Enzymatic reactivity and anti-tumor activity of 1-(beta-D-arabinofuranosyl)-2-thiocytosine derivatives.

Sixteen derivatives of 1-(beta-D-arabinofuranosyl)-2-thiocytosine (araSC), including five 5'-esters, three 3'-esters, five N4-amides and three 5'-phosphodiesters, were synthesized and their reactivity to mouse tissue homogenates, including plasma, liver and intestine, and antitumor activity in mice bearing P388 cells were measured. The ester derivatives had a potent effect on the enzyme systems while the amide and phosphodiester derivatives were less active. The reactivity of ester derivatives was highly dependent on their chemical structure. The reactivity of amides and phosphodiester derivatives on mouse plasma and intestinal homogenate was also dependent on the chemical structure, although their action on intestinal enzymes was very similar. Two of eight ester derivatives showed considerable antitumor activity in vivo, although they also showed serious toxicity indicated by a weight loss in the mice. Four out of five amides and two out of three phosphodiesters showed antitumor activity, and two were highly effective (>200% in T/C, the ratio of the mean survival time of the treated group to that of the control group) with only a very slight weight loss.

Suppression of ascites formation and re-accumulation associated with human ovarian cancer by an anti-VPF monoclonal antibody in vivo.

Ascites formation is often observed in ovarian cancer patients. Vascular permeability factor (VPF) may induce ascites formation. We established an animal model of ascites formation and re-accumulation by i.p. transplantation of a human ovarian adenocarcinoma cell line, NOS2, into nude mice. The formation of ascites was observed after 10 days of tumor inoculation and continued for up to 4 weeks. In the ascitic fluid, biologically active VPF was detected. The repeated i.p. administration of an immunoneutralizing monoclonal antibody (MAb) to VPF, MV833, significantly inhibited the formation of ascites throughout the experiments. Re-accumulation of ascites occurred quickly in control mice after aspiration of ascites and these mice died within 20 days. MV833 again inhibited the re-accumulation of ascites and significantly prolonged the life span of mice without any side effect. These results indicate that VPF plays an important role in the accumulation of ascites induced by ovarian cancer and an anti-VPF MAb is a new specific drug to suppress the formation and re-accumulation of ascites. This MAb may contribute to ameliorating quality of life of cancer patients as well as prolong their survival.

Antitumor effect on human gastric cancer and induction of apoptosis by vascular endothelial growth factor neutralizing antibody.

Induction of apoptosis by antiangiogenic therapy has been suggested as a new anticancer strategy. To clarify the mechanism of the antitumor effect achieved by inhibition of vascular endothelial growth factor (VEGF), which is a major mediator of angiogenesis, we used an orthotopic transplantation model of human gastric carcinoma line (MT2) treated with a monoclonal VEGF neutralizing antibody (VEGF Ab). We histologically examined the microvessel density (MVD) and the apoptotic index (AI) in this model. Transplanted tumor growth was significantly inhibited by the VEGF Ab (P = 0.03), and there was a significant decrease in the number of mice with liver metastasis (P = 0.004). The MVD detected by immunohistochemical staining with ER-MP12 antibody was 33.6 +/- 8.0 in the control group and 21.1 +/- 5.4 in the treated group, and the difference was significant (P < 0.0001). The AI values of the control and treated groups were 4.73 +/- 1.11 and 7.26 +/- 1.62, respectively, and this difference is also significant (P < 0.0001). However, the expression of VEGF mRNA in transplanted tumors did not show a significant difference between the control and treated groups. These results suggest that the antitumor effect of the VEGF Ab on human gastric carcinoma is exerted by inducing mild hypoxia followed by apoptosis, which does not influence VEGF mRNA expression in the carcinoma.

Wide spectrum of antitumor activity of a neutralizing monoclonal antibody to human vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is known as an angiogenic factor for tumor angiogenesis. We developed a neutralizing anti-VEGF monoclonal antibody (MAb), MV833, and examined its antitumor activity against 27 human tumor cell lines transplanted in nude mice. All the tumor cell lines used in this study secreted various amounts of VEGF into culture medium in vitro. However, the growth of the cell lines, including three which expressed VEGF receptor, was not affected by exogenously added MV833 in vitro. All tumor cell lines including colon, lung, breast, pancreas, and melanoma, grew subcutaneously in nude mice. The growth of HeLa/v5, which had been transformed by human VEGF121 gene and secreted large amounts of VEGF, was significantly faster than that of the control vector transformant. Although the amounts of VEGF secreted from two HeLa transformants differed greatly, MV833 completely inhibited the growth of both tumors. Moreover, the growth of the other 25 human tumor cell lines transplanted into nude mice was also strongly suppressed by MV833. Neither the amount of VEGF secreted from each tumor cell line in vitro nor the expression of VEGF receptor correlated with the antitumor activity of MV833. MV833, administered when tumor volumes reached 400 mm3, completely inhibited the growth of some tumor lines. The results show VEGF to be a critical angiogenic factor for many tumors. VEGF-neutralizing antibody could be applicable as an antitumor agent for a wide range of tumors.

