Synergistic Anti-glioma Effects in Vitro and in Vivo of Enediyne Antibiotic Neocarzinostatin and Paclitaxel via Enhanced Growth Delay and Apoptosis-Induction.

Neocarzinostatin (NCS) is a member of enediyne antibiotics with high anticancer potential. Our study was performed to explore the synergistic anti-glioma effects of NCS and paclitaxel (PTX) in vitro and in vivo. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicities of the drugs to human glioma cells U87MG and rat glioma cells C6 were evaluated. The results showed that the combinations of NCS and PTX can synergistically inhibit glioma cells survival. Cell apoptosis was detected by flow cytometry, and the results showed that the combinations of NCS and PTX synergistically enhanced apoptosis ratio of glioma cells. Western blot revealed that the cell signaling pathways of proliferation and apoptosis were synergistically regulated, in which Akt was synergistically inactivated, p53 was up-regulated with down-regulation of bcl-2. Meanwhile, with the subcutaneous model of U87MG cells and intracerebral implantation model of C6 cells, the combination strategy could synergistically delay the glioma growth and significantly prolong the survival of rats bearing orthotopic glioma. This study demonstrates that the combination of NCS and PTX can potentiate the effect on survival and apoptosis of glioma cells via suppression of Akt, bcl-2, and activations of p53; Meanwhile, the in vivo studies also confirmed that the combination of NCS and PTX synergistically inhibit the gliom growth. Our data about the combinational effects of NCS with PTX may provide an alternative strategy for glioma therapy.

[Hepatocellular carcinoma with multiple lung metastasis resulting in long-term disease-free survival by transcatheter arterial infusion chemotherapy of SMANCS].

The patient was a 61-year-old female with alcoholic liver cirrhosis, who was admitted to our hospital due to elevation of AFP.During the evaluation, both abdominal ultrasound and enhanced abdominal CT revealed a hepatocellular carcinoma measuring 4 cm in the S6-7 region, complicated with an arteriovenous shunt.Additionally, the lung CT examination showed 20 isolated bilateral lung tumors, all of which were less than 1.4 cm in diameter. Following the diagnosis, we performed a transcatheter arterial infusion chemotherapy of SMANCS at 3 mg through the right heptic artery. Thereafter, the AFP level returned to normal. Additionally, the tumors previously observed in both liver and lung, and exhibited by both lung CT and enhanced abdominal MRI, had disappeared.The patient has been in clinical remission more than 10 years to date.

Styrene maleic acid neocarzinostatin treatment for hepatocellular carcinoma.

A variety of treatments have recently been introduced to improve the prognosis of hepatocellular carcinoma (HCC). These anticancer therapies include the oily carcinostatic agent styrene maleic acid neocarzinostatin (SMANCS). SMANCS is a chemical conjugate of a synthetic copolymer of styrene maleic acid (SMA) and the proteinaceous anti-cancer agent neocarzinostatin (NCS), which dissolves in organic solvents such as pyridine and acetone, and particularly in Lipiodol. NCS is a simple protein capable of inhibiting DNA synthesis and inducing DNA degradation. Lipiodol is an ethyl ester of iodinated poppy seed oil in which most of the unsaturated double bonds in oleic, linoleic and linolenic acid are almost completely iodinated. When a homogeneous suspension of SMANCS with Lipiodol (SMANCS/Lipiodol) is administered intra-arterially, Lipiodol acts as a carrier of SMANCS. Many studies have demonstrated the clinical efficacy of SMANCS/Lipiodol in the treatment of HCC. We have shown that transcatheter arterial infusion (TAI) with SMANCS/Lipiodol has a more favorable focal therapeutic effect than does epirubicin in Lipiodol in the initial treatment of HCC. However, recent clinical studies have indicated that SMANCS causes severe adverse reactions and complications. We have also reported a case of HCC in which multifocal hepatic infarction developed after TAI with SMANCS/Lipiodol. Arterial administration of SMANCS/Lipiodol, therefore, should be given as peripherally as possible via the tumor feeding arteries, to enhance the efficacy of the agent and to reduce the adverse effects.

