Leptomeningeal dissemination is one of the major causes which increase the morbidity and mortality of the patients with malignant brain tumors. The incidence of this complication is increasing, however, no sufficient treatment is available at present. Therefore, in an attempt to establish a new treatment, we studied toxicity and therapeutic effect of intrathecal ACNU (nimustine hydrochloride) using experimental animals. Systemic and local toxicity was tested in normal rats that received ACNU intracisternally. The animals given ACNU more than 3.0 mg/kg progressively lost their body weight, and ACNU 6.0 mg/kg was fatal in 80% of animals. Animals given ACNU less than 1.5 mg/kg gained weight in the same rate as in control animals. Increased capillary permeability to intravenous Evans blue was observed in the subpial region of the brain in the rats given ACNU 6.0 mg/kg intracisternally. The increase of capillary permeability was dominant along the ambient cistern, hypocampal fissure, and at the base of the brain. Demyelinization and loss of neurons were seen in the same areas as well. These changes were not observed in the animal given ACNU less than 1.5 mg/kg. Therapeutic effect of intrathecal ACNU against the leptomeningeal tumor was studied in rats with meningeal carcinomatosis which was induced by intracisternal inoculation of 1 X 10(4) cells of Walker 256 carcinosarcoma. The median survival times of the animal given ACNU 1.5 mg/kg intrathecally on day 2 or 5 after tumor inoculation were prolonged by 55 to 64%, and 64 to 145%, respectively, as compared to those of untreated control animals (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane-modifying agents such as reserpine, calcium antagonists (nicardipine, verapamil) and calmodulin inhibitor (trifluoperazine) were found to enhance the cytotoxicity of ACNU in vitro and in vivo in ACNU-resistant C 6 (C 6/ACNU) glioma. In in vitro experiments, uptake and retention studies with [14C] ACNU revealed that intracellular uptake and retention of ACNU in C 6 cells were larger than those in C 6/ACNU cells, and that these membrane-modifying agents increased the cellular uptake and retention of ACNU in C 6, especially in C 6/ACNU cells. The amount of ACNU in C 6/ACNU cells reached the same level as that detected in C 6/ACNU cells. When these drugs were added along with ACNU at the concentration of 10 to 20 microM to the culture in vitro, ACNU resistance was completely overcome. In in vivo experiment, reserpine, nicardipine, verapamil and trifluoperazine in doses 250 to 500 micrograms/kg intrathecally administered with 1 mg/kg ACNU 1 day after the tumor inoculation significantly enhanced the chemotherapeutic effect of ACNU in C 6/ACNU bearing (C 6/ACNU-MG) rats. It might be concluded that the mechanism of enhancement of ACNU cytotoxicity presented in in vitro and in vivo is explained by the enhanced accumulation of ACNU by these membrane-modifying agents in C 6, especially in ACNU-resistant (C 6/ACNU) cells, and, furthermore, that combination chemotherapy with ACNU and such membrane interacting drugs as reserpine, calcium antagonists (nicardipine, verapamil) and calmodulin inhibitor (trifluoperazine) could lead to the capability of overcoming resistance to ACNU in glioma.

Prognostic indicators in the clinical study were discussed at the cellular level in relation to the number of tumor stem cell, the rate of repopulation during the treatment course and cellular radio-chemosensitivity. The number of tumor stem cells seems to correlate to tumor volume in the narrowly defined tumor type, but may not in the mixed group of tumor types. Thymidine or BUdR labeling index may be an useful indicator of rate of repopulation and also of tumor aggressiveness. Cellular radio-chemosensitivity varies not only among tumor types but also among subpopulations within a tumor. Further investigations of tumor stem cell assay in the response to radio-chemotherapy using fresh tumor specimens from patients are certainly required before the assay methods are established.