Cytotoxicity and pharmacokinetics of 1-beta-D-arabinofuranosyl-2-thiocytosine, a 2-sulphur substituted derivative of cytarabine.

1-(beta-D-Arabinofuranosyl)-2-thiocytosine (araSC), a 2-substituted derivative of cytarabine (araC), has been investigated for its cytotoxicity, enzymatic stability, plasma concentration-time profile in mice, and cytokinetics. This derivative showed strong cytotoxicity in several mammalian cell lines, although activity (IC50s) was weaker than araC. Greater stability to mouse cytidine deaminase was observed; the half-life in the presence of the enzyme was about 4-times longer than that of araC. The plasma concentration-time profile in mice in vivo showed prolonged retention of araSC when compared with araC. Cytokinetic study using flow cytometry indicated a non-S-phase specific effect of this compound.

Antitumor effect of a neutralizing antibody to vascular endothelial growth factor on liver metastasis of endocrine neoplasm.

Distant metastasis of gastrointestinal endocrine neoplasm is resistant to currently available treatments. Because hematogenic metastasis is dominant, anti-angiogenic drugs are expected to be a novel therapy for this neoplasm. In the present study, the therapeutic effect of vascular endothelial growth factor neutralizing antibody (VEGFAb) on liver metastasis of an endocrine neoplasm was investigated experimentally. Cecal transplantation into nude mice of small pieces of EN-1, a xenotransplanted human intestinal endocrine neoplasm, resulted in liver metastasis. A treated group (n = 19) received 100 micrograms/mouse of VEGFAb intraperitoneally on alternate days from day 10 after tumor transplantation, and the control group (n = 19) received saline. Five of the 19 control mice died of tumor progression, of which 2 could not be evaluated. The cecal tumor weighed 6316 +/- 2333 mg (n = 17) in the control group and 1209 +/- 837 mg (n = 19) in the treated group (P < 0.01) 6 weeks after transplantation. Liver metastasis developed in 16 of 17 control mice and in 2 of 19 treated mice (P < 0.01). The VEGF level of the whole cecal tumor in the control group was significantly higher than that in the treated group (305.1 +/- 174.1 vs. 54.7 +/- 41.2 mg; P < 0.001). VEGFAb did not cause any body weight loss (28.52 +/- 1.63 in the control vs. 28.44 +/- 1.71 g in the treated group). These results indicate that VEGFAb may be a novel therapeutic agent for endocrine neoplasm with distant metastasis.

Structure-cytotoxic activity relationship for the toad poison bufadienolides.

The toad poison bufadienolides including natural and derivatized compounds were tested for their cytotoxic effects on primary liver carcinoma cells PLC/PRF/5 and their structure-cytotoxic activity relationships were studied. For this study, a ligand-binding model was developed by using a pharmacophore mapping program, Distance Comparisons (DISCO). The structural features that are common to the 3D structures of active bufadienolides were identified to provide approach to a 3D QSAR method by using Comparative Molecular Field Analysis (CoMFA) study and to correlate the steric and electrostatic fields of the molecules to their activities. A valuable model which enables prediction of their activities was obtained from the CoMFA analysis, which may be employed for the drug designs of new bufadienolide analogues.

Anti-tumor and anti-metastatic effects of human-vascular-endothelial-growth-factor-neutralizing antibody on human colon and gastric carcinoma xenotransplanted orthotopically into nude mice.

In order to determine whether the inhibition of vascular-endothelial-growth-factor (VEGF) activity by administration of an immunoneutralizing antibody could suppress tumor growth and metastasis in spontaneous metastatic models of human colon and gastric carcinoma, 4 human carcinoma xenografts, 2 human colon carcinomas (TK4 and TK 13) and 2 gastric carcinomas (MT2 and MT5) were transplanted orthotopically into nude mice. The anti-VEGF antibody (MV833, 100 microg/mouse) or the same volume of saline was administered i.p. on alternative days from day 10 after transplantation. With each of the 4 xenografts, administration of MV833 significantly inhibited not only primary tumor growth but also macroscopic liver metastasis, although the growth rate varied. The inhibitory effect of MV833 on primary tumor growth appeared to have no correlation with the level of VEGF in tumor. Body-weight gain in each treated group was comparable with that in the control group. No toxicity of the antibody was observed. These results suggest that an anti-VEGF antibody can be effective against a wide variety of cancers, and that VEGF may be a possible target for cancer therapy.

