After a long and energetic world-wide search for antibiotics from actinomycetes, the chances of finding new classes of biologically active compounds from these organisms seem to have drastically diminished. In this paper, sophisticated procedures for finding new potentialities of antibiotic-producing actinomycetes and isolating a novel class of antitumor antibiotics, the saframycins, are described. These antibiotics are satellite antibiotics which are co-produced in minute quantities with streptothricin by a strain of Streptomyces lavendulae. Saframycins, cyano-containing saframycin A in particular, are promising antitumor antibiotics because of their low toxicity to immunologically competent cells and organs, especially the bone marrow. Significant amplification of the yield of saframycin A was attained by the quite unexpected observation that the addition of cyanide to the culture broth significantly increased its production. Subsequent isolation of the saframycin A precursor, decyanosaframycin A or saframycin S, enabled us to prepare two kinds of labeled saframycin A which were critical for the elucidation of the unique molecular action mechanism of the antibiotic. On the basis of studies on saframycin biosynthesis, a method of preparing new saframycin derivatives using resting cells of the producing strain has been developed, and new saframycins with amino functional groups on their N-pyruvoyl side chain were obtained. Their HCl salts are readily soluble in water and are expected to exhibit pharmacodynamic properties different from those of other saframycins.

A phase II study of KW 2083 [7-N-(p-Hydroxyphenyl)-Mitomycin C] was carried out in 14 cases of stomach cancer, 5 of lung cancer, 5 of colon cancer and 5 other types of cancer. KW 2083 was intravenously injected at a dose of 40 mg/body weekly in 26 cases. Among 23 evaluable cases, partial response was obtained in 6 cases (26%). The PR cases were 4 of stomach cancer and 2 of lung cancer, the former being all undifferentiated adenocarcinoma. Regarding hematologic toxicities, thrombocytopenia was the most principal toxicity and an important weak point of KW 2083. Thrombocytopenia (less than 75,000/mm3) was observed in 13 cases (50%). Recovery took about 4 weeks, but by that time 3 cases had still not recovered to 75,000/mm3. leukocytopenia (less than 3,000/mm3) was observed in 17 cases (65%). Concerning gastrointestinal symptoms, anorexia occurred in 11 cases (42%), nausea and vomiting in 11 cases (42%), diarrhea in 1 case and stomatitis in 1 case. T1/2 (beta-phase) of KW 2083 was half that of Mitomycin C.

The antitumor antibiotic sporamycin is composed of polypeptide and non-chromophore subunits and shows remarkable antitumor activity against various animal tumors. The mechanism of action of this compound involves host-mediated antitumor activity as well as direct cytotoxic activity due to the inhibition of nucleic acid synthesis. Pretreatment of mice with sporamycin produced not only a remarkable inhibition of tumor growth but also strengthened the resistance of the host to infection with Pseudomonas aeruginosa. The immunological activity described above was also observed using the isolated polypeptide moiety of sporamycin. The antitumor antibiotic stubomycin possesses a marked cytotoxic activity against mammalian cells in vitro and in vivo. Stubomycin showed inhibitory activity against DNA, RNA and protein synthesis to almost the same degree. It was also shown that these activities were significantly reduced by lipids such as phosphatidylserine, olive oil and cardiolipin. On the other hand, stubomycin did not show any mutagenic effects in mammalian cells or bacteria. It seems that the primary action of stubomycin is due to change and ultimate lysis of the cell surface. It was observed that a new antibiotic, kazusamycin, possessed very strong cytotoxic activity against mammalian cells in vitro. However this compound exhibited no antibacterial activity against gram-positive and gram-negative bacteria. We are presently investigating the biological activities of a monoclonal antibody combined with kazusamycin.

