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A quadruple combination chemotherapy with ACNU, adriamycin, methotrexate and prednisolone (AAAP), was performed on 9 patients with non-Hodgkin's lymphoma, who had been previously treated with a VEPA (P) regimen. Complete remission rate was 44% (4/9) and, in four other patients, the tumor was decreased in size. Complete remission duration was from 30 days to over 390 days. Median survival time was 105 days. Myelosuppressive toxicity was severe. In eight patients, WBC counts were less than 3,000/mm3. In six patients, platelet counts were less than 100,000/mm3. An AAAP regimen is useful in relapse or for resistant forms of non-Hodgkin's lymphoma.

A 60-year-old woman was referred to us because of tumors on the occipital and the bilateral submaxillary areas. Biopsy proved them to be well-differentiated lymphosarcoma. On admission, systemic lymphadenopathy was noted and there was 26% of plasmacytoid cells in the bone marrow of the sternum. Monoclonal gammopathy of IgM,K type was found; her disease was diagnosed as a macroglobulinemia (IgM: 8,460 mg/dl). VENP-therapy consisted of vincristine 1 mg/w, cyclophosphamide 50 mg/d procarbazine 50 mg/d and prednisolone 30 mg/d was applied for about four weeks, but in vain. Transaminase levels were elevated (GOT 575 U, GPT 480 U) and the superficial lymphnodes did hardly diminish. Therefore, after improvement of the liver dysfunctions, 5 courses of AAAP-therapy, which was consisted of ACNU 50 mg/d (IV drip over 4 hrs), adriamycin 20 mg/d (IV push), methotrexate 25 mg/d (IV push) and prednisolone 60 mg/d (IV push) once a week or three were employed with excellent clinical effects. The superficial lymphnodes disappeared, M-protein and plasmacytoid cells in the bone marrow markedly decreased. An interval of the initial remission reached to 17 months. As previously reported, AAAP-therapy was also effective to multiple myeloma and acute lymphocytic leukemia of B-cell type. Therefore, AAAP-therapy would be one of the best chemotherapies for B-cell malignancy including macroglobulinemia.

Using our new in vitro antitumor sensitivity assay, the basis of which depends on predictive analysis of morphological findings of L1210 leukemia cells under the influence of antitumor agents, 5 kinds of nitrosoureas including MCNU were comparatively tested for antitumor activity. A cell killing effect became apparent very soon under BCNU and CCNU, rather late under Methyl-CCNU and MCNU, and intermediately under ACNU. Various cellular biological effects were apparently induced in L1210 cells by MCNU and its mechanism of action seemed to be broader than that of any other members of the nitrosoureas.

For the early diagnosis of recurrence of acute leukemia, differential count of the 5000 leukocytes was found very effective. In 29 cases out of 30 cases whose leukemic cells were elevated to the level higher than 10/5000 from the level of 0-4/5000 in stable stage of remission, overt recurrence was diagnosed by peripheral blood or bone marrow examination after 3-12 weeks. Prevention of overt recurrence was observed in several cases, in which an intensive treatment was administered at the early stage of recurrence. For the treatment of cases with overt recurrence, it is important to plan the treatment with drugs different from those used in previous treatment. AAAP therapy or new drugs such as mitoxantrone might be considered. In cases in which leukemic cells proliferate earlier than normal cells after intensive treatment, a sustained small dose Ara-C therapy was often effective.

A nitrosourea derivative, ACNU (nimustine hydrochloride), is often used in the chemotherapy of brain tumors and shows considerable efficacy, since it crosses the blood-brain barrier (B.B.B.). This drug is also considered to be useful for intrathecal treatment of meningeal gliomatosis (MG) because of its short half-life in the blood or cerebrospinal fluid (CSF) and its strong cytotoxicity for glioma cells. In order to evaluate the efficacy of intrathecal therapy of MG with ACNU, MG models, which were produced by intracisternal inoculation of rat C6 glioma, were treated with intrathecal or intravenous administration of ACNU. When intrathecally administered 1 day or 3 days after tumor inoculation, ACNU (1 mg/kg) significantly prolonged the survival time of MG rats, where ILS was 35.7 to 42.9% and 24.1 to 25.0%, respectively. In MG rats which were treated intrathecally with ACNU (1 mg/kg) 5 days after tumor inoculation or intravenously with ACNU (15 mg/kg), ACNU failed to prolong survival time compared with the controls. It might therefore be suggested that intrathecal chemotherapy with a low dose of ACNU is effective in the early stages of MG, in which intravenous treatment with a high dose of ACNU is ineffective.

The effect of multidisciplinary treatment of small cell carcinoma of the lung was tested. The response rates to induction chemotherapy were 69.2% (9/13) and 57.1% (12/22) in small cell carcinoma of the lung with limited and extensive disease, respectively. The response rates of oat and intermediate cells were 63.6% (14/22) and 50% (5/10), respectively. Radiation therapy to primary site and mediastinum after induction chemotherapy was also effective, producing the response rates of 87.8% (7/8) and 58.3% (7/12) in limited disease and extensive disease, respectively. The non-responder and recurrent cases responded to chemotherapy with epipodophyllotoxin and/or cisplatin but not with mitomycin C and/or adriamycin. The median survival of all 37 cases was 12.2+ months, and those with limited and extensive cases were 14.8+ and 8.7 months, respectively.

Effects of a water-soluble nitrosourea derivative, 1-(4-amino-2-methylpyrimidin-5-yl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride [ACNU]. on survival and cell progression of HaLe S3 cells was investigated. The survival of exponentially growing cells exposed to increasing concentrations of the drug was characterized by a threshold-type survival curve (D0 = 7.0 micrograms/ml X 1 hr, Dq = 3.5 micrograms/ml X 1 hr). ACNU exerted its main killing effect on cells in G1 and G2 + M phases, whereas cells in S phase were resistant to the drug. Changes in survival response as a function of cell cycle were mainly dependent upon the extent of the exponential slope of the survival curve. Cell progression effects were examined by using a low concentration of ACNU in which 80% of treated cells could survive. Cells in G1 and early S phases at the time of treatment were not prevented from entering S phase but prolonged in duration of S phase followed by a marked delay in progression through G2 phase. However, such a delay in cell progression time was reduced in cells treated in mid S phase as compared with G1 and early S phases. Cells treated in late S and G2 phases could normally progress into mitosis.

