BCNU Research Articles

Mechanism of action of the nitrosoureas—III: Reaction of bis-chloroethyl nitrosourea and bis-fluoroethyl nitrosourea with adenosine

Previous papers in this series have provided evidence for the formation of haloethyl nucleoside derivatives from the interaction of the therapeutic nitrosoureas with cytidine and guanosine. Such derivatives could be important in explaining the cytotoxic action of bis-chloroethyl nitrosourea (BCNU), bis-fluoroethyl nitrosourea (BFNU), and related therapeutic agents. We now report the formation of 1-haloethyl adenosines from the reaction of BCNU and BFNU with adenosine. These 1-substituted haloethyl adenosines cyclize to form 1, N 6-ethanoadenosine: 1-chloroethyladenosine with a half-life of 20 min in neutral aqueous solution at 37°, and 1-fluoroethyladenosine with a half-life of 20 hr under the same conditions. 1-Hydroxyethyladenosine is also a major product of the reaction of either BCNU or BFNU with adenosine, but it is not formed from the hydrolysis of either 1-haloethyladenosine. Accordingly, a reaction mechanism involving a cyclized nitrosourea derivative is proposed to explain the formation of this and other hydroxyethyl nucleosides.

Ablation of murine jejunal crypts by alkylating agents

The gut microcolony assay has been used to measure damage to intestinal crypts by single and split doses of 3 alkylating agents: mechlorethamine hydrochloride (HN2), bis-chloroethyl-nitrosourea (BCNU) and isopropyl methane sulphonate (IMS). The single-dose survival curves for whole crypts were distinguished by extrapolation numbers (3.0, 176 and 1.5 respectively) that were lower than most previously published values for assay by irradiation. Significant sparing of crypts occurred when doses of HN2 or BCNU, but not IMS, were given in 2 equal fractions separated by more than 2 h. Deduced D0 values for those cells from which crypts regenerate were 1.9 mg/kg HN2, 19 mg/kg BCNU and 487 mg/kg IMS.

Action of radiation or alkylating agents on LSA lymphoma during growth

The effect of 60Co irradiation and the alkylating agents mustargen, cyclophosphamide, and bis-2-chloroethyl-1-nitrosourea (BCNU) was investigated on the LSA ascites tumor. Single exposures of radiation or alkylating agent was given at different stages of tumor growth. This tumor system was shown to have markedly changing tumor cell age cohort compositions at different tumor stages with differing 5 Fluorouracil (5-FU) sensitivities for cell killing and treatment-induced increase in host survival time, at different tumor stages. Similar variation in altering survival time also was found for the alkylating agents with all showing greater activity against the early, rapidly growing tumor. Stationary phase and advanced tumors also showed markedly different tumor response. The different agents had greatly different potencies against the advanced G1-arrested non-proliferating tumor. 60Co radiation showed much less variation in effectiveness than the alkylating agents based upon the host survival time. The mode of action of treatment-induced in vivo life prolongation by these agents is complex and tumor age dependent.

Pulmonary Toxicity Associated with Bischloroethylnitrosourea (BCNU)

Ten patients developed pulmonary fibrosis after bischloroethylnitrosourea (BCNU) therapy for malignancy. This was lethal in seven patients, four of whom had no evidence of tumor at autopsy. Presenting symptoms were either the insidious onset of cough and dyspnea or the sudden onset of respiratory failure. Physical findings were unremarkable. Chest roentgenogram usually showed interstitial infiltrates. Pulmonary function studies showed resting hypoxia with diffusion and restrictive defects. This complication of therapy does not appear to be dose related and may be made more likely by the concomitant administration of cyclophosphamide. Prednisone therapy did not benefit most patients. The literature and the implications of the use of BCNU alone or in combination are reviewed.

