Motohiro Kobayashi

Antitumor activity of TZT-1027, a novel dolastatin 10 derivative.

Dolastatin 10, a pentapeptide isolated from the marine mollusk Dolabella auricularia, has antitumor activity. TZT‐1027, a dolastatin 10 derivative, is a newly synthesized antitumor compound. We evaluated its antitumor activity against a variety of transplantable tumors in mice. Intermittent injections of TZT‐1027 were more effective than single or repeated injections in rake with P388 leukemia and B16 melanoma. Consequently, TZT‐1027 shows schedule dependency. TZT‐1027 was effective against P388 leukemia not only when administered i.p., but also when given i.v. However, although TZT‐1027 given i.v. was active against murine solid tumors, TZT‐1027 administered i.p. was ineffective against all the tumors tested with the exception of colon 26 adenocarcinoma. The i.v. injection of TZT‐1027 at a dose of 2.0 mg/Ag remarkably inhibited the growth of three murine solid tumors; colon 26 adenocarcinoma, B16 melanoma and M5076 sarcoma, with T/C values of less than 6%. The antitumor activities of TZT‐1027 against these tumors were superior or comparable to those of the reference agents; dolastatin 10, cisplatin, vincristine, 5‐fluorouracil (5‐FU) and E7010. In experiments with drug‐resistant P388 leukemia, TZT‐1027 showed good activity against cisplatin‐resistant P388 and moderate activity against vincristine‐ and 5‐fluorouracil‐resistant P388, but no activity against adriamycin‐resistant P388. TZT‐1027 was also effective against human xenografts, that is, tumor regression was observed in mice bearing MX‐1 breast and LX‐1 lμng carcinomas. TZT‐1027 at 10 μM almost completely inhibited the assembly of porcine brain microtubules. Therefore, its mechanism of antitumor action seems to he, at least in part, ascrihable to the inhibition of microtubule assembly. Because of its good preclinical activity, TZT‐1027 has been entered into phase I clinical trials.

TZT-1027, an antimicrotubule agent, attacks tumor vasculature and induces tumor cell death

TZT‐1027, a dolastatin 10 derivative, is an antimicrotubule agent with potent antitumor activity both in vitro and in vivo. In this study, we performed biochemical and histopathological examinations, and evaluated TZT‐1027‐induced tumoral vascular collapse and tumor cell death in an advanced tumor model, murine colon 26 adenocarcinoma. In addition, we studied the effects of TZT‐1027 on cultured human umbilical vein endothelial cells (HUVEC). Tolerable doses of TZT‐1027 induced tumor‐selective hemorrhage within 1 h. This hemorrhage occurred mainly in the peripheral area of the tumor mass. Measurements of tumoral hemoglobin content and dye permeation revealed that the hemorrhage occurred firstly and tumor blood flow stopped secondarily. The vascular damage was followed by continuous induction of apoptosis of the tumor cells, tumor tissue necrosis, and tumor regression. In cultured HUVEC, TZT‐1027 induced marked cell contraction with membrane blebbing in 30 min. These cell changes were completely inhibited by K252a, a broad‐spectrum inhibitor of protein kinases. These effects of TZT‐1027 on both tumor vasculature and HUVEC were greater than those of vincristine. In conclusion, TZT‐1027 quickly attacked the well‐developed vascular system of advanced tumors by a putative protein kinase‐dependent mechanism, and then blocked tumor blood flow. Therefore, TZT‐1027 has both a conventional antitumor activity and a unique anti‐tumoral vascular activity, making it a potentially powerful tool for clinical cancer therapy.

Characterization of the Interaction of TZT-1027, a Potent Antitumor Agent, with Tubulin

TZT‐1027, a derivative of dolastatin 10 isolated from the Indian Ocean sea hare Dolabella auricularia in 1987 by Pettit et al., is a potent antimicrotubule agent. We have compared the activity of TZT‐1027 with that of dolastatin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS). TZT‐1027 and dolastatin 10 inhibited microtubule polymerization concentration‐dependently at 1–100 μM with IC50 values of 2.2±0.6 and 2.3±0.7 μM, respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1–3 μM with IC50 values of 2.7±0.6, 1.6±0.4 and 1.6±0.2 μM, respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 μM or more. TZT‐1027 also inhibited monosodium glutamate‐induced tubulin polymerization concentration‐dependently at 0.3–10 μM, with an IC50 of 1.2 μM, whereas VLB was only effective at 0.3–3 μM, with an IC50 of 0.6 μM, and caused so‐called “aggregation” of tubulin at 10 μM. Scatchard analysis of the binding data for [3H]VLB suggested one binding site (Kd 0.2±0.04 μM and Bmax 6.0±0.26 nM/mg protein), while that for [3H]TZT‐1027 suggested two binding sites, one of high affinity (Kd 0.2±0.01 μM and Bmax 1.7±0.012 nM/mg protein) and the other of low affinity (Kd 10.3±1.46 μM, and Bmax 11.6±0.83 nM/mg protein). [3H]TZT‐1027 was completely displaced by dolastatin 10 but only incompletely by VLB. [3H]VLB was completely displaced by dolastatin 10 and TZT‐1027. Furthermore, TZT‐1027 prevented [3H]VLB from binding to tubulin in a non‐competitive manner according to Lineweaver‐Burk analysis. TZT‐1027 concentrationdependently inhibited both [3H]guanosine 5′‐triphosphate (GTP) binding to and GTP hydrolysis on tubulin. VLB inhibited the hydrolysis of GTP on tubulin concentration‐dependently to a lesser extent than TZT‐1027, but no inhibitory effect of VLB on [3H]GTP binding to tubulin was evident even at 100 μM. Thus, TZT‐1027 affected the binding of VLB to tubulin, but its binding site was not completely identical to that of VLB. TZT‐1027 had a potent inhibitory effect on tubulin polymerization and differed from vinca alkaloids in its mode of action against tubulin polymerization.