A new ovarian cancer cell line has been established from the dispersed cells taken from a heterotransplanted tumor at the 6th passage in nude mice which had been serially transplanted with a primary lesion of undifferentiated carcinoma (FIGO) of the ovary. Selection of cancerous epithelioid cells was completed by the 4th passage by means of the colonial cloning method. The cell line designated TYK-nu was subcultured serially in Eagle's MEM with 10% FCS more than 100 times over 31 months at March, 1986 and was observed to produce a tumor on implantation into nude mice histologically similar to the original tumor. These cells form a monolayer in a pavement-like arrangement with polygonal cells including giant cells and reveal a lack of contact inhibition. PAS positive substance can be seen in the cytoplasm. Desmosome structure, well-developed cell organelles and glycogen granules can be found by electron microscopy. The population doubling time was about 37 hours and the plating efficiency was 13-17%. All cells were seen to be of the human karyotype on chromosomal analysis and the number of chromosome in the majority of cells was hyperdiploidy with a mode of 56 at the 6th and 84th passages. A submetacentric marker chromosome was present. Using in vitro colony assay, TYK-nu was studied for drug sensitivity to cisplatin, adriamycin, vincristine, 5-fluorouracil and etoposide, and found to be sensitive to cisplatin.

Experimental single-agent chemotherapies using 11 anticancer agents for 15 human cancers xenografted into nude mice revealed that each cancer line seemed to retain a degree of individuality in its spectrum of chemosensitivity irrespective of whether it originated from the same organ or whether it was of a similar histologic type. As possibly factors relevant to this chemosensitivity, we investigated the following 9 parameters in cancer tissue or cancer-bearing mice, i.e., grade of differentiation, vascularity, percentage necrosis, volume doubling time, labeling index (LI), LDH activity, tissue/serum LDH ratio, thymidine phosphorylase activity, and serum CEA. Values of these parameters remained markedly constant within each cancer line. The relationships between these 9 parameters and chemosensitivities to the 11 drugs were investigated in 15 cancer lines. A statistically significant relationship was noted in 22% of the 99 such combinations. Parameters showing a close correlation to chemosensitivity differed from drug to drug, such as ADR vs LI and vascularity, 5'-DFUR vs CEA and vascularity, and MMC vs LDH ratio and grade of differentiation. It was concluded that the chemosensitivity of cancer lines to each drug is a result of various interacting factors.

Various degrees of hepatic failure and renal abnormality may occur as complications of anti-neoplastic chemotherapy. The etiologies of alterations in liver function and the clinical pictures of chemotherapeutic agents as potential hepatotoxins, and of drugs with hepatic metabolism, and their combination uses, are discussed. As strategies are available for prevention and amelioration of these problems, management by periodic reevaluation of liver function is most important. In renal failure, myeloma kidney as tumor-associated nephropathy, hyperuricemic nephropathy and treatment-related nephrotoxicity are discussed with regard to their etiologies and clinical pictures. Aggressive management with intravenous hydration can ameliorate these complications of therapy, and careful monitoring of renal function and serum electrolytes are essential during administration of these agents.

One of the serious problems in chemotherapy of brain tumors is that tumor cells are able to acquire resistance to initially effective cytotoxic agents. In order to study the mechanism of this resistance against chemotherapeutic agents, especially ACNU, two variant cell lines (C6/ACNU and 9L/ACNU) resistant to ACNU [1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride] were selected previously in vivo from rat C6 glioma and 9L gliosarcoma, respectively. Uptake and retention of ACNU in these resistant cells were studied with [14C]ACNU. The result indicated that the resistance exhibited by both sublines of C6/ACNU and 9L/ACNU cells were due to the reduced uptake and retention of the drug. The study of the effects of oxidative phosphorylation inhibitor, DNP (2, 4-dinitrophenol), on the uptake and retention of ACNU suggested that there is an active outward transport mechanism for ACNU in 9L/ACNU cells. It might be concluded that ACNU-resistant brain tumor cells are resistant to ACNU by virtue of both the reduced uptake of the drug and the increased active efflux.