[A preliminary report on the treatment of pleural carcinomatosis with SMANCS].

To clarify the effect of SMANCS on malignant pleural carcinomatosis, seven patients with malignant pleural effusion were treated with SMANCS administered via an intracavitary route. Five patients showed improvement after one or two injections of SMANCS into the thoracic cavity, although 2 patients needed further therapy with the immunopotentiating agent picibanil (OK-432). No serious adverse effects were observed. This simple therapeutic tactic with SMANCS may be effective in cases of malignant pleural carcinomatosis.

Multiple hepatic infarction after transcatheter arterial infusion with SMANCS.

We report a patient with hepatocellular carcinoma who developed multiple hepatic infarction after transcatheter arterial infusion (TAI) with a suspension of styrene maleic acid neocarzinostatin (SMANCS) and Lipiodol (SMANCS/Lipiodol). The parameters of hepatic functional reserve were apparently decreased after the second TAI with SMANCS/Lipiodol, and the patient died of hepatic failure 103 days after the second TAI. The autopsy liver specimen revealed multiple hepatic infarctions associated with peripheral arterial stenosis or occlusion, and portal thrombosis. It is speculated that both the arterial occlusion and the portal thrombosis caused the hepatic infarction, based on a long-term insufficiency of blood supply to the hepatocytes arising from toxic arteritis caused by SMANCS/Lipiodol.

SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy.

This review discusses the development and therapeutic potential of prototype macromolecular drugs for use in cancer chemotherapy, in particular the development and use of SMANCS, a conjugate of neocarzinostatin and poly(styrene-comaleic acid). The various topics covered include a brief description of the chemistry and polymer conjugation, the binding of the conjugate to albumin and the biological behaviour in vitro and in vivo after arterial injection in animals, including plasma half-life, and the lipid solubility of SMANCS in medium chain triglycerides and Lipiodol, a lipid contrast medium suitable for use in X-ray-computed tomography. The biological response-modifying effects and the tumor-targeting mechanism of SMANCS and other macromolecular drugs are also discussed. The latter mechanism is accounted for in terms of a tumor 'enhanced permeability and retention' (or EPR) effect. A principal advantage in the use of SMANCS or other macromolecular drugs is the potential for a reduction or elimination of toxicity. Macromolecular drugs such as a pyran copolymer-NCS conjugate show a marked reduction in bone marrow toxicity normally associated with the use of NCS. This is believed to be due to a hypothetical blood-bone marrow 'barrier' which, relative to NCS, restricts or limits access of the macromolecular drug to the bone marrow. In addition, the clinical possibilities for SMANCS are discussed, including the suggestion that angiotensin II-induced hypertension has clinical potential in improving the selective delivery of macromolecular drugs (i.e. SMANCS) to tumors. Aqueous SMANCS formulations have been tested in pilot studies in patients with solid tumors of the ovary, esophagus, lung, stomach, adrenal gland and in the brain. Formulations based on SMANCS/Lipiodol have been shown to be effective both as a diagnostic tool and for therapeutic use in solid tumors where the formulations are given arterially via a catheter. In a pilot study in primary unresectable hepatoma, an objective reduction in tumor size was observed for about 90% of cases when an adequate amount of the macromolecular drug was administered. A patient receiving such treatment with no active liver cirrhosis and tumor nodules/lesion confined within one liver segment might expect to have a 90% chance of survival after treatment for at least 5 years.

Effective cancer targeting using an anti-tumor tissue vascular endothelium-specific monoclonal antibody (TES-23).