Antitumor activity of TZT-1027 (Soblidotin) against vascular endothelial growth factor-secreting human lung cancer in vivo

TZT‐1027 (Soblidotin), an antimicrotubule agent, has been demonstrated to show potent antitumor effects, though the relationships among antitumor effect, cytotoxicity and anti‐vascular effect of TZT‐1027 have not been studied. We established in vivo human lung vascular‐rich tumor models using a vascular endothelial growth factor‐secreting tumor (SBC‐3/VEGF). SBC‐3/VEGF tumors exhibited a high degree of angiogenesis in comparison with the mock transfectant (SBC‐3/Neo) tumors in a dorsal skinfold chamber model and grew much faster and larger than SBC‐3/Neo tumors in the tumor growth study. The antitumor activity of antimicrotubule agents, including TZT‐1027, was evaluated in both early‐ and advanced‐stage SBC‐3/Neo and SBC‐3/VEGF tumor models to elucidate the relationship between the antitumor activity and anti‐vascular effect of these agents. TZT‐1027 exhibited potent antitumor activity against both early‐ and advanced‐stage SBC‐3/Neo and SBC‐3/VEGF tumors, whereas combretastatin A4 phosphate did not. Vincristine and docetaxel exhibited potent antitumor activity against early‐stage SBC‐3/Neo and SBC‐3/VEGF tumors, and advanced‐stage SBC‐3/Neo tumors, but did not exhibit activity against advanced‐stage SBC‐3/VEGF tumors. The difference in antitumor activity between these agents could be ascribed to differences in direct cytotoxicity and anti‐vascular effect. Furthermore, a prominent accumulation of erythrocytes in the tumor vasculature, followed by leakage and scattering of these erythrocytes from the tumor vasculature, was observed after TZT‐1027 administration to mice bearing advanced‐stage SBC‐3/VEGF tumors. These findings strongly suggest that TZT‐1027 has a potent anti‐vascular effect, in addition to direct cytotoxicity.

Comparison of the antivascular and cytotoxic activities of TZT-1027 (Soblidotin) with those of other anticancer agents

TZT-1027 (Soblidotin), a microtubule-depolymerizing agent exerts both a direct cytotoxic activity against cancer cells and an indirect antivascular activity against tumor vascular endothelial cells. We compared both activities of TZT-1027 with those of various anticancer agents having different mechanisms of action, including vinca alkaloids, a vascular targeting agent, a taxane and nonmicrotubule-binding agents. In the MTT assay, TZT-1027 most potently inhibited the growth of both murine colon C26 cancer cells and human umbilical vein endothelial cells, implying its potent antivascular activity against tumor vasculature in addition to its cytotoxic activity against cancer cells. Treatment with 0.1 μg/ml TZT-1027 significantly enhanced vascular permeability in human umbilical vein endothelial cell monolayers and a single intravenous administration of 2 mg/kg TZT-1027 significantly reduced the perfusion of Colon26 tumors implanted into mice, with efficacies superior to vinca alkaloids and comparable to a known vascular targeting agent. These results strongly suggest that TZT-1027 exerts marked antivascular activity. Next, to clarify the mechanism of the antivascular activity, we have taken a novel approach, and analyzed the relationships among human umbilical vein endothelial cells cytotoxicity, vascular permeability and tumor perfusion, on the basis of efficacies of each agent. Analyses revealed strong and significant correlations, and indicated that the vascular endothelial cell damage leads to endothelial barrier dysfunction and, thereby, tumor vascular shutdown. In summary, TZT-1027 was verified to have not only an excellent cytotoxic activity, but also an attractive antivascular activity through the induction of damage to vascular endothelial cells. We believe that these dual activities may make TZT-1027 useful for treating solid tumors.

Tumor-specific antivascular effect of TZT-1027 (Soblidotin) elucidated by magnetic resonance imaging and confocal laser scanning microscopy.

