We have developed a new anticancer drug sensitivity test using the short-term microplate culture and viable cell staining method, and the details of this method are reported. The tumor cells are cultured with the anticancer drugs for 2 days and 4 days. After culture, the microplate is centrifuged. The surviving tumor cells are fixed with ethanol and stained with crystal violet. After washing out the remaining crystal violet, 200 microliters of sodium lauryl sulfate is put into each microwell and the absorbance of each is measured. When we examined this method using leukemic cell lines, we found that the numbers of cells were in proportion to the absorbance, and that the surviving cells could be counted by the absorbance within the range of 0.4 to 1.7. On the assumption that the effective range is more than 60% of the cytotoxicity index (CI), tables of CI classified into drugs, concentrations and duration of culture for each leukemic cell line were made. With these tables, comparison of the anticancer drugs and subsequent selection of the effective ones became easier. This method is simple, rapid and convenient for handling a large volume of material. Its application to further clinical practice is therefore expected.

Immuno-enhancing actions of carnosine, beta-alanine, homocarnosine, and gamma-aminobutyric acid were studied in ddY mice by evaluating plaque-forming cell reaction against sheep red blood cells. Animals were administered the test agents in prior to, or simultaneously with, various treatments that are known to reduce immune function such as administration of the anti-tumor agents, mitomycin C and 5-fluorouracil, immunosuppressant cyclophosphamide, antiinflammatory agent hydrocortisone, or cancer implantation and gamma-irradiation. Experiments were performed also in aged mice with reduced immune function. The administration of these drugs showed non-specific immuno-enhancing effects under all conditions examined and on all cell groups that may have been affected by these immunosuppressive stimulus.

In Vitro Colony Assay was carried out to examine chemosensitivity in 57 specimens from gynecologic malignancies, and following results were obtained. Total of 34 (59.6%) formed adequate colonies for sensitive assay. Among ten anticancer drugs that were tested, Cis-platinum, Adriamycin, 5-FU and Bleomycin were relatively effective for ovarian malignancies. In vivo chemosensitive tests were also performed using five xenograft systems of human gynecologic tumor. These in vivo results were compared with in vitro data, and a 75% true positive and an 87.5% true negative rate were observed. Retrospective correlations between in vitro chemosensitive data and patient responses to chemotherapy were analyzed from 15 evaluable patients, then a 71.5% true positive and a 75% true negative rate were observed. To obtain pharmacokinetic data on anticancer drugs, we measured concentrations of plasma and tumor tissue for Cis-platinum, Adriamycin and Bleomycin in operated patients and xenografts. The in vivo tissue concentrations were higher than the in vitro drug concentrations usually employed in colony assay. By comparing the inhibitory effects on tumor colony growth of 1-hr and continuous drug exposures in colony assay, in vitro continuous exposure schedules were made available for cell cycle specific anticancer drugs.

Serum specimens from mice treated orally with Ge-132 (100 mg/kg) exhibited antitumor activity against Ehrlich (allogeneic) and RL 1 (syngeneic) ascites tumors in BALB/c mice. Sera obtained from mice 24 hours after Ge-132 administration displayed the highest antitumor effect and the antitumor activity was dose-dependent. Sera prepared from mice 12, 36 or 48 hours after Ge-132 treatment had no protective effect. Circulating interferon (IFN) was induced at 24 hours after administration. The antiviral activity of serum from Ge-132-treated mice was inactivated by treatment with trypsin, low pH, and anti-IFN-gamma antiserum. The inactivated preparations of serum IFN induced by Ge-132 did not show antitumor activity when administered to mice bearing Ehrlich ascites tumors. These results suggest that the antitumor activity in the sera of Ge-132-treated mice may have been expressed through IFN-gamma which was induced by Ge-132.

Each anticancer agent has its own pharmacologic activities. Effect of anticancer agent may be influenced by the concomitant administration of another drug. In addition, patients with advanced cancer occasionally require other medications as well. These drugs can alter both the effects and adverse reactions of anticancer agents. Therefore, much attention must be placed on the interaction with anticancer agents. This paper summarizes drug interactions between anticancer agents and other drugs. Furthermore, clinical significance of these interactions will be discussed. It is hoped that this paper will provide more attention for the study of this problem.

A new in vitro chemosensitivity test was developed from comparative studies on the cytotoxicity of anticancer drugs against human tumor tissues xenografted into nude mice and their cultivated cells in vitro. Half a gram of the material was sufficient to examine the sensitivity of the tissues to 10 kinds of potential anticancer drugs and the results were obtained within 24 hours. The test was applied to all of 20 patients with advanced ovarian cancer. The predictive accuracy was 58% in 12 evaluable patients. This response rate was higher than those of conventional combination chemotherapy with or without cisplatin and adriamycin. Individual ovarian cancers showed different sensitivities to the drugs. These results indicate that heterogeneity of sensitivity to anticancer drugs exists among individual ovarian cancers and that our new type of in vitro chemosensitivity test is useful for selecting the most effective drugs for each individual type of ovarian cancer.