Immunoconjugate targeting of solid tumors has not been routinely successful because the endo-thelial cells of blood vessels act as a physical barrier against the transport of macromolecules, such as antibodies. In the present study, we attempted to achieve tumor vascular targeting with an anti-tumor tissue endothelium-specific monoclonal antibody (TES-23). TES-23, an IgG1 monoclonal antibody raised against rat KMT-17 fibrosarcoma-derived endothelial cells, was covalently conjugated with neocarzinostatin (NCS) in a previous study. The TES-23-NCS conjugate induced tumor hemorrhagic necrosis, and showed marked anti-tumor effects against rat KMT-17 fibrosarcoma. This result prompted us to investigate whether this approach would be applicable to various other types of solid tumors. One hour after injection of (125)I-labeled TES-23 into BALB / c mice bearing Meth-A fibrosarcoma and Colon 26 adenocarcinoma, the tumor accumulation of TES-23 was greater than that of the control IgG. In the present study, we report the anti-tumor effects of this monoclonal antibody in mice bearing Meth-A fibrosarcoma. Mice treated with the immunoconjugate showed improved survival with no side effects. This result indicates that common antigens may be found in different kinds of tumor endothelial cells, and that TES-23 might recognize these antigens.

[Targeting therapy using a monoclonal antibody against tumor vascular endothelium].

Recent studies have revealed that the targeting therapy using monoclonal antibody against tumor associated antigens did not have a clinically satisfactory effect due to various physiological characters of tumor. We propose a novel approach targeting tumor vascular endothelium to solve the inefficiency of common tumor missile therapy. In this study, the tissue distribution of anti-tumor vascular endothelium monoclonal antibody (TES-23) produced by immunizing with plasma membrane vesicles obtained from isolated rat tumor-derived endothelial cells (TECs) was assessed in various tumor-bearing animals. Radiolabeled TES-23 dramatically accumulated in KMT-17 fibrosarcoma, a source of isolated TECs after intravenous injection. In Meth-A fibrosarcoma, Colon-26 adenocarcinoma in BALB/c mice and HT-1080 human tumor tissue in nude mice, radioactivities of 125I-TES-23 were also up to fifty times higher than those of control antibody with little distribution to normal tissues. Furthermore, immunostaining of human tissue sections showed specific binding of TES-23 on endothelium in esophagus and colon cancers. These results indicate that tumor vascular endothelial cells express a common antigen in different tumor types of various animal species. In order to clarify the efficacy of TES-23 as a drug carrier, an immunoconjugate, composed of TES-23 and neocarzinostatin, was tested for its antitumor effect in vivo. The immunoconjugate (TES-23-NCS) caused a marked regression of the tumor, KMT-17 in rats and Meth-A in mice. Thus, from a clinical view, TES-23 would be a novel drug carrier because of its high specificity to tumor vascular endothelium and its application to many types of cancer.

Medullary thyroid carcinoma with multiple hepatic metastases: treatment with transcatheter arterial embolization and percutaneous ethanol injection.

A 54-year-old man with medullary thyroid carcinoma in the thyroid gland was unable to undergo total thyroidectomy because the tumor had invaded the mediastinum. Radiation therapy and chemotherapy were given. Seven years later, intractable diarrhea and abdominal pain appeared, and computed tomography demonstrated hypervascular tumors in the thyroid gland and in the liver. The tumors were successfully treated with percutaneous ethanol injection to a lesion in the thyroid gland and transcatheter arterial embolization followed by percutaneous ethanol injection to tumors in the liver. Transcatheter arterial embolization and percutaneous ethanol injection may be valuable in treating medullary thyroid carcinoma.

Intracavitary administration: pharmacokinetic advantages of macromolecular anticancer agents against peritoneal and pleural carcinomatoses.

BACKGROUND: Pleural and peritoneal carcinomatoses are quite difficult to control in patients with advanced cancer. We have devised a suitable formulation of anticancer agents to be injected by the intracavitary route. MATERIALS AND METHODS: The pharmacokinetics of macromolecular anticancer agent, copoly(styrene/maleic acid)-conjugated neocarzinostatin (smancs) and radiolabeled albumin were studied after intraperitoneal administration to ascitic tumor-bearing rats and mice, and were compared with the pharmacokinetics of other low-molecular-weight anticancer agents, mitomycin C (MMC) and doxorubicin (DOX). RESULTS: Pharmacokinetic analyses indicated that smancs showed a much higher drug concentration for a longer time in the peritoneal cavity, and a much lower drug concentration in the blood circulation than did MMC or DOX. The cavity/blood ratios of the area under the concentration curve (AUC), of smancs, bovine serum albumin (BSA), DOX, and MMC were 9.69, 7.06, 1.38, 1.15, respectively. CONCLUSION: These results suggest that macromolecular agents are cleared more slowly from the cavitary compartment and remain there at a high concentration while the blood concentration remains low.

