Evaluation of antitumor activities of hyaluronate binding antitumor drugs: synthesis, characterization and antitumor activity.

To enhance the selective delivery of antitumor drugs into regional lymph nodes and cancerous tissues via a hyaluronate (HA) receptor (CD44), we synthesized HA-mitomycin C complex and HA-epirubicin complex. To investigate the specific distribution of HA into regional lymph nodes and to evaluate the HA receptor on lewis lung carcinoma cells, we also synthesized 14C-labelled HA and fluorescent HA (FR-HA). The metabolic studies of 14C-HA and HA-epirubicin complex were performed in rats. The specific distribution of both compounds to the lymph nodes were observed after sc treatment. Internalization mechanisms of HA into carcinoma cells (lewis lung carcinoma) via HA receptor was investigated using fluorescent HA (FR-HA) in vitro. Internalization of FR-HA following binding to the cell surfaces was observed. HA-Mitomycin C (MMC) exhibited potent anti-metastatic effects against lewis lung carcinoma implanted in mice at an extremely low dose (0.01 mg/kg) whereas free MMC had no effects.

[Studies on metabolism and disposition of sizofiran (SPG), an anti-tumor polysaccharide. III. Degradation and excretion of SPG in rats].

Degradation and excretion of Sizofiran (SPG), an anti-tumor polysaccharide, were studied in rats after a single or multiple administration. After a single intravenous injection of [14C]SPG (3 mg/kg), SPG distributed in the liver was degraded at very slow rate to SPG-like substances (SPGLS) having lower molecular weight than that of SPG, while SPG in the spleen and mesenteric lymph node was metabolized at much slower rate than that in the liver. In the experiment with multiple subcutaneous administration, SPG was also found to be present mainly as SPGLS in the liver, but almost as an unchanged SPG in the spleen. SPG was excreted in the urine mainly as metabolites with a molecular weight of less than 10000. These results indicate that degradation of SPG to lower molecular weight-SPGLS is a prerequisite for efficient urinary excretion and the degradation occurs mainly in the liver.

[Studies on metabolism and disposition of sizofiran (SPG), an anti-tumor polysaccharide. II. Effects on hepatic drug-metabolizing enzyme system in rats].

The effect of a single or multiple administration of sizofiran (SPG), an anti-tumor polysaccharide, on a hepatic drug-metabolizing enzyme system was studied in rats. When SPG was given intravenously at a single dose of 0.5 or 10 mg/kg, no alteration was observed in activities of aminopyrine (AP) N-demethylase and aniline hydroxylase, and in cytochrome P-450 (P-450) content in the livers of rats 48 h after dosing. However, only AP demethylase activity decreased by 34% after the administration of 200 mg/kg. Similarly, no change in the hepatic enzyme activities and P-450 content was observed for up to 180 d after a single dose of 10 mg/kg. Subcutaneous treatment of animals with either 10 or 40 mg/kg dose for 3 and 6 months resulted in no alteration in the enzyme activities and P-450 content. These results may indicate that the therapeutically effective dose of SPG has no effect on a hepatic drug-metabolizing enzyme system in rats.

[Analysis of human sera obtained from lung cancer patients by two-dimensional electrophoresis after schizophyllan (SPG) treatment].

Schizophyllan (SPG) was administered to 13 lung cancer patients (i.m. 20mg X 2/week) for 3 weeks without chemo or irradiation therapies, and serum proteins were analyzed by two-dimensional electrophoresis (TDE). Additionally, immunosuppressive acidic protein (IAP) was quantitatively determined by single radial immunodiffusion (SRID). By TDE analysis, human serum proteins were separated into more than 100 spots, and about 14 spots were found to show quantitative changes in cancer patients. Quantitative examination was therefore conducted on changes of 8 components among these spots, including alpha 1-acidic glycoprotein (alpha 1 AG), acidic alpha 2-macroglobulin (acidic alpha 2 M), haptoglobin (Hp) and IAP. The protein which showed the most marked decrease in cancer patients, located between transferrin and IgG on the above TDE patterns, was ascertained to have a molecular weight of about 150,000 using a gel filtration method. This protein was increased in 7 of 13 patients after SPG treatment.

Purification, physicochemical characterization, and antitumor activity of a cancer-associated human serum protein that is increased by treatment with schizophyllan, an antitumor polysaccharide.

A serum protein was purified from normal human sera by several steps of purification, and tentatively designated as cancer-associated serum protein (CAP-135), since the content of the protein was remarkably decreased in cancer patients. The purified CAP-135 has an approximate molecular weight of 135,000 daltons, and is composed of three subunits of 78,000, 34,500 and 24,800 daltons. CAP-135 showed an isoelectric point of pH 5.5-5.8 and contained a small amount (1.38%) of neutral sugar. CAP-135 is assumed to be modified complement C3 on the basis that it reacted only with anticomplement C3 in immunodiffusion assay, and one of its subunits had a molecular weight similar to that of the beta-chain of human complement C3. The ip injection of CAP-135 apparently inhibited the growth of sarcoma-180 implanted into the groin of Jc1:ICR female mice. The present results indicate that CAP-135 may be of diagnostic value.

Enzyme immunoassay for mabuterol, a selective beta 2-adrenergic stimulant in the trachea.

A sensitive double antibody and heterologous enzyme immunoassay for mabuterol was established. For competitive reactions, antibody raised against diazotized mabuterol-human serum albumin was incubated with a mixture of diazotized mabuterol analog (RC-1) labeled with beta-D-galactosidase and standard or sample. Free and antibody-bound enzyme hapten were separated using anti-rabbit IgG immobilized on polystyrene balls. Activity of the enzyme on the solid phase was fluorometrically determined. The present immunoassay allows detection of 0.5 to 100 pg/tube of mabuterol. Pharmacokinetic behavior of this agent in human plasma and urine was studied after a single oral administration (50 micrograms). The maximum level was achieved after 2-3 hrs with approximately 280 pg mabuterol /ml of plasma and the half life of mabuterol was estimated to be 19.5 hrs. Cumulative amount of mabuterol in the first 72 hrs urine was 64.3 +/- 13.2% of the administered dose.