In a murine model it has been shown that the antitumor activity of carboxyethylgermanium sesquioxide (Ge-132) can be depleted by administration of macrophage (M phi) blockers. In the present study, the role that M phi play in the antitumor activity of the compound was investigated. Oral administration of Ge-132 in mice was demonstrated to be effective in activating M phi (Ge-132-cytotoxic M phi), and the cytotoxic activity of these M phi appeared in the peritoneal cavity of mice 48 hours after the oral administration of the compound. Co-cultivation of RL male-1 leukemia or Ehrlich carcinoma cells with Ge-132-cytotoxic M phi in vitro resulted in marked suppression of the growth of tumor cells. The transfer of peritoneal exudate cells (PEC), or purified M phi fractions of PEC from Ge-132-treated mice to mice bearing Ehrlich or RL male-1 ascites tumors resulted in significant protection. However, when the cytotoxic M phi were depleted by carbonyl-iron treatment in vitro, no antitumor effect was demonstrated in mice bearing Ehrlich or RL male-1 ascites tumors. Macrophage fractions obtained from PEC of Ge-132-treated mice exhibited an inhibitory effect against certain tumors both in vivo and in vitro suggesting that the antitumor effect of Ge-132 observed in vivo resulted from the activation of M phi.

A case of toxic leucoencephalopathy induced by 5 FU derivatives is reported. A 46-year-old woman was diagnosed as having breast cancer, and radical mastectomy was performed on May, 1982. After operation, she was given irradiation and 5FU derivative (tegafur or carmofur) 600 mg and Nolvadex 20 mg (tamoxifen citrate) were administered every day. After taking the medication for a month, she began to stagger and developed a tremor in both arms. She was admitted to our hospital on August 16, because she showed evidence of dysarthria and memory disturbance in addition to her initial complaints. Soon after admission, she developed akinetic mutism, and metastasis in the brain stem was suspected. In spite of her severe condition, she was given radiation over the posterior fossa and continued the medication. On September 22, CT disclosed low density area in the centrum semiovale bilaterally. She died of DIC on November 30. An autopsy was performed. The brain weight was 1110 g and the outer surface of the brain was normal. In frontal cut surfaces stained with K.B., bilateral degeneration of the centrum semiovale was apparent. Microscopically, the degree of myelin degeneration was stronger than that of axon, and numerous fatty granular cells were found in the degenerated area. There were no bizarre shaped astrocytes, inclusion body or cellular infiltration. Fibrillary gliosis was scanty. No metastasis was found in the central nervous system or other organs. Based on these pathological findings and clinical history, toxic leucoencephalopathy induced by 5 FU derivatives was suggested.

4'-Epi-adriamycin (EPI) is a new derivative of adriamycin (ADM). The cytotoxic effect of EPI on the T24 cell line, an established cell line from human urinary bladder carcinoma, the distribution of the drug in blood, urine and tissues of various organs and histopathological change in the bladder mucosa in dogs following intravesical instillation of the drug, were studied. The cytotoxicity of EPI on the cultured T24 cells was examined by a colony formation method. After 2 hours exposure, EPI was slightly less cytotoxic than ADM, but showed higher cytotoxicity than mitomycin C or aclacinomycin A. The drug levels in blood, urine and tissues were measured by HPLC following bladder instillation in Beagle dogs with bilateral cutaneous ureterostomy. They were elevated in proportion to the drug concentration instilled intravesically. After 50 mg of EPI dissolved in 10 ml of physiological saline was instilled intravesically, the blood levels of EPI were not elevated significantly and reached the maximum levels of only 0.222 microgram/ml. The total amount of EPI excreted into the urine during the 10 hours after instillation was 389 micrograms which was equivalent to 0.78% of instilled EPI. The tissue levels of 50 mg of EPI after 6 hours retention were 1216 +/- 1094 micrograms/g in the bladder mucosa, 259 +/- 250 micrograms/g in the bladder muscular layer, 7.65 +/- 1.19 micrograms/g in the iliac node, 22.1 +/- 4.8 micrograms/g in the cortex of kidney, 15.1 +/- 3.8 micrograms/g in the medulla of kidney, 11.3 +/- 1.0 micrograms/g in liver and 5.80 +/- 1.20 micrograms/g in the heart. To examine the effect of the drug on the bladder mucosa, 50 mg of EPI was instilled intravesically. After 6 hours retention, bladder mucosa was observed through a microscope and a scanning electron microscope. Only exfoliation in the mucosa was observed sporadically and no histological change was observed in the submucosal layer. The above results suggest that EPI is a suitable drug for intravesical chemotherapy to bladder cancers.

Phase II study of 5'-DFUR was conducted in 195 patients with malignant tumors by the Osaka Chemotherapy Cooperative Study Group. Five CR and 20 PR cases were obtained out 133 evaluable cases and the total response rate was 18.8% (15.8% in gastric, 38.1% in breast) One PR was observed for each of head & neck and esophageal cancer. It was noteworthy that four CR were observed in breast cancer. Adverse reaction was observed in 61 out of 151 cases (40.4%), and major side effects were digestive symptoms such as diarrhea (22.5%), nausea-vomiting (11.9%) and anorexia (10.6%). These results suggest that 5'-DFUR can be useful for the treatment of malignant tumors.

Clinical effects of daily oral administration of 5'-DFUR were studied in patients with advanced or recurrent gastrointestinal cancer and breast cancer. Cases included 5 gastric cancer, 18 colorectal cancer, 27 breast cancer and 1 malignant melanoma. Out of 38 cases who completed the treatment, CR was observed in 2 and PR in 7, the response rate being 23.7%. Out of 23 cases with breast cancer who completed the treatment, CR was observed in 2 and PR in 6, the response rate being 34.8%. Safety was evaluated in 42 cases and diarrhea was found in 17.1% of cases; however, it was easily reduced by decreasing the dosage or discontinuing administration of the drug.