An anti-human VEGF monoclonal antibody, MV833, that exhibits potent anti-tumor activity in vivo.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor for tumor angiogenesis and growth. We previously established the immunoneutralizing monoclonal antibodies (MAbs) to human VEGF, and showed that MV101 (IgG1) and MV303 (IgG2a) inhibited the growth of human solid tumor xenografts in nude mice. Then, we tried to develop another immunoneutralizing anti-VEGF MAb that exhibited more potent antitumor activity than MV101 or MV303. We obtained more than 140 clones of hybridomas that were producing anti-VEGF MAb from the mice immunized with recombinant human VEGF121. Among them, 26 clones showed the immunoneutralizing activity and MV833 possessed the most potent antitumor activity in vivo. A total of 9 i.p. administrations of 25 microg of MV833 inhibited the growth of human fibrosarcoma HT-1080 solid tumor xenografted in nude mice more potently than MV101 or MV303. Moreover, only 1 i.v. administration of 100 microg of MV833 on Day 1 after tumor inoculation also significantly inhibited the growth of HT-1080 in vivo, whereas MV101 and MV303 did not. All three MAbs inhibited the growth of human umbilical vein endothelial cells (HUVEC) induced by VEGF121 and the binding of 125I-labeled VEGF121 to HUVEC to a similar extent. The binding of MV101 and MV303 to VEGF121 was cross-competitive; however, MV833 weakly competed with the binding of MV101 to VEGF121. These findings indicated that MV833 recognized the region(s) of VEGF differently than MV101 or MV303, and this difference contributed to the superiority of antitumor activity of MV833.

Inhibition of tumor growth and metastasis by an immunoneutralizing monoclonal antibody to human vascular endothelial growth factor/vascular permeability factor121.

We elucidated the relationship between vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), which is a potent angiogenic factor, and the growth of primary and metastatic tumors using an immunoneutralizing monoclonal antibody against human VEGF/VPF121. The monoclonal antibody, MV303, suppressed the growth of human umbilical vein endothelial cells (HUVEC) induced by VEGF/VPF121 or VEGF/VPF165 but did not inhibit its growth induced by basic fibroblast growth factor. MV303 inhibited the binding of 125I-VEGF/VPF121 to HUVEC. We examined the effects of MV303 on tumor angiogenesis using a membrane chamber packed with the human fibrosarcoma cell line HT-1080 and implanted s.c. into BALB/c mice. The neovascularization induced by HT-1080 was inhibited by the i.v. injection of MV303 at a dose of 100 micrograms/mouse. Furthermore, the growth of solid tumors of s.c. implanted HT-1080 in BALB/c nude mice was almost completely inhibited by the i.v. and s.c. administration of MV303 ten times from day 1 at a dose of 100 micrograms/mouse (T/C values of tumor volume at day 18 were 0.20 and 0.18, respectively). Tumor growth was suppressed when MV303 was administered, even from eight days after tumor inoculation. MV303 suppressed the increase in lung weight caused by experimental metastasis with i.v. inoculation of cultured HT-1080 cells to BALB/c nude mice. The life spans of the mice treated with MV303 were significantly prolonged. These results indicated that VEGF/VPF played an important role in both primary and metastatic tumor growth as a tumor angiogenesis factor. MV303, an immunoneutralizing monoclonal antibody against VEGF/VPF, potently inhibited both primary and metastatic tumor growth with no marked side effects.

Isolation and characterization of neutralizing monoclonal antibodies to human vascular endothelial growth factor/vascular permeability factor121 (VEGF/VPF121).

We have established monoclonal antibodies (MAbs) against human vascular endothelial growth factor/vascular permeability factor121 (VEGF/VPF121). Two (MV101 and MV303) of the 28 MAbs neutralized the mitogenic activity of VEGF/VPF121 on human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. Both of the MAbs reacted to VEGF/VPF121 and also VEGF/VPF165 with somewhat different binding properties in a sandwich-type enzyme-linked immunosorbent assay (ELSIA). The binding of MV101 and MV303 to VEGF/VPF121 was competitive, but MV415, another anti-VEGF/VPF121 MAb without neutralizing activity, did not complete with either of the antibodies. MV101 and MV303 specifically recognized the native form of VEGF/VPF121 and VEGF/VPF165 in Western blotting. They did not react with VEGF/VPF when the antigens were fractionated under reducing conditions. These observations suggested that MV101 and MV303 might recognize the epitopes closely located on the configuration of VEGF/VPF121 molecule and the epitopes recognized by MV101 and MV303 may play an important role in the VEGF/VPF-receptor signal transduction. These MAbs significantly suppressed the growth of a human hepatoma, PLC/PRF/5, in vivo.