Transarterial chemotherapy with zinostatin stimalamer for hepatocellular carcinoma.

Zinostatin stimalamer (SMANCS) is a lipophilic intra-arterial chemotherapeutic agent for hepatocellular carcinoma (HCC). Thirty HCC patients underwent transcatheter arterial injection of 4 mg SMANCS-lipiodol emulsion. Their responses were evaluated by computed tomography 1 month after treatment. Complete response (CR) was defined as disappearance or 100% necrosis of all tumors. Partial response (PR) was defined as > or = 50% reduction and/or > or = 50% necrosis. We regarded the lipiodol accumulation in tumors as being necrotic. CR and PR were observed in 8 patients (27%) and 4 patients (13%), respectively, and the overall response rate (CR + PR/all patients) was 40% (12/30). Of 12 patients whose serum alpha-fetoprotein levels had been more than 200 ng/ml before treatment, 5 patients (42%) showed more than 50% reduction in this level within 1 month after treatment. Toxicity was quite acceptable, although grade 4 toxicity (WHO) was observed as liver dysfunction in 1 patient. Transarterial chemotherapy with SMANCS, which is well tolerated, appears to have moderate antitumor effect in patients with HCC.

A novel dosage approach for evaluation of SMANCS [poly-(styrene-co-maleyl-half-n-butylate) - neocarzinostatin] in the treatment of primary hepatocellular carcinoma.

We report a Phase I/II clinical trial of poly-(styrene-co-maleyl-half-n-butylate)-neocarzinostatin (SMANCS) for intra-arterial treatment of hepatoma. Early patients received 4 or 8 mg SMANCS dissolved in Lipiodol; later patients were treated according to tumour size and degree of filling achieved. SMANCS/Lipiodol drained rapidly from normal liver but was retained within tumour interstitium. Tumour nodules filled with SMANCS/Lipiodol usually stabilised and often regressed. No UICC criteria-defined responses were achieved, partly due to difficulties of filling several lesions simultaneously. Signs of therapeutic activity suggest a more extensive clinical study is warranted.

Targeted chemotherapy in mice with peritoneally disseminated gastric cancer using monoclonal antibody-drug conjugate.

The murine monoclonal antibody A7 (MAb A7) is reactive against most human gastric cancer cell lines. Using a nude mouse peritoneal dissemination model of human gastric cancer, we investigated targeted chemotherapy using a conjugate of neocarzinostatin (NCS) with MAb A7 (A7-NCS). After demonstrating cytotoxicity of the complex against the human gastric cancer cell line MKN45 in vitro, we intraperitoneally injected A7-NCS, NCS or saline into nude mice bearing peritoneally disseminated human gastric cancer. A7-NCS inhibited peritoneal dissemination significantly more effectively than NCS. MAb A7 may prove to be an effective carrier for antineoplastic drugs in patients with peritoneal dissemination of gastric cancer.

Hepatic infarction following percutaneous ethanol injection therapy for hepatocellular carcinoma.

We report on two patients who developed hepatic infarction after undergoing percutaneous ethanol injection therapy (PEIT) for hepatocellular carcinoma (HCC). In both cases, liver function parameters deteriorated immediately after the ethanol injection, and enhanced computed tomography images showed a wedge-shaped avascular low-density area due to hepatic infarction. In one patient, PEIT was performed for a nodule treated with transcatheter arterial infusion (TAI) using a suspension of styrene maleic acid neocarzinostatin (SMANCS) 4 weeks before. In the other patient, TAI with SMANCS had been carried out 14 months previously for a different nodule in the same segment where the nodule treated with PEIT was located. When PEIT is used for patients with HCC who have previously undergone TAI, especially with SMANCS, PEIT may induce hepatic infarction.