A phase II clinical trial of mitoxantrone in refractory or relapsed malignant lymphomas was conducted by a cooperative study involving 17 institutions. Of 46 patients entered, 33 were evaluable for responses and toxicity. Thirty-one of the 33 had been previously exposed to adriamycin at a median dose of 220 mg/m2 (range 21-489 mg/m2), and two additional patients had each been given THP-adriamycin at a dose of 80 mg/m2 or 4'-epi adriamycin at a dose of 69 mg/m2. Mitoxantrone was administered in 3 different schedules: 8-12 mg/m2, every 3-4 weeks in 23 patients; 4-6 mg/m2, weekly, in 3 patients; and 2-4 mg/m2, for 5 days, in 7 patients. Summarizing the responses obtained in the 3 schedules, there were 2 partial responders among 5 with Hodgkin's disease, while there were 8 complete responders and 4 partial responders among 28 with non-Hodgkin's lymphoma. The overall response rate for all the evaluable patients was 42% with a complete response rate of 24%. The median response duration was 7+ weeks (range 4-27+ weeks) for complete responders and 7 weeks (range 4-46+ weeks) for partial responders. The major toxicity was myelosuppression: leukocytopenia less than 3,000/microliter occurred in 79% of patients, and thrombocytopenia less than 75,000/microliter in 35%. Other toxic effects were minimal, mild nausea and/or vomiting occurred in 39%, and diarrhea in 3%. Possible drug-related liver and renal dysfunctions were observed in 19% and 10%, respectively. The favorable response to mitoxantrone in patients with prior anthracycline antibiotic therapy suggests that the drug is not fully cross-resistant with anthracycline antibiotics, and that this drug is of value in combination with other drugs as a salvage therapy for patients with refractory or relapsed malignant lymphomas.

One of the most serious problems in chemotherapy of brain tumors is that tumor cells are able to acquire resistance to initially effective cytotoxic agents. In order to study the mechanism of such resistance to ACNU and the means to overcome it, two variant cell lines (C6/ACNU and 9L/ACNU) resistant to ACNU were selected in vivo. Uptake and retention of ACNU in these resistant cells were studied with [14C] ACNU. The results indicated that the resistance exhibited by both sublines of C6/ACNU and 9L/ACNU were due to both reduced uptake and retention of the drug. In an attempt to clarify the more detailed biochemical mechanism of resistance in these cells, we surveyed various membrane-modifying agents which potentiate the sensitivity of these resistant cells to ACNU. Among a number of membrane-modifying agents, calcium antagonists, especially nicardipine and verapamil, were found to cause retention of ACNU in the resistant cells and to enhance the effect of ACNU on these resistant cell lines. It might therefore be concluded that ACNU resistance can be overcome by membrane-modifying agents, such as nicardipine and verapamil.

The relationship between morphological manifestations and cell kinetic changes of three lung cancer cell lines after exposure with chemotherapeutic agents was studied. After treatment with Cis-dichloro diammine platinum (II) (CDDP), an increase in cells of G2/M compartment at first, and then of S compartment was observed. As for the morphological manifestation, enlarged nuclear cells were more frequently observed. These cells seemed to be in S-G2/M compartment and to die finally. However a part of cells escaped from complete blockade may show multiple nuclei. Also after treatment with Etoposide (VP-16), an increase of G2/M compartment was observed, and on the morphological manifestation enlarged nuclear cells or double-or multiple-nuclear cells were observed. As these cells seemed to enter into G2/M compartment immediately. Cell destruction was thought to be started earlier compared with other two drugs. After treatment with Peplomycin (PEP), its effects on cell cycle traverse were only minimum accumulation of G2/M compartment in high PEP concentration. However concerning the morphological manifestation, many cells treated with PEP revealed enlarged, double or multiple nuclei. This suggests that morphological manifestation may reflect cytocydal effects more dominantly than cell cycle traverse. Each chemotherapeutic agent influenced the morphological manifestation and the cell kinetics of human lung cancer cells characteristically. It seemed to be important to study these relations in order to estimate the effect of chemotherapeutic agents and the therapeutic efficacy on cancer cells.