Characteristics of Drug-Resistant Cell Sublines L5178Y: We isolated aclarubicin (ACR)-, adriamycin (ADM)-, bleomycin (BLM-, and macromomycin (MCR)-resistant (r) cell sublines. The BLMr cell line did not show cross-resistance to other drugs. The ACRr and ADMr cell lines displayed cross-resistance to other anthracyclines. The drug-resistance of these cell lines was due to changes in membrane transport. All four resistant cell lines showed higher activity of membrane alkaline phosphodiesterase (APD) than the parental cells. The APD of the BLMr scell line differed from that of the parental line in molecular size. 2-Crotonyloxymethyl-4, 5, 6-trihydroxycyclohex-2-enone: We isolated an inhibitor of APD from a Streptomyces species. This substance inhibited the drug-resistant cell lines of L5178Y more markedly than the parental line in culture and showed synergistic effects with ACR against the ACRr cell line. It was an SH-inhibitor, and prevented DNA polymerase alpha and some mitotic processes. Transplantability of Drug-Resistant L5178Y Cells: DBA/2 mice, the syngeneic host, exhibited more resistance to ip transplantation of drug-resistant cell lines than parental cells. The animals showed the strongest resistance to the ACRr cell line. Treatment with cyclophosphamide markedly reversed the host resistance, suggesting that the immune mechanism was involved in the resistance. The ACRr cells were sensitive to NK cells, but the parental cells were not. Injection with anti-asialo GM1 markedly decreased host resistance. The results suggested that NK cells were involved in the transplantation resistance of mice to the ACRr cells. 230-Kilodalton Membrane Protein of ACRr Cells Identified by Monoclonal Antibody: We prepared monoclonal antibodies to the ACRr cells, and found that a monoclonal antibody, designated SC438, specifically agglutinated the ACRr cells. A specific 230K membrane protein was found in the ACRr cells by immunoprecipitation. Natural BLM Resistance of Chinese Hamster V79 Cells: V79 cells were more resistant to BLM than CHO cells. This natural drug-resistance was is due to higher BLM hydrolase activity. We isolated BLM cell lines, and found that BLM supersensitivity was not due to BLM hydrolase, but to decreased repairing activity of DNA damage.

In vitro chemosensitivity of lung cancer and other chest tumors was evaluated by human tumor colony assay (HTCA). From 61 specimens 33 (54%) grew more than 30 colonies from which evaluation of chemosensitivity could be performed. Of 41 specimens of lung cancer, 26 (63%) yielded adequate growth for drug testing. Nine out of 26 specimens of non-small cell lung cancer showed more than 50% reduction in colony formation, and in 4 of the 26 specimens, more than 70% reduction was obtained with more than one of the drugs tested. Specimens obtained from metastatic lesions of lung cancer showed higher plating efficiency and drug sensitivity than those from primary lesions. Plating efficiency of non-epithelial tumors was lower than that of epithelial tumors. HTCA has a potential value for screening anticancer agents against lung cancer and other chest tumors. However, the assay still has many problems to be resolved, such as difficulty in obtaining single-cell suspensions and poor plating efficiency.

1-Hexylcarbamoyl-5-fluorouracil (HCFU) was administered orally to 63 patients with hepatocellular carcinoma (HCC) at a daily dose of 200 to 600 mg. Twenty three of those patients who received HCFU over 4 weeks without any other therapies were studied for its antitumor effect. Eighteen patients were evaluated for tumor size. The results showed a response rate of 16.7% (3/18) based on the criteria of Koyama et al. Patients with minute hepatoma had an especially good response; the response rate being 40% (2/5), or 60% (3/5) including minor response. Tests showed no aggravation of liver function. HCFU was considered to be useful in the treatment of HCC. However, unexpected adverse neurological effects were encountered in 6 patients. They began with slurred speech, bradypragia and gait disturbance, and finally progressed to unconsciousness in 4 out of 6 patients, in which the clinical picture resembled hepatic encephalopathy. Though reversible, these symptoms necessitate immediate withdrawal of HCFU therapy.

The cardiotoxicities of 4'-epidoxorubicin (4-epiDX) and doxorubicin (DX) were compared in rabbits. Thirty-one male NZW rabbits weighing 2.4 to 2.6 kg were used for each compound and 15 were kept as controls. DX or 4'-epiDX was administered intravenously with a dose of 0.8 mg/kg for 3 consecutive days a week for 5-10 weeks. Rabbits were sacrificed at 5, 8 and 10 weeks after initial administration. Under nembutal anesthesia, electrocardiogram, phonocardiogram and carotidgram were recorded. Light and electron microscopic studies of the heart were performed according to conventional methods. Myocardial alterations were semi-quantitatively evaluated with light microscopy, using a modified grading system derived from Bertazzoli. Twelve of the DX-treated rabbits and 4 of the 4'-epiDX-treated rabbits died after 5 weeks or later. In the electrocardiogram study, ST-T changes were less frequently observed in rabbits treated with 4'-epiDX than those with DX. Significant increases of PEP/LVET were observed in rabbits treated with DX for 8 and 10 weeks, similar increases were only observed while in rabbits treated with 4'-epiDX for 10 weeks. Histologically, both drugs produced myocardial degeneration and necrosis. The lesions were characterized by multiple vacuolization of myocardial cells, atrophy of myocardial cells, interstitial edema and fibrosis. Myocardial alterations induced by 4'-epiDX were qualitatively similar to those induced by DX, but were much less severe at the same cumulative dose. These results demonstrate that 4'-epiDX is less cardiotoxic than DX.