T and B lymphocyte populations of the spleen and thymus in normal and immunosuppressed BALB/c mice

The antituinor agent 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) has been studied in order to determine its effect on thymic and splenic T and B lymphocytes in normal and immunosuppressed BALB/c mice. Utilizing indirect immunofluorescence and lymphocyte proliferation studies we detected an initial reduction of splenic T and B cells as a result of the administration of an optimal dose, 30 mg/kg, of BCNU. The population dynamics of the thymic lymphocytes are totally different in their mitogenic reactivity than that of the splenic lymphocytes. An initial decline in the PHA and LPS-sensitive splenic lymphocytes of BCNU-treated mice was temporary. However, there was no return to normal levels detected for the Con A-sensitive splenic lymphocytes. On the other hand, the PHA-sensitive thymic lymphocytes of BCNU-treated mice not only failed to repopulate but were totally depleted by the tenth day.

Sequential and adjuvant chemotherapy for malignant astrocytoma of the brain

Twenty-nine patients with grade III or IV astrocytomas were treated with 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU), 75--100 mg/M2 intravenously (IV) for two consecutive days every four to six weeks, in an effort to improve the quality and length of their survival following surgical resection. Twenty-one of these patients received adjunctive chemotherapy as soon as they recovered from surgery and 18 received concomitant adjunctive radiation therapy as well. Eight patients were treated with radiation therapy after surgery, and BCNU was not added until evidence of tumor growth appeared. BCNU was discontinued and the epipodophyllotoxin, VM-26, was given at a dose of 100 mg/M2 IV weekly when further tumor growth occurred. Patients receiving adjuvant chemotherapy had a shorter mean and median survival than patients receiving BCNU when progression appeared following radiation therapy. Six patients with progressive disease following surgical resection and radiation therapy for grade II astrocytoma were treated with BCNU. Improvement or stabilization of neurologic symptoms occurred in four of these patients. This trial of lower-dose BCNU therapy produced survival rates similar to higher-dose studies and permitted fewer physician visits by the patient.

Hepatocellular carcinoma in a young woman with prolonged exposure to oral contraceptives

A case of hepatocellular carcinoma in a young female is presented in which the apparent etiology was the use of oral contraceptives for 5 1/2 years. Sequential therapeutic trials with intraarterial 5-fluorouracil, intraarterial adriamycin, and intravenous 1,3 bis[2-chloroethyl]-1-nitrosourea (BCNU) were unsuccesful. The adverse effects of oral contraceptives on the structure and function of hepatic tissue are reviewed.

Severe generalized glutathione reductase deficiency after antitumor chemotherapy with BCNU" [1,3-bis(chloroethyl)-1-nitrosourea

Patients receiving BCNU [1,3-bis(2 chloroethyl)-1-nitrosourea] acquire a profound deficiency of erythrocytic oxidized glutathione reductase (GSSG-R) within minutes after the first intravenous injection of a single therapeutic dose (75 mg/M.2) of the drug. This effect is not accompanied by changes in the activites of 19 additional erythrocytic enzymes tested, is reproducible in vitro in a dose-related manner, and is not caused by the antitumor agents administered concurrently with the nitrosourea. The inactivation of erythrocytic GSSG-R results in decreased levels of reduced glutathione (GSH), marked GSH instability and disturbed hydrogen peroxide removal with a positibe ascorbate cyanide test and leads to increased susceptibility to oxidative hemolysis, particularly in glucose-6-phosphate dehydrogenase (G-6-D)-deficient patients. BCNU inhibits GSSG-R irreversibly, probably through alkylation rather than carbamylation, and the reappearance of enzyme activity in vivo after each chemotherapy pulse depends on the capacity of the marrow to release erythrocytes with normal activity formed during the drug-free interval. BCNU inhibits GSSG-R not only in erythrocytes but also in human leukocytes and platelets, as well as in yeast, monkey erythrocytes, and all the organs tested in the mouse. This generalized, severe, and specific GSSG-R deficiency caused by therapeutic doses of BCNU may enhance or mediate the toxic and antitumor effects of the nitrosourea and provides a simple yet sensitive biochemical means of monitoring bone marrow reserve in patients receiving multiple courses of chemotherapy with this agent.