FU-O-G, the O-glucuronide methylester of 5-fluorouracil (5-FU), is a compound with a unique chemical structure. It is an antitumor agent of the prodrug type which exerts its activity by enzymatically liberating 5-FU in tumor tissues. It has been reported that the antitumor activity of this compound is superior to those of 5-FU and Tegafur (FT-207) in the treatment of various transplantable tumors. In this study, long-term administration of FU-O-G to mice bearing relatively slow-growing tumors such as Lewis lung carcinoma, mammary tumor FM3 A and hepatoma MH-134 was carried out. Consequently, FU-O-G was shown to be remarkably effective against those tumors, whereas 5-FU and FT-207 were hardly effective. Long-term daily administration was shown to be more effective than intermittent dosage in the treatment of Lewis lung carcinoma. In combination therapies of FU-O-G with other antitumor drugs, FU-O-G exhibited a synergistic effect against Lewis lung carcinoma when combined with Carboquone (CQ) or Nimustine hydrochloride (ACNU).

Human motor endplate acetylcholinesterase was inhibited in vitro by alkylating antineoplastic agents, most strongly by mechlorethamine, followed by DTIC, ACNU, cyclophosphamide and ifosfamide. Eleven other antineoplastic agents did not inhibit the enzyme substantially nor interfered with cholinesterase measurement. Cyclophosphamide and mechlorethamine inhibited human plasma pseudocholinesterase most strongly, followed by thiotepa, ACNU, DTIC, ifosfamide and BCNU. Mechlorethamine, ACNU and ifosfamide inhibited the motor endplate and plasma cholinesterase practically equally, DTIC inhibited motor endplate cholinesterase more strongly, while cyclophosphamide was a more selective inhibitor of plasma cholinesterase. Inhibition of human red blood cell acetylcholinesterase was identical to that of motor endplate acetylcholinesterase; therefore, red cells would be a preferable indicator in monitoring cholinesterase inhibition by antineoplastic agents.

This study, based on a cooperative group project involving 4 major medical institutes in Japan, presents the second survey of malignant melanoma patients (198 cases) where an attempt is made to systemically evaluate the survival rates of these patients with respect to the tumor thickness and location of primary lesions, and the response to chemoimmunotherapy. More than 50% of total collected cases showed the primary lesions on the limbs. The most common type and site of involvement is the acral lentiginous melanomas involving the plantar areas (more than 30). The survival rates affecting the limbs were better than those affecting the non-limb areas. However, the comparison of the survival rates did not reveal any difference between those cases affecting the plantar and non-plantar areas. The difference in the prognosis of melanoma patients appeared to be related to the tumor thickness. By historical comparison, the DAV (DTIC, ACNU, VCR) treated group exhibited a better survival rate than the non-DAV treated group. Furthermore, the DAV group with immunoadjuvant therapy (mainly OK-432) showed a better prognosis than the DAV group without any immunoadjuvant therapy.

Flow cytometric analysis was used to study the effect of a calcium influx blocker, nicardipine hydrochloride, on the cytotoxicity of anti-cancer drugs in tumor cells. The parameters used for this study were DNA- and protein-cell distribution histograms. Cells and drugs used for this study were C6 cells from CD Fisher rat glioma and VCR, ADM and ACNU, respectively. The growth inhibitory effect estimated by concentration at ID50 on C6 cells was indicated as follows; VCR showed an 89-fold enhancement, while ACNU showed little enhancement following addition of nicardipine hydrochloride. DNA and protein histograms obtained by flow cytometry revealed almost the same effects as cells which were studied using high concentration of VCR without nicardipine hydrochloride. For the other drugs, ADM showed a small enhancement on histograms, while with ACNU, little enhancement was noted as well as the inhibitory effect of each drug described above. The results indicate that nicardipine hydrochloride greatly affects the cell cytotoxicity of VCR but not so much with ADM and ACNU. From these results, it appears that this drug enhances the action of anti-cancer drugs not only by merely blocking the efflux of drugs from cells but also by other mechanisms which remain to be clarified.

The serum carcinoembryonic antigens (CEA) levels in 177 advanced lung cancer patients were studied to assess their value for the prognosis and indicating the effectiveness of chemotherapy. The relationship of pretreatment CEA levels with histology and stage of disease was also examined. Levels in excess of 5 ng/ml and 20 ng/ml were found in 55% and 32% of lung cancer patients, respectively. The elevated CEA levels were more frequently observed in patients with adenocarcinoma (65% in excess of 5 ng/ml) and extensive disease, but pretreatment CEA levels were not significantly correlated with the histology and clinical stage of disease. In 102 patients with adenocarcinoma, there was no significant difference of survival time in each patient with CEA levels less than 5 ng/ml, 5.0 less than or equal to - less than 20 ng/ml and in excess of 20 ng/ml; median survival time was 7, 7, 8 mo, respectively, and response to chemotherapy was not significant in each of these groups. Serial serum CEA measurements in patients with pretreatment levels in excess of 20 ng/ml correlated well with changes in disease status reflecting clinical response to chemotherapy. Mean percent changes of CEA levels to pretreatment levels were-77.4% in patients with partial response (PR), -55.6% in those with minor response (MR), -4.0% in patients with no change (NC) and +79.0% in patients showing progressive disease (PD). There was a significant difference in the percent changes of CEA levels between patients with an objective response (PR) and patients who had none (MR + NC) (p less than 0.02). CEA levels of all patients who had PD increased or unchanged. Serial measurements of serum CEA are useful in patients whose pretreatment levels are more than 20 ng/ml for monitoring the response to chemotherapy, and may be a useful noninvasive technique for patients with unmeasurable disease as a monitor of tumor burden in response to chemotherapy and recurrent disease.