Various types of human gynecological cultured tumor cells were tested for the sensitivity to Peplomycin (PEP), an effective antitumor antibiotic for squamous cell carcinomas, by the regrowth assay method together with morphological observation. Bleomycin-hydrolase activity of these cell lines was also compared in cell-free extracts by assaying the conversion of Bleomycin into its deamidated from (HPLC method). SKG-I, SKG-II, SKG-IIIb cells derived from squamous cell carcinoma of the cervix and RKN cells derived from myosarcoma of the ovary were much more sensitive to PEP than other cell lines. PEP was found to be mainly a time-dependent drug, but also concentration dependent. The effect of PEP on cell morphology was characterized by the appearance of enlarged cells and swelling nuclei. The specific activities of Bleomycin-hydrolase in SKG-I, SKG-II, SKG-IIIb cells were shown to be relatively lower than that in other cell lines. These results suggested that cervical squamous carcinoma cells and ovarian myosarcoma cells were sensitive to PEP and Bleomycin-hydrolase activity was one of factors which decided the PEP sensitivity of human cultured tumor cells.

Thirty-nine patients with superficial bladder cancer underwent intravesical instillation therapy of Aclacinomycin-A (ACM). Antitumor effect of ACM was evaluated in 19 patients and objective responses (CR + PR) of tumor were observed in 84.2% of these patients. Prophylactic instillation therapy of ACM was carried out on 20 patients and the results were compared with those obtained for 10 control patients who had first episode of bladder tumor and received no instillation therapy. No significant difference in the recurrent rate was observed between these two groups. The major side effect for instillation therapy with ACM was bladder irritation which appeared in 38.5% of all the patients.

Recently, there has been much concern that cancer chemotherapy may have undesirable consequences in the form of secondary malignancies. At present, secondary leukemia or therapy-linked leukemia constitutes a well delineated clinical syndrome characterized by several distinct symptoms, that was illustrated by the presentation of cases recently observed. In an attempt to understand the basis for this therapy-linked leukemia, mutagenic activity of various anticancer agents were studied by the induction of sister chromatid exchanges and risk of a certain drug as a possible candidate causing secondary leukemia was discussed from the molecular cytogenetic point of view. From both clinical and cytogenetic aspects it seemed that bifunctional alkylating agents were potentially relevant to the development of secondary leukemia.

A phase I-II study of KW2083, an analog of mitomycin C (MMC) was performed in a total of 22 patients. KW2083 was escalated by single intravenous administration of 40, 50, 60, 70, and 80 mg/m2 doses. Treatments were repeated every 4-6 weeks unless unacceptable toxicities occurred. The median times taken to reach nadir for each dose level were 9-12 days for leukocytes and 7-13 days for platelets respectively. The median times taken for recovery were 8-22 days for leukopenia and 10-37 days for thrombocytopenia. Variable and non-predictable hematological toxicity was observed in some cases. Biphasic hematological toxicity was observed in 4 courses. Acute toxicity occurred in 17 courses for 11 patients and consisted of nausea (44%), vomiting (13%), diarrhea (2.7%) and stomatitis (2.7%). Nephrotoxicity (elevation of BUN, 8.1% and proteinuria, 5.4%) occurred in 3 patients who had no previous impairment of renal function. Alopecia (8.1%) was also observed. Marked elevations of hepatic enzymes were noted in one patient who developed acute fulminant hepatitis with the second dose of KW2083. Objective response was observed in one of 20 evaluable patients. From this preliminary study, the maximum tolerable dose is 70 mg/m2 and the optimal dose of KW2083 was determined to be 50 mg/m2 at 6-week intervals. KW2083 has been introduced as an MMC analog of potential interest. However, hematological and non of hematological toxicities are very similar to those of MMC and does not appear that KW2083 will supersede MMC.

The influence of PSK on the metabolism of FT-207 was studied in patients with gastric cancer. The 5-FU concentration in the blood was determined 15 min, 30 min, 1 hour and 3 hours after intravenous injection of FT-207, 400mg. The blood level of 5-FU remained constant in 86% of patients after administration of PSK for 7 days, but decreased in 14% of patients. After administration of PSK for 8 to 14 months, no change in the blood level of 5-FU was detected. These results suggest that PSK has no distinct influence on the activation of FT-207.