Effect of arterial administration of a high molecular weight anti-tumor agent, styrene maleic acid neocarzinostatin, for multiple small liver cancer--a pilot study.

To assess the efficacy of the zinostatin derivative, the anti-tumor agent, styrene-maleic acid neocarzinostatin, in treating multiple small liver cancers, 29 patients with multiple hepatocellular carcinoma of 3 cm or less in diameter were treated with intraarterial injections of this high molecular weight agent, mixed with Lipiodol. Computed tomography 3 months after the first therapy showed complete deposition of Lipiodol in the entire area of the original tumor in 8 patients (27.6%), 50%-99% deposition in 4 (13.8%), 10%-49% in 10 (34.5%), and less than 10% in 7 (24.1%). After repeated injections, Lipiodol deposition in the entire area of the original tumor was found in 11 patients (37.9%). The degree of Lipiodol deposition depended on the angiographic vascularity of the tumor and on the images of the computed tomogram during arterial portography. Although complete deposition of Lipiodol was found in all tumors in 10 (58.8%) of the 17 patients with well demarcated round hypervascularity, only 1 (8.3%) of 12 patients with ill demarcated tumors showed complete deposition of Lipiodol in the tumors. Taking into account that hypervascularity on angiograms was closely correlated with the degree of Lipiodol accumulation on computed tomograms taken later, it appears that well demarcated round-shaped liver cancer is the best candidate for styrene-maleic acid neocarzinostatin therapy.

Targeting immunotherapy using the avidin-biotin system for a human colon adenocarcinoma in vitro.

A new method of targeting immunotherapy using the avidin-biotin system in vitro was investigated. Both an anti-carcinoembryonic antigen monoclonal antibody (anti-CEA MAb) and an anti-cancer drug, neocarzinostatin (NCS), were biotinylated. A human colon adenocarcinoma cell line (LoVo) was immunized with biotinylated anti-CEA MAb; avidin was added, and the cell line was incubated with various concentrations of biotinylated NCS for either 72 h or 7 min. In the incubation for 72 h, the IC50 was similar (approximately 0.45 microgram/ml) for biotinylated NCS for LoVo cells immunized with biotinylated anti-CEA MAb and those without immunization. In the incubation for 7 min, the IC50 (concentration producing 50% cytotoxicity) of biotinylated NCS for LoVo cells immunized with biotinylated anti-CEA MAb (0.35 microgram/ml) was five times less than that of non-immunized LoVo cells (1.8 micrograms/ml). Thus the present system has the potential to reduce the dosage of anti-cancer drugs needed, and this strategy seems likely to be a valuable clinical tool in targeting immunotherapy.

Changes in cellular components of spleen and lymph node cells and the effector cells responsible for Meth A tumor eradication induced by zinostatin stimalamer.

We reported previously that pretreatment with zinostatin stimalamer (ZSS) eradicated Meth A tumors in BALB/c mice. We herein investigated cellular components of spleen and lymph node cells of Meth A-bearing ZSS-pretreated mice by flow cytometry; the antitumor effector cells by in vivo depletion of T cells, NK cells, or macrophages; and host-mediated antitumor activity associated with ZSS treatment after tumor transplantation. ZSS given on day-3 transiently decreased the number of spleen cells. The percentage of T cells increased, but B cells and macrophages decreased. B cells decreased in inguinal lymph nodes in Meth A-bearing ZSS-pretreated mice, but increased in Meth A-bearing control mice. In vivo depletion experiments using antibodies or carrageenan showed that antitumor effector cells for tumor eradication are Thy1.2+/Lyt2.2+ and that at least a part of them are asialo GM1+. Thy1.2+/Lyt2.2+/asialoGM1- cells are important in generation of the antitumor activity of ZSS; however, L3T4+ T cells are also involved in initiation of tumor eradication. The result of ZSS treatment after tumor transplantation suggests that ZSS might exhibit antitumor activity by augementating host-mediated antitumor resistance, as well as its intrinsic cytocidal activity.