A combination chemotherapy "VEMA" consisting of vincristine (VCR), cyclophosphamide (Endoxan, EX), methotrexate (MTX) and nimustine (ACNU) has been carried out for the treatment of small cell bronchogenic carcinoma since September, 1978. "VEMA" regimen consists of VCR 1.3 mg/m2 iv push on day 1, EX 500 mg/m2 iv infusion on day 1 and 2, MTX 28 mg/m2 iv push on day 1, 2 and 3, and ACNU 67 mg/m2 iv push on day 3. This dose schedule was repeated every 3 to 4 weeks. The regimen was given to 14 patients and 12 patients were evaluable. In the 12 evaluable cases, 2 case of complete response (CR), 7 cases of partial response (PR) and 2 cases of effusion effective were obtained. Response rate of CR + PR was 90%. Response rate including CR, PR and effusion effective was 91.7%. The major clinical toxicity of "VEMA" therapy was bone marrow suppression. Other side effects were anorexia, nausea, vomiting, alopecia and stomatitis: etc; however, these side effects were not life threatening to terminate "VEMA" therapy. In conclusion, "VEMA" regimen is a new potent combination chemotherapy in the treatment of small cell bronchogenic carcinoma.

Malignant melanoma is one of the most malignant froms of cancer, for which the prognosis is still very grave. However, it need not necessarily be regarded as such a fearful tumor if it is found at an early stage and treated thoroughly and adequately. An understanding of the early clinical changes in the size, color and shape of the tumor is required, and early discovery can save a patient's life. Histopathological examination should be done for diagnosis, and S-100 protein should be checked together with monoclonal antibody studies sometimes. The main principle of treatment in the early stage of malignant melanoma is to make an incision 2 to 5 cm away from the edge of the tumor. As a rule, exploratory excision and minor resection should not be performed. It may be said, accordingly, that multidisciplinary treatment should only be given for malignant melanoma of stage 1b and more advanced stages. At present, chemotherapeutic combinations used as the first choice in Japan are two regimens, one of which is DAV (DTIC, ACNU and vincristine), and the other, PAV (peplomycin, ACNU and vincristine). The effectiveness rate with DAV and PAV have been approximately 30% in various clinical studies. Favorable clinical responses obtained by radiotherapy have often been observed after fast neutron irradiation and hyperthermia therapy. Immunotherapy and interferon treatment are also often applied for malignant melanoma.

Comparative studies of alternating non-cross resistant chemotherapy, A.V.N.-D.V.C. (ACNU + VCR + PCZ-ADM + VCR + CPA) was carried out on 19 patients with small cell lung cancer. A.V.N. (A. V.F.) therapy on 45 patients and D.V.C. therapy on 19 patients were used as historical control, respectively. A.V.N. (A.V.F.) therapy was composed of ACNU (2.5 mg/kg, day 1), VCR (0.02 mg/kg, once every week) and PCZ (1 mg/kg, daily) (or FT-207 (15 mg/kg, daily], and duration of one course was 6 to 8 weeks. D.V.C. therapy was composed of ADM (1 mg/kg, day 1) VCR (0.02 mg/kg, days 1 and 5) and CPA (2 mg/kg, days 1 to 5), and repeated every 4 weeks. A.V.N.-D.V.C. therapy was done by a timely alternation of one course of A.V.N. and two course of D.V.C. Reduction rate of tumor size was 84% in A.V.N.-D.V.C. therapy, 62% in A.V.N. (A.V.F.) therapy and 26% in D.V.C. therapy, respectively. Median survival time was 13 months on A.V.N.-D.N.C. therapy, 8.5 months and A.V.N. (A. V.F.) therapy and 4 months on D.V.C. therapy. Significant prolongation of median survival time was obtained in A.V.N.-D.V.C. therapy in comparison with that of historical controls. Major toxicity in A.V.N.-D.V.C. therapy was slight bone marrow suppression.

Local chemotherapy by intra-arterial administration of ACNU was performed in 2 cases with CNS leukemia, which responded well to this therapy. The first patient was a 42-year-old male who was diagnosed as having chronic myelogenous leukemia with local infiltration of leukemic cells to the optic nerves. By intra-arterial infusion of ACNU (60mg), symptoms and results of ophthalomological examinations were improved remarkably. The second case was a 16-year-old male diagnosed as having acute lymphoblastic leukemia, and complicating with meningeal involvement during the course of chemotherapy. Complete remission was achieved by intra-thecal administration ACNU. Intra-arterial infusion of ACNU would be an effective local chemotherapy of CNS leukemia and its effectiveness could be achieved by less dose of ACNU compared to that of intravenous infusion. Therefore, side effects, e.g., delayed myelosuppression caused by ACNU could be decreased by this method.

The effect of a combination therapy of hyperthermia (43 degrees C, 30 min) and ACNU (10 mg/kg) on B 16 melanoma and C 24 melanoma in vivo was studied. This combination therapy was effective in both melanoma. In C 24 melanoma, remarkably the tumor regression and the partial destruction of the histological structure, were observed.

A new water-soluble nitrosourea ( MCNU ) was tested for its antitumor activity against fourteen human glioma cell lines and two neuroblastoma cell lines. Four experiments were performed to determine its antitumor activity: inhibition of cell growth, comparison with ACNU, morphological observation, and analysis of DNA histogram with flowcytometry . Seven out of 14 gliomas (50%) and one neuroblastoma cell lines showed more than 50% inhibition of cell growth in vitro, appearance of giant multinucleated cell morphologically, and DNA accumulation in G2+M and/or S phase of cell cycle in the medium of 10 micrograms/ml MCNU . Antitumor activity and spectrum of MCNU against human brain tumors were almost the same as with ACNU.