We examined the effect of PSK on involution of the thymus in tumor-bearing mice. The weight and cell number of the thymus decreased and the size distribution (scatter profile) of thymus cells was changed in X5563-bearing C3H/He mice. Also in these mice, 3H-thymidine incorporation into the thymus was reduced compared with that in control mice, as evaluated not only by per organ but also by per 1 mg of thymus tissue. Such an involution was also observed in tumor-free mice injected with immunosuppressive substance, which had been obtained from ascites of X5563-bearing mice and revealed suppressive activity against lymphocyte proliferation. PSK administration prevented such modulation in the thymus of tumor-bearing mice and immunosuppressive substance-injected tumor-free mice. In other words, it is considered that the various changes in the thymus demonstrated in tumor-bearing mice may be attributable to the suppression of cell proliferation in the thymus, that such suppression is caused at least partly by an immunosuppressive substance which possesses inhibitory activity against lymphocyte proliferation, and that PSK has an antagonistic activity against such a substance so as to restore the function of the thymus in tumor-bearing hosts.

In vitro chemosensitivity was evaluated by succinate dehydrogenase inhibition (SDI) test in 94 human tumors including 59 gastric cancers, 27 colo-rectal cancers and 8 malignant lymphomas. Tumor fragments were exposed to 12 kinds of antitumor drugs at ten times peak plasma concentration. Evaluable rates were 86/94 (91%) for all cases, 56/59 (95%) for gastric cancers, 22/27 (81%) for colo-rectal cancers and 8/8 (100%) for malignant lymphomas. The mean of SD activity was decreased to 48% of that of control cells with aclacinomycin, 49% with carboquone, 53% with actinomycin D, 54% with mitomycin C and 54% with daunomycin for gastric cancers, 59% with adriamycin for colo-rectal cancers and 33% with cyclophosphamide (40487 S), and 33% with actinomycin D, 37% with vinblastine and 39% with adriamycin for malignant lymphomas. When the SD activity was reduced to below 50% by antitumor drugs, the chemosensitivity was defined as positive. The antitumor drugs which had a higher chemosensitive-positive rate were aclacinomycin, carboquone and mitomycin C for gastric cancers, adriamycin for colo-rectal cancers and 40487 S, daunomycin and vinblastine for malignant lymphomas. Our results suggest that the origin of a tumor is a critical factor in its chemosensitivity.

In vitro chemosensitivity tests of anticancer agents for 119 fresh human tumors were performed by the human tumor clonogenic assay (HTCA) technique and the following results were obtained. Colony growth (greater than or equal to 5 colonies/dish) was observed in 35 of 50 gastric cancers (70.0%), 10 of 17 colon cancers (58.8%), 13 of 14 breast cancers (92.9%), two of six esophageal cancers (33.3%), three of six sarcomas (50.0%), three of 16 hematological malignancies (18.8%) and seven of 10 other tumors (70.0%). Colony growth rate differed according to the type of tumor. Fifty four tumors formed adequate colony growth (greater than or equal to 30 colonies/dish) for the chemosensitivity test. Mitomycin C (MMC), 5-Fluorouracil (5-FU), 4-hydroperoxy cyclophosphamide (CPM), Adriamycin (ADM), Cis-dichlorodiammineplatinum (CDDP), and alpha-interferon (IFN-alpha) were tested. The average positive rates of MMC, 5-FU, CPM, CDDP, and IFN-alpha were 26.9, 21.6, 10.5, 26.9, 36.8, and 23.3% respectively for all the tumors tested. In gastric cancer, the positive rates of MMC and 5-FU were 24.0 and 21.6% respectively, whereas the rates were 33.3 and 33.3% in colon cancer and 18.2 and 16.7% in breast cancer respectively. Each tumor exhibited its own chemosensitivity rates against various anticancer agents. Eighteen of the results obtained were comparable to clinical responses. The true positive rate was 50.0% (2/4) and the true negative rate 92.9% (13/14). A statistically significant correlation was observed between the results of HTCA and clinical responses (chi 2 test, p less than 0.05). The combined effects of IFN-alpha and MMC were surveyed against 20 gastroenterological tumors. Nine tumors exhibited synergistic effect, though antagonistic effect was observed in three tumors. The effects of oxygen tension (2%, 5%, 20%) on colony growth were investigated. The greatest development of colonies occurred at an oxygen of five percent, which is considered to be physiological oxygen tension, and statistically significant increases of plating efficiencies at 5% O2 as compared to those at 20% O2 were observed (t-test, p less than 0.025).