TZT‐1027 (soblidotin), an antimicrotubule agent, has previously been evaluated in terms of its antivascular effects. In this study, Evans blue perfusion, magnetic resonance imaging (MRI), and confocal laser scanning microscopy (CLSM) were utilized to further elucidate the antivascular effect of TZT‐1027 in female nude mice and rats bearing human breast tumor MX‐1, as well as in female Sprague‐Dawley rats that developed breast tumors induced by dimethylbenz(a)anthracene (DMBA). Therapeutic doses of TZT‐1027 caused nearly complete regression of implanted MX‐1 tumors in nude mice and rats as well as DMBA‐induced tumors in rats. The perfusion in MX‐1 tumor implanted in nude mice was drastically reduced within 30 min after TZT‐1027 administration and was completely inhibited after 6 h or more, although not reduced in normal tissue of kidney. The study using MRI demonstrated that rich blood flow within tumors was remarkably reduced 1–3 h after TZT‐1027 administration both in nude rats bearing MX‐1 tumors and in rats with DMBA‐induced tumors. Furthermore, the study with CLSM in nude mice bearing MX‐1 tumors revealed a disruption of tumor microvessels at 1 h and a destruction of tumor microvessel network at 3 h after TZT‐1027 administration. In contrast, these types of vascular disorders were not observed in heart and kidney. These results suggest that TZT‐1027 specifically damages tumor vasculatures, leading to extensive tumor necrosis within tolerable dose range, and confirms earlier observations that TZT‐1027 exerts a considerable antivascular effect in addition to an excellent cytotoxic effect. (Cancer Sci 2007; 98: 598–604)

Antivascular effects of TZT-1027 (Soblidotin) on murine Colon26 adenocarcinoma

We investigated the ability of TZT‐1027 (Soblidotin), a novel antimicrotubule agent, to induce antivascular effects, because most vascular targeting agents that selectively disrupt tumor vasculature also inhibit tubulin polymerization. Treatment with 10−7 g/mL TZT‐1027 rapidly disrupted the microtubule cytoskeleton in human umbilical vascular endothelial cells (HUVEC), and significantly enhanced vascular permeability in HUVEC monolayers. In addition, single intravenous administration of 2 mg/kg TZT‐1027 to mice bearing Colon26 tumors significantly reduced tumor perfusion and caused extravascular leakage of erythrocytes 1 h after administration. Subsequently, thrombus formation with deposition of fibrin and tumor necrosis was observed 3 and 24 h after administration, respectively. These results strongly suggest that TZT‐1027 possesses antivascular effects. TZT‐1027 induced apoptosis not only in HUVEC but also in C26 cancer cells (cell line of Colon26 solid tumor) in vitro, suggesting it exerts direct cytotoxicity against tumor cells in addition to its antivascular effects. A single intravenous administration of 1, 2 and 4 mg/kg TZT‐1027 significantly prolonged the survival of mice with advanced‐stage Colon26 tumors in a dose‐dependent manner. Furthermore, TZT‐1027 itself less markedly enhanced the permeability of normal vessels, but was additive with vascular endothelial growth factor, indicating the possibility that TZT‐1027 selectively exerts its activity on tumor vessels. In summary, these results suggest that TZT‐1027 exerts both an indirect antivascular effect and a direct cytotoxic effect, resulting in strong antitumor activity against advanced‐stage tumors, and that TZT‐1027 may be useful clinically for treating solid tumors. (Cancer Sci 2006; 97: 1410–1416)

Gene Expression Profiling of Exposure to TZT-1027, a Novel Microtubule-Interfering Agent, in Non-Small Cell Lung Cancer PC-14 Cells and Astrocytes

Clinical use of TZT-1027, amicrotubule-interfering agent that inhibitsthe polymerization of tubulin, is expectedbecause of its potent effects on solidtumors. TZT-1027 is thought to act directlyon cellular microtubules, and arrest cellmitosis, however, the molecular mechanismsof the microtubule damage by TZT-1027 havenot been fully identified. To investigatethe possible novel mechanisms of action ofTZT-1027, we used the cDNA macroarraytechnique to examine its effect on theexpression of hundreds of tightlytranscriptionally controlled genes. We usedtwo cell lines, one was human non-smallcell lung carcinoma PC-14 cells as a modelfor cancer cells, and the other was humanastrocytes as a model for normal neuronalcells, because the dose-limiting-factor ofmicrotubule-interfering agents is mainlyperipheral neurotoxicity. mRNAs preparedfrom the PC-14 and astrocyte cell linestreated with TZT-1027 were compared with588 genes spotted onto the filter, andwhich gene groups TZT-1027 modulatedbetween the two cell lines wasinvestigated. TZT-1027 exposure modulatedexpression of a variety of genes includingthe genes encoding cell-cycle and growthregulators, receptors, angiogenesis andinvasion regulators, rho family smallGTPases and their regulators and growthfactors and cytokines. However, the way ofgene regulation by TZT-1027 exposure wasdifferent between PC-14 cells andastrocytes. Genes up-regulated in bothPC-14 cells and astrocytes were those forRAR-ε, TNFR 1 and 2 and so on.Specifically altered genes in PC-14 cells,such as the genes coding for cytokeratin 8,XPG, fau and the genes regulated only inPC-14 cells may be involved in theantitumor activity of TZT-1027. On theother hand, growth factor receptorprecursors was upregulated specifically inastrocytes by TZT-1027 and this generegulation only in astrocytes may becandidates related with neurotoxicity.