T cells bearing IgG Fc receptors (Tr cells) were determined in 40 cases of brain tumor using the double rosette method, and compared with the changes of PHA reaction, immunoglobulin (IgG A M), performance status, and mass effect on CT in 20 pre-and post-operative and/or radio-clinico-immunotherapeutic patients. The proportion of control Tr cells was 8-19% in 20 healthy adults. Values of Tr cells over 20% were correlated with a poor grade of performance status, and large mass effects on CT. Post-therapeutic change in the value was well correlated with change in performance status, mass effects, and IgG. Our results suggest a correlation between the value of Tr cells and clinico-radiological findings. Immunological analysis of Tr cells serves as a parameter of tumor expansion or prognosis, and could be of significant importance clinically.

Effect of intraarterial infusion chemotherapy on metastatic liver tumor (28 cases) of colo-rectal cancer was compared with that of oral or intravenous chemotherapy (8 cases). The efficacy of the selective intraarterial chemotherapy was 40%, non-selective intraarterial chemotherapy, 11.1% and oral or intravenous chemotherapy 0%, respectively. Mean survival time of the intraarterial chemotherapy was 11.1 months, and that of oral or intravenous chemotherapy was 6.0 months, suggesting greater efficacy of the intraarterial infusion chemotherapy. Especially, the selective intraarterial infusion chemotherapy will be an effective therapy for inoperable metastatic liver tumor.

The use of drugs in combination for the management of cancer patients aims at the increased therapeutic advantage by elimination the problem of the heterogeneous sensitivity of cancer cells to anticancer drugs, and by delaying or preventing the development of drug resistance within given tumors. Theoretically, the effects of drugs in combination are classified as antagonistic, subadditive, additive and synergistic. Since these results hold for both tumors and hosts, the effects of combined drugs should be considered in terms of therapeutic index. From this viewpoint, the choice and administration schedule of drugs in combination must be synergistic or additive for the tumors and antagonistic or subadditive for the hosts in regard to combined effects. Thus, the rationale for combined drug therapy should be considered from the aspects of biochemical basis, drug resistance, cytokinetic and pharmacologic rationales, and toxicologic basis.

In the present paper we discussed whether a new method of chemosensitivity test could be developed using FCM (flow cytometry) and applied to malignant brain tumor cells labelled with BrdU monoclonal antibody. For this purpose, a basic study was performed with an ACNU-resistant C6 cell line and a sensitive one to see if this method was able to detect the difference of sensitivity between these cell lines. After 8 hours treatment with ACNU (concentration: 10 micrograms/ml), ACNU-sensitive cells revealed on LI (labeling index) of 46%, which was very high in comparison with controls, whereas the figure for ACNU-resistant cells was 34%, which was almost the same LI value as non-treated control cells. This means that change in the BrdU labeling index after chemotherapy in vitro can be used to determine the chemosensitivity of malignant brain tumors when FCM is employed. Furthermore, this method can detect the chemosensitivity of each clone in polyclonal human tumors which is impossible for the HCSA method, since the latter can reveal only the chemosensitivity as a whole in such tumors.

A case of erythroleukemia coexistent with pulmonary emphysema is reported. A 67-year-old male was admitted to our hospital in May 1981, with a few year history of cough, sputum and fatigue. He had already been diagnosed as having pulmonary emphysema and moderate anemia. On physical examination, except for pallor, no other findings were remarkable. The initial hematological examination showed hemoglobin, 9.6 g/dl, red cell count, 251 x 10(4)/microliters, platelet count, 7.3 x 10(4)/microliters, white cell count, 2600/microliters with neither myeloblasts nor erythroblasts. A sternal marrow aspiration revealed 21% myeloblasts and 40% erythroblasts including 7.5% megaloblastoids. Periodic Acid Schiff staining was strongly positive for a part of erythroblasts. A chest X-P finding was typical for pulmonary emphysema. Pulmonary function was moderately damaged. He was started on chemotherapy with AAAP (ACNU 50 mg/d i.v. drip over 4 hr x 4d, adriamycin 20 mg/d i.v. push x 4d, Methotrexate 20 mg i.v. push x 4d). The first course of AAAP brought him a complete remission with both disappearance of myeloblasts and erythroid precursors with megaloblastoid nuclei in the marrow and the normalization of white cell count and platelet count in the blood. He was discharged in September 1981 after completion of a consolidation chemotherapy with AAAP. Since then, he received two courses of AAAP as an intensification chemotherapy and has been in complete remission for more than 13 months. His pulmonary function has not been affected and no myocardial damage has been seen throughout AAAP therapy. Thus, AAAP therapy seems to be an excellent chemotherapy even for an aged patient with erythroleukemia.

Between August 1982 and February 1985, 13 patients with pathological stage I non-small cell lung cancer received postoperative chemotherapy with ACNU, ADM and 5-FU. Nine patients had squamous cell carcinoma, two adenocarcinoma, and two large cell carcinoma. All patients were alive in August, 1985, but four of them had recurrent disease. The site of recurrence was the brain in three and the contralateral lung in one. Of these four patients, three had t2n0(-) p0 squamous cell carcinoma and one t1n0(-) p1 large cell carcinoma. In a control group consisting of six squamous cell carcinoma and six adenocarcinoma, recurrence occurred in one patient with t1n0(-) p0 squamous cell carcinoma. Although the treated group had unfavorable conditions of cell type and tumor size, there was no evidence that this combination chemotherapy could control recurrence of the tumor under these conditions.