BCNU-modification of the in vitro radiation response in 9L brain tumor cells of rats

Cultured rat 9L brain tumor cells were treated for 1 hr with low concentrations of 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) [1 μg/ml, 95% survival; 3 μg/ml, 40% survival] before, during or after X-irradiation. Posttreatment survival was assessed by the ability of treated single cells to form colonies with more than 50 cells after 14 days of incubation. All survival values obtained with the combination treatment schedules were normalized for the BCNU cell kill and then compared to the survival value obtained after X-irradiation alone. Survival curves constructed from these data were analyzed by both the multitarget model 2 and the quadratic model. 1,4,9 These low concentrations of BCNU tended to reduce the shoulder of the X-ray survival curve but had little effect on the final slope. This was reflected by significant time-dependent variations in the parameter α (the probability of killing a cell from single events) and no significant time-dependent variations in β (the probability of killing a cell from double events). In general, BCNU was more effective when it was given at relatively long time intervals before X-irradiation than when it was given after, during or just before X-irradiation. BCNU was most effective when it was given 15 hr before X-irradiation. A potentiation factor (the ratio of the per cent survival after treatment with X-rays to the per cent survival after the combination treatment normalized for the BCNU kill) was calculated at 200, 300 and 400 rad for all schedules investigated. An enhancement ratio (the ratio of X-ray doses required to produce the same level of survival) also was calculated. Under the most effective conditions the potentiation factor varied from 1.2 to 1.5 and the enhancement ratio was greater than 3.0. Since the equivalent of these low BCNU concentrations could be administered to animals and probably humans before every radiation treatment without significant systemic toxicity, an increase of 4–5 logs in the tumor cell kill might be obtained with the best combination schedule. If this combination schedule proved effective in animal solid tumors, it might be adapted successfully for the treatment of some solid tumors, particularly the malignant gliomas.

Comparative studies on the antitumor activity and the bone marrow toxicity of 1-(beta-D-glucopyranosyl)-3-(2-chloroethyl)-3-nitrosourea and 2-(3-(2-chloroethyl)-3-nitrosoureido)-D-glucopyranose.

Antitumor activity and bone marrow toxicity of a new nitrosourea analog, 1-(beta-D-glucopyranosyl)-3-(2-chloroethyl)-3-nitrosourea (GANU), were compared with those of 2-(3-(2-chloroethyl)-3-nitrosoureido)-D-glucopyranose (Chlorozotocin) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). GANU was found to be highly active against mouse leukemia L-1210 when administered intraperitoneally or intravenously, being the same degree as Chlorozotocin. Even by oral route, GANU exhibited significant activities, whereas Chlorozotocin failed to show any activities by this route. Studies on the bone marrow toxicity as measured by the depression of white blood cell counts and the marrow cellularity revealed that both GANU and Chlorozotocin were less toxic than BCNU. GANU, however, weemed to be rather more toxic than Chlorotocin.

Comparative effects of alkylating agents and other anti-tumour agents on the intracellular level of adenosine 3′,5′-monophosphate in Walker carcinoma

