a mg/kg basis) dose levels of both evaluated po versus iv P388 leukemia; MTD levels were not achieved, and hence not compared, for either compound using the po route of administration. Both etoposide and BMY-40481-30 yielded comparable maximum effects against ic P388 leukemia, ic L1210 leukemia, and sc B16 melanoma, but etoposide was more efficacious versus sc M5076 sarcoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical and biological characterization of 4'-iodo-4'-deoxydoxorubicin.

4'-Iodo-4'-deoxydoxorubicin is a doxorubicin (DXR) analogue with greater lipophilicity and reduced basicity of the amino group. In vitro 4'-iodo-4'-deoxydoxorubicin is more cytotoxic than DXR against a panel of human and murine cell lines and is characterized by a higher and faster uptake. In vivo, the spectrum of activity of 4'-iodo-4'-deoxydoxorubicin is comparable to that of DXR, but the new compound has higher activity against murine P388 leukemia resistant to DXR and against pulmonary metastases from Lewis lung carcinoma. Moreover, the new analogue exhibits antitumor activity also after p.o. administration and shows no cardiotoxicity in experimental systems.

Biochemical and biological activity of the anthracycline analog, 4-demethyl-6-O-methyl-doxorubicin.

The chromophore-modified derivative of doxorubicin, 4-demethyl-6-O-methyl-doxorubicin, has been tested for antitumor activity in a range of experimental murine tumor systems. In contrast to the inactive 6-O-methyl derivative of daunorubicin, 4-demethyl-6-O-methyl-doxorubicin provided antitumor effects comparable to that of the parent compound. In addition, detailed DNA-interaction studies showed that the doxorubicin derivative retains the ability to bind DNA by the intercalation mechanism. However, the binding affinity was appreciably reduced following structural modification in the anthraquinone chromophore. On the basis of the proposed models of intercalation, these results could be rationalized in terms of steric influence of the bulky methoxy group. The results of this study are in agreement with the correlation already observed between DNA binding and relative antitumor activity of anthracyclines.