The effects of heat and antitumor drugs on malignant brain tumor cell lines were studied. A human glioblastoma cell line (SKMG1) and rat malignant brain tumor cell lines (T9, EB 679 and TR 481) were used in this experiment. Five different modalities of treatment with heat and drugs were used as follows: (Group 1) exposure to heat alone at 42 degrees C for one hour; (Group 2) exposure to antitumor drug alone for one hour (ACNU 2.5 or 5 micrograms/ml, ACR 0.02 micrograms/ml and CDDP 1 microgram/ml); (Group 3) simultaneous exposure to heat at 42 degrees C and drug for one hour; (Group 4) heat at 42 degrees C given first for one hour, followed by one hour exposure to drug one hour later ("preheating"); (Group 5) drug given first for one hour, followed by one hour exposure to heat at 42 degrees C one hour later ("postheating"). After each treatment, the inhibition rate at 4 days was evaluated and compared for each group. A synergistic effect was observed in Group 3. For example, when T9 cells were exposed to ACNU and to heat at 42 degrees C at the same time for one hour, inhibition rate was 78%, while the rates for Group 1 and Group 2 were 7% and 21%, respectively. The cytotoxicity of simultaneous treatment with antitumor drugs (ACNU, ACR and CDDP) and hyperthermia at 42 degrees C was apparently superior to that of other treatment modalities.

Augmentation of cytotoxicity against malignant glioma using a combination of Hu IFN-beta and ACNU was analyzed in vitro from the points of cell growth inhibition and alteration of the DNA histogram. Cytotoxicity was tested by exposing 13 human glioma cell lines to ACNU at a concentration of 5 micrograms/ml and/or Hu IFN-beta at a concentration of 10(3) IU/microliters. These concentrations were considered to be clinical doses. Additive or synergistic effects of the combination of the two agents were observed in all cell lines tested. The cytotoxic effect of two log kill was seen in two cell lines given ACNU treatment alone, one cell line treated with Hu IFN-beta alone, and nine treated with the combination. Flow-cytometry studies of the DNA histogram showed accumulation in the G2+M phase with ACNU and in the S phase with Hu IFN-beta. On the other hand, marked accumulation using the combination in the S phase followed by the G2+M phase was observed and this accumulation lasted for over a week. The present results show an augmentation of antitumor activity and suggest the effectiveness of combined Hu IFN-beta/ACNU therapy in the treatment of patients with malignant glioma.

A randomized comparative study of anticancer chemotherapy CE (CDDP plus etoposide) and CE-AVN (ACNU, VCR plus procarbazine) was carried out on 27 patients with small cell lung cancer (SCLC) without previous chemotherapy. In CE therapy, 12 patients received injection of CDDP (80 mg/m2 on day 1) and etoposide (75 mg/m2 on day 1-5) every 4 weeks (Protocol 1). Fifteen patients received 2 courses of CE and 1 course of AVN therapy (ACNU 100 mg/m2 on day 1, vincristine 0.7 mg/m2 once a week and procarbazine 50 mg/day, daily) for 6 to 8 weeks (Protocol 2). One patient (8%) and 2 patients (20%) achieved complete response with Protocol 1 and 2, respectively. Seven patients (58%) and 9 patients (60%) achieved partial response with Protocol 1 and 2, respectively. The median survival time (MST) was 12 and 14 months, and duration of remission was 4.5 and 7 months in patients treated with Protocol 1 and 2, respectively. No significant difference in MST and duration of remission in each group was observed. However, 2 patients (13%) treated with Protocol 2 survived more than 3 years. Both protocols were well tolerated with only moderate gastrointestinal symptoms, mild bone marrow toxicity and alopecia.

This paper provides an overview of cancer chemotherapy with special reference to the pharmacokinetics of the nitrosoureas. At physiological PH, the chloroethylnitrosoureas can be decomposed into an isocyanate and 2-chloroethyl diazene hydroxide. Therefore, it is clear that they have both alkylation and carbamoylation actions. In addition to the spontaneous chemical dissociation, the nitrosoureas can be metabolized by liver microsomal enzymes to more polar hydroxylated products, and certain nitrosoureas can be denitrosated by these enzymes to the parent urea. Since the lipid-soluble nitrosoureas and some of the water-soluble nitrosoureas such as ACNU and MCNU demonstrated to cross the blood-brain barrier, they have been used in the treatment of primary brain tumors and tumors and tumors of metastatic origin. It has been demonstrated from the results of our study and other reports that the alkylation of DNA by ACNU progresses more slowly as compared with that of other alkylating agents. This is an important finding in relation to the appearance of delayed myelosuppression of the nitrosoureas and in the design of dose schedules of these agents. The major clinical emphasis has been directed towards the more active chloroethylnitrosoureas with reduced myelosuppression, and attempts are now made for this purpose. Unfortunately, the results of phase I and II trials of the newly developed nitrosoureas suggest that these agents produce delayed and cumulative bone marrow toxicity. Antitumor activity of the nitrosoureas is frequestly observed in chronic myelocytic leukemia, malignant lymphoma, brain tumors and small cell carcinoma of the lung, and less frequently in gastrointestinal carcinoma, multiple myeloma and malignant melanoma. In order to enhance clinical effects of the nitrosoureas, further investigation of the design in therapeutic schedules on the basis of their pharmacokinetic characteristics will be needed.

Cytotoxic and cytokinetic effects of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl) 3-nitrosourea hydrochloride (ACNU) on cultured rat and human glioma cells (C-6 and KC) were studied in vitro. Exponentially growing culture cells were exposed to ACNU at the final concentrations of 5 micrograms/ml, 20 micrograms/ml, and 80 micrograms/ml, respectively. The cytotoxic effect was evaluated by inhibition of cell growth and the cytokinetic effect was analyzed by DNA histogram using a flow cytometer. Inhibition of cell growth was dose-dependent in ACNU and C-6 cells were more resistant than KC cells. The growth of C-6 and KC cells were not inhibited at all by low concentrations of ACNU (5 micrograms/ml, 20 micrograms/ml), however, at these concentrations a marked accumulation of treated cells in S and G2+ M phases was evident. The accumulation in S and G2+M phases was dose-dependent and it was more prominent in KC than C-6 cells. ACNU-treated cells accumulated initially in S phase and then in G2+M phase. After maximum accumulation in G2+M phase, the cells seemed to be released into G1 or G0 phase. These results indicate that the cytokinetic effect of ACNU (5 micrograms/ml, 20 micrograms/ml) is more conspicuous than the cytotoxic effect on C-6 and KC cells.