The ability of a number of anti-tumour agents to elevate the intracellular level of adenosine 3′,5′-monophosphate (cyclic AMP) in Walker carcinoma has been investigated. Of these agents HN2, chlorambucil, merophan, CB 1954, and cis-dichloro diammine Pt II [ cis-Cl 2(NH 3) 2 Pt II) caused appreciable elevations of the cyclic nucleotide. These compounds are all regarded as having a typical alkylating agent spectrum of action. A linear relationship was obtained between the intracellular cyclic AMP level produced by a given dose of cis-Cl 2(NH 3) 2 Pt II and the observed percentage inhibition of cell growth. There was a rapid elevation of cyclic AMP in response to chlorambucil which reached its peak within 1 hr of treatment. This preceded the inhibition of thymidine incorporation which reached 50 per cent by 7 hr. 1,3- Bis(2-chloroethyl)1-nitrosourea (BCNU) and (±) 1,2- bis-(3,5-dioxopiperazin-1-yl)propane (ICRF 159) had no effect on the intracellular level of cyclic AMP in Walker carcinoma at any dose level studied. The cyclic 3′,5′-nucleotide phosphodiesterase from bone marrow and intestinal mucosa, two tissues susceptible to alkylating agents, has been investigated. This enzyme behaves kinetically as if two separate activities exist, one with a low affinity for the substrate and the other with a high affinity. The distribution of the two forms of the enzyme from these tissues agrees with the correlation previously obtained between sensitivity to alkylating agents and a high percentage of the low K m form of the enzyme.

A clinical trial of procarbazine plus vincristine plus bis-chloroethyl-nitrosourea plus imidazole carboxamide dimethyl triazeno in metastatic malignant melanoma.

Only a limited percentage of patients with metastatic malignant melanoma respond to single-agent chemotherapy. Vincristine, procarbazine, imidazole carboxamide dimethyl triazeno (DTIC) and bis-chloroethyl-nitrosourea (BCNU) have been used as single agents by various investigators. A response rate of +/- 20% was seen with vincristine (1-3) and BCNU (3, 4), and a response rate of 20-30% has been observed with DTIC (3, 5-8) and procarbazine (3, 9, 10). Newer agents such as triazeno imidazole carboxamide mustard (TIC mustard) (11), chloroethyl cyclohexyl nitrosourea (CCNU) (12), and chloroethyl methylcyclohexyl nitrosourea (methyl-CCNU) (13) have not so far proved superior. During the last years various drug combinations have been tried in an effort to improve the results of treatment in patients with metastatic malignant melanoma. In 1959 Moon reported objective response in 9 out of 20 patients who received a minimum of two courses of a combination of BCNU and vincristine (14). In a randomized trial performed by Acute Leukemia Group B (15), only 24% responded to this combination as compared to 32% and 29% for two different DTIC schedules. There were 120 patients who entered onto this protocol. A combination of DTIC plus cyclophosphamide plus vincristine was reported to give a 25% response (16). Workers at the Mayo Clinic reported objective response in 4 out of 18 patients treated with a combination of DTIC plus vincristine as compared to 1 out of 19 using CCNU (12). Costanza and co-workers (Eastern Co-Operative Oncology Group study) reported that 12 out of 61 patients responded to treatment with DTIC plus BCNU as compared to 9 out of 51 on DTIC alone (17); while Cohen (18) and co-workers reported that 10 out of 16 patients treated with the triple combination of vincristine plus BCNU plus DTIC showed significant response and concluded that this combined approach in the treatment of disseminated malignant melanoma warranted further study. Preliminary analysis of an Eastern Co-Operative Oncology Group protocol showed no statistical advantage of DTIC plus methyl-CCNU over each drug on its own (19). The present study was undertaken to investigate the possible advantage of combining DTIC plus vincristine plus BCNU plus procarbazine in courses of treatment. All of these agents are of some value on their own and all four have possible different mechanisms of action.

Combination Therapy for Myelomatosis

Twenty-eight patients with multiple myeloma have been treated with a quadruple chemotherapeutic regimen consisting of 1, 3 bis (2-chloroethyl)-1-nitrosourea (BCNU), cyclophosphamide, melphalan, and prednisolone. Nineteen new patients and nine who had escaped from previous single-agent therapy were included in the study. The results to date, on eight criteria of response, seem to be superior to those obtained from previous chemotherapeutic regimens. The study has been in progress for 18 months and only three patients have died. Only one who had not received previous therapy died, and she had complicating hyperparathyroidism, which almost certainly contributed to her death.