A phase I study on TA-077, a water-soluble nitrosourea, was performed by a 9-institution clinical group using 89 patients with various malignant tumors. The study consisted of single-dose i.v. administration and daily i.v. administration for 3-6 consecutive days. The dose-limiting factor was delayed leukopenia and thrombocytopenia which reached nadirs about 5 weeks and about 4 weeks after the initiation of administration and recovered in 2 to 3 weeks, respectively. Gastrointestinal toxicity, such as nausea, vomiting and anorexia, appeared in the early stage of treatment, although most of these symptoms were mild or moderate. Other side effects, including transient liver and renal dysfunctions observed in a few cases, were mild and appeared not to be dose-dependent. M.T.D. in single administration was considered to be more than 3,000 mg/m2 and M.T.D. in 5-day consecutive administration was considered to be 1,000 mg/m2/day. The recommended dose schedule for initiation of the phase II study was assumed to be 700 to 900 mg/m2/day for 5 consecutive days.

In vitro chemosensitivity was evaluated by bioluminescence ATP assay in 12 human tumors including 7 gastric cancers and 5 colo-rectal cancers. Tumor fragments minced with scissors were exposed to 6 kinds of antitumor drugs: carboquone, adriamycin, aclacinomycin A, mitomycin C, cisplatin and 5-FU at peak plasma concentration or ten times peak plasma concentration. After 3 days at 37 degrees C, each tumor fragment suspension was washed with phosphate-buffered saline, boiled for 3 min and assayed for its intracellular ATP level using the luciferin-luciferase method. The ATP level of the drug-untreated group was 6.01 +/- 4.55 X 10(-10) moles/mg tissue protein in gastric cancers and 9.77 +/- 8.46 X 10(-10) moles/mg tissue protein in colo-rectal cancers. The percentages of cases in which the ATP level was reduced to less than 50% by the antitumor drugs were 70% for carboquone, 11% for adriamycin, 30% for aclacinomycin A, 45% for mitomycin C, 13% for cisplatin and 18% for 5-FU at peak plasma concentration, and 90% for carboquone, 78% for adriamycin, 80% for aclacinomycin A, 82% for mitomycin C, 63% for cisplatin and 82% for 5-FU at ten times peak plasma concentration. The test for chemosensitivity prediction is thus available at peak plasma concentration of antitumor drugs in ATP assay.

Reserpine was shown to enhance the cytotoxicity of ACNU in both C6 and C6/ACNU rat glioma cells in vitro and also to enhance the chemotherapeutic effect of ACNU in C6/ACNU-bearing rats (C6/ACNU meningeal gliomatosis rats), in which ACNU resistance could be partially overcome by reserpine. When reserpine was added to the culture at a concentration of 10 microM, the IC50 of ACNU for C6/ACNU cells decreased to the level of that for C6 cells. Intracellular uptake of ACNU in C6/ACNU cells increased and the efflux from the cells decreased when 20 microM reserpine was added to the culture. In in vivo experiments, combined ACNU (1 mg/kg) and reserpine (250 micrograms/kg) therapy by intrathecal injection of these drugs improved % ILS (increased life span) with statistical significance compared with that after treatment with ACNU alone. The probable explanation of the enhanced cytotoxic-effect of ACNU in ACNU-resistant glioma cells presented in in vitro and in vivo is increased intracellular ACNU concentration resulting from inhibition of the efflux of ACNU from the resistant cells.