A combination chemotherapy (PAV) consisting of peplomycin, ACNU and vincristine (VCR) was given to 30 patients with malignant melanoma and its therapeutic evaluation was performed. The objective response rate was 42.9% for the patients with stage IV metastatic lesions; three of 7 patients showed improvement. This regimen was particularly effective for both cutaneous and subcutaneous metastatic lesions. When PAV was applied as an adjuvant therapy to the operable cases with stage Ib and II, a five-year survival rate was 50% and the result was far better than that of operation alone. Our results in PAV regimen almost identical with those of DAV(DTIC, ACNU, and VCR) regimen as an adjuvant therapy. The result indicates that PAV regimen is useful for the treatment of malignant melanoma since toxic reactions were mild. Further studies are necessary to assess the efficacy of PAV regimen.

A 60-year old woman with metastatic malignant melanoma who was well responded to a combination chemotherapy including DTIC was reported. She was noted a lentigo in the left first toe and histological examination revealed malignant melanoma in October 1978. Amputation of the left lower leg and dissection of the left inguinal lymph nodes had been done. OK-432 was injected as postoperative immunotherapy. She was readmitted to our hospital with the symptoms of pain and numbness of the left arm. Physical examination revealed a palpable mass in the left supraclavicular region. Incisional biopsy of the supraclavicular mass revealed metastatic malignant melanoma. She received a combination of 100mg DTIC i.v. for 5 days, 100 mg ACNU i.v. for one day and 1 mg VCR i.v. for one day (DAV chemotherapy) postoperatively. Subcutaneous injection of OK-432 with the dose of 5 KE per week was continued. Major side effects of DAV chemotherapy were nausea and transient leukocytopenia. No serious side effects were observed. On completion of the first course of DAV chemotherapy, abnormal shadow of the left apex was completely disappeared and on completion of the third course of DAV chemotherapy, high density area was markedly decreased in the cervical CT. She gained symptomatic reliefs and was discharged in August 1983. The combination chemotherapy including DTIC appeared to be effective in the treatment of metastatic malignant melanoma.

Five patients with malignant pleural effusion were treated with intrapleural administration of ACNU. Three of 5 patients showed cytologically negative in the pleural fluid and the fluid was eliminated in two patients. In 5 patients, 200 mg of ACNU was injected into the pleural space and pharmacokinetic behavior was studied. The clearance curves of ACNU in pleural fluid were described by either one-compartment model or two-compartment model. The mean half life of slow phase was 0.75 hour. These results indicate that ACNU disappears rapidly from the pleural space after the intrapleural administration. The effect of ACNU on the pleura was studied histologically in rabbits. At a dose of 3mg per kg body weight, the mesothelial cells were swelling and small areas of cellular desquamation appeared over the pleural surface. With increase in dosage, these findings were more pronounced and in the submesothelial tissue there was edema as well as cellular infiltration. In rabbits given two injections with interval of one week, the pleura showed a stronger reaction than that produced by single injection.

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.

Hyperthermic treatment using ACNU combined with a thermosensitizing drug, methylglyoxal-bis-guanylhydrazone (MGBG), was studied in human gastric cancer xenotransplanted into nude mice. In order to increase the intra-cellular MGBG content, intraperitoneal injection of alpha-difluoromethylornithine(DFMO) 1000 mg/kg was performed twice with an interval of 6 hours and 50 mg/kg of MGBG was given at the time of the second administration of DFMO. After 6 hours of MGBG administration, ACNU 20 mg/kg was given intraperitoneally and, subsequently a 23-minute hyperthermia was carried out in a water bath at 43.5 degrees C. After 48 hours a second hyperthermia was performed by the same method. Tumor weight was estimated using Battelle's Columbus Institute protocol and the inoculated tumors, which were extirpated 60 minutes after 3H-thymidine injection at a prescribed interval after cessation of hyperthermia, were assayed biochemically for the determination of DNA biosynthesis. In mice given ACNU, DFMO, MGBG plus heat, considerably superior results were obtained. Although the DFMO plus MGBG group was inferior in antitumor activity to the ACNU only or heat only group, the DFMO, MGBG plus heat group showed much the same antitumor effects, compared to the ACNU plus heat group. These data suggest that the thermosensitizing efficacy of MGBG may be applicable for clinical use.

Skin metastasis of malignant melanoma has been difficult to control by chemoimmunotherapy. We report a case of melanoma with marked reduction of multiple skin and lung metastasis and an improved cell-mediated immunity using combined DAV (DTIC, ACNU, Vincristine) and OK-432 chemoimmunotherapy in association with doses of indomethacin administered over a long period (250 mg/day, 6 months) to relieve the cancerous pain.

Various nitrosoureas are widely used effectively for chemotherapy of brain tumors, clinically and experimentally. Fourteen cases of malignant brain tumor received intravenous injection of 100-150 mg/body of ACNU, and the concentration of ACNU in blood and cerebrospinal fluid (CSF) was measured. The conclusion drawn from this study on penetration of ACNU is summarized as follows: 1. ACNU concentration in CSF and its ratio to that of serum (CSF/serum %) showed the various values of 0-0.076 micrograms/ml (0-63%), 30 min. after administration; 2. Concentration of ACNU in spinal CSF differed from that in ventricular CSF and CSF/serum ratio was 19.9% in spinal CSF and 42% in ventricular CSF, respectively; 3. Statistically no correlation between ACNU CSF/serum ratio and the postoperative time was found. Transition of ACNU into cerebrospinal fluid during radiation therapy was noted with high value of CSF/serum ratio.

Since interferon (IFN) has a mechanism of action very different from chemotherapeutic agents, it is possible that a combination of two may be of therapeutic value. The authors studied increased cytotoxic effects of anticancer drugs by IFN on human tumors xenografts in nude mice. Tumor used in this study were "SH-10", "S-7379" (gastric cancer) and "O-7294" (malignant melanoma), serially transplanted subcutaneously. IFN was injected, 5 X 10(5) mu/mouse, every day for 2 weeks and a single drug was administered 3 times every fourth day. Cytotoxic effect was determined by tumor size on day 16 after treatment. Of the 7 drugs, MMC and ADM were most effective. Other drugs showed a slight inhibition of tumor growth by combination therapy with drugs and IFN.

Experimental models of meningeal gliomatosis (MG) have been produced by intracisternal inoculation of C6 and 9L glioma cells into Wistar and Fisher 344 rats, respectively. Chemotherapy of these models and in vitro chemosensitivity assay for these cell lines were studied with ACNU, BCNU and VM-26. In vitro chemosensitivity assay revealed that 9L cells were sensitive to all of the anticancer drugs above, and that C6 cells were resistant to ACNU and BCNU, but not to VM-26. In vivo experiment, the survival time of the rats inoculated with 9L glioma cells (9LMG) was prolonged by both ACNU and BCNU but not by VM-26. None of these drugs were effective against the rats inoculated with C6 glioma cells (C6MG). It is concluded that the result of in vitro chemosensitivity assay is not always correlative with that of in vivo. This implies that an in vivo chemosensitivity assay system including MG models is indispensable in researching into chemotherapy of brain tumor.

In order to evaluate the efficacy of surgical adjuvant chemotherapy in patients undergoing gross curative resection for colorectal cancer (excluding m and sm cancer), a randomized controlled study was conducted from January, 1982 to October, 1983. Four hundred and ninety-one institutions participated in this study. The schedules for drug administration differed according to each district. In the Hokkaido and Shikoku districts, the patients were divided into the following two groups, one was a combination of ACNU and Futraful (FT) and the other was FT only. In the Chubu and Kinki districts, three groups were studied, namely those receiving a combination of ACNU and FT, those receiving FT only and those given no adjuvant chemotherapy. In the Tohoku and Kanto districts, a combination of MMC and FT and administration of FT only were studied. In the Chugoku and Kyushu districts, the patients were divided into a combination of ADM and FT, and FT only group. Among the 3,926 registered cases, 3,421 cases were valid for the study. As to the background factors, there were no significant differences among the groups in each district. There were no significant differences in one-year survival rates and one-year disease-free rates. No serious adverse effects were observed in any of the groups.

There have been many attempts to treat patients with malignant brain tumors represented by glioblastomas using nitrosourea (NU) derivatives such as BCNU and CCNU but the clinical results are not so remarkable compared with previous reports concerning experimental studies. The reason for an efficacy of NU derivatives in brain tumors is considered to be its higher lipid solubility which makes the drug crossing the BBB easily. On the other hand, there is some evidence that higher lipid solubility did not guarantee NU to reach always to all portions of solid tumor after systemic administration. We have performed a chemotherapy of malignant brain tumors using ACNU for five years. This drug is not only lipid but also water-soluble in some grade; therefore, the drug is administrated intravascularly and locally with ease. Nine cases of malignant gliomas were treated with local chemotherapy employing ACNU and two cases of glioblastomas are surviving now over five years and about four years, respectively. From the anatomical standpoint of view, it is considered to be necessary for local chemotherapy of brain tumors to possess some pathognomonic characters such as cyst formations, central necrosis and localized cortico-meningeal adhesions, which are rather frequently found in malignant gliomas and are suspected also easily by CT examination preoperatively. A local chemotherapy in the present study has been performed using 10 to 50 mg of ACNU through an indwelling catheter inserted during operation. No general toxicity occurred in all patients except one, who experienced purulent meningitis after long-term drainage. In our study on concentration of ACNU, intracarotid injection resulted in higher concentration in brain tumor tissue than intravenous injection, but these concentration considered to be not high enough to suppress the tumor cell growth. On the other hand, the intracavitary concentration of ACNU at 24 hr after drug administration was high enough to suppress the tumor cell growth.

A 33-year-old woman with malignant melanoma well responded to a chemotherapy including DTIC, ACNU, and VCR, and intralegional injection of OK-432. Four years prior to admission, a lentigo of 5 mm diameter at her left frontal chest was found, but, histological examination resulted in no distinctly malignant findings. In November 1979, multiple subcutaneous tumors appeared over posterior surface of the chest and left axillar region, which were gradually increasing in number and size, then she was admitted to our hospital in June, 1980. Biopsy of subcutaneous tumors revealed a malignant melanoma and its metastasis to skin. Immunochemotherapy was started immediately based upon this diagnoses. The patient received 100mg DTIC i.v. for 4 days, 100mg ACNU i.v. for one day and 1 mg VCR i.v. for one day. OK-432 was locally injected into some tumors as an immunotherapy. On completion of the third course of chemotherapy, all subcutaneous tumors were decreased in size, especially the tumors injected with OK-432. However, patient didn't respond to a repeated chemotherapy, thereafter malignant melanoma was metasized to uterus and peritoneum in May, 1981, and she died in September, 1981. Major side effects of this chemotherapy were nausea and mild transient leukocytopenia. The combination of chemotherapy including DTIC and local injection of OK-432 appeared to be effective for malignant melanoma.

The eligibilities for chemotherapy, prognostic factors and effects of chemotherapy were evaluated in pulmonary metastasis from uterine cervical carcinoma. There was no difference in median survival between patients treated with surgery and those with chemotherapy. The mediastinal lymph node involvement and vascular invasion were demonstrated to be important prognostic factors in patients treated with surgery, and it was suggested that systemic chemotherapy should be given to the patients with mediastinal lymph node involvement. The median survivals of patients treated with chemotherapy were 12.3 months in patients with pulmonary metastasis alone and 5 months in those with both pulmonary and other visceral metastasis, respectively. Responders to chemotherapy survived longer than non-responders. Overall response rate was 42.7% (44/103), and the response rate of MDU (Mitomycin C+Dextran sulfate+Urokinase) (62.3%) was higher than other chemotherapeutic regimens, which suggested that appropriate chemotherapy would prolong the survival of patients with pulmonary metastasis from uterine carcinoma.

Reserpine was found to enhance the effect of ACNU on ACNU-resistant C6 glioma (C6/ACNU). When reserpine was added to the culture medium at the concentration of 10 microM, the IC50 of ACNU for C6/ACNU was decreased to the level of that for C6. Intracellular uptake of ACNU increased in both resistant and sensitive cells when 10 microM reserpine was added to the culture medium. This phenomenon is more remarkable in C6/ACNU than in C6.

Forty-one non-selected patients with malignant brain tumors have been treated since 1976 with chemoimmunotherapy using ACNU and PSK postoperatively. Eleven (27%) of these cases have survived usefully over five years, including 6 cases (30%) out of 20 with glioblastoma (astrocytoma III, IV approximately Kernohan). This long-term survival response for glioblastoma was considered to be better than any previous figures in the literature. Among these 6 cases, one child of 11 years old with brain stem tumor was found to be so significantly improved by chemoimmunotherapy that no surgery was needed. In another patient with astrocytoma III of the bilateral thalamus, the tumor was seen to be diminished so completely on CT examination after administration of ACNU(100 mg x 4), that no surgery was needed except for external decompression and was needed except for external decompression and biopsy. In three other cases local (intracavitary) chemotherapy was performed successfully. Local concentration of ACNU in these latter cases 24 h after its insertion was verified as being markedly higher than that in peripheral blood and brain tissues after intravascular administration. In an immunological study of these brain tumors, parameters such as lymphocytes and T cells in peripheral blood and Ig(G.A.M) in serum were examined. The results showed that these parameters were almost at normal levels in long-term survival patients (Group I), low in short-term survivals patients (Group II died) and normal in all control cases (Group III benign brain tumors, traumatic disorders etc.). Combined administration of PSK was considered to be very effective for the improvement of these parameters.

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.

Twenty-two patients with advanced adenocarcinoma of the lung were treated with the combination chemotherapy "EACAM" consisting of cyclophosphamide (333mg/m2 X 1), adriamycin (27mg/m2 X 1), cisplatin (25mg X 5), nimustine (33mg/m2 X 1), and methotrexate (27mg/m2 X 3). This regimen was repeated once every 4 or 5 weeks. One complete response (CR) and 8 partial responses (PR) were obtained in 21 evaluable patients and the response rate was 42.9%. It has not been possible to calculate the median survival time for all of the evaluable cases, since 13 of them are still alive up to the present time. The side effects observes were as follows: nausea and vomiting (81.8%), alopecia (81.8%), stomatitis (22.7%), leukocytopenia less than 2,000/mm3 (45.5%), and thrombocytopenia less than 5 X 10(4)/mm3 (18.2%). Apart from strong myelosuppression, no severe infection or bleeding tendency was noticed. A mild elevation of serum createnine was observed in one patient, and no patients developed renal insufficiency. The combination chemotherapy "EACAM" is therefore considered to be a very effective and tolerable treatment for adenocarcinoma of the lung.

Fourteen patients with advanced malignant melanoma were treated with a combination chemotherapy consisting of ACNU 100 mg/m2 i.v. on Day 1 in 6 week intervals and DTIC 200 mg/m2 i.v. on Days 1 to 5 at 3 week intervals. Four patients had prior chemotherapy and 2 had prior immunotherapy. Excluding 4 patients received the regimen for adjuvant chemotherapy, 10 of 14 patients were evaluable for response. There were 3 patients of partial responses, 3 minor responses, 1 no change, and 3 progressive diseases. The durations of partial responses were 1, 1, and 8 months, respectively, while the survival times in these patients were 5, 21, and 10 months, respectively. Leukopenia less than 4,000/cmm occurred in 10 of 14 patients (71%) and thrombocytopenia less than 100 X 10(3)/cmm in 9 of 14 patients (64%), moreover, these hematologic toxicities were cumulative. Serum GOT and GPT elevated to 3,460 mu/ml and 1,365 mu/ml, respectively in one patient, but this returned to a normal level one month later. Nausea and vomiting were mild to severe in 12 of 14 patients, being most marked on Day 1 and decreasing intensity during the next several days. Other non-hematologic toxicities including skin rash, fever, and phlebitis were noted in each one patient, respectively. Hematologic toxicity of this regimen was a dose limiting toxicity; therefore, intensive supportive therapy to prevent infection and hemorrhage is essential for the management of the patients during this chemotherapy.

From 1974 to 1984, we have treated 70 patients with SCLC using radiotherapy (RT) alone or in combination with chemotherapy (CT). It was demonstrated that a heavy CT regimen causing a severe toxicity did not always cause good results. On the other hand, there was a long-term survivor in those patients who were treated with RT alone. Considering from our results as well as from reviews of literatures, it is evident that RT can reduce a significant number of local relapses but has little effect on systemic disease. Therefore, RT added to CT seems to improve a long-term survival specifically in those patients in whom CR is induced from CT. The characteristics of the long-term survivors treated with combined treatment are: limited disease, thoracic RT, good PS, only single site of metastases. In conclusion, there is no doubt that CT plays an important role in the management of SCLC. However, its efficacy is still far from desired, and a new type of CT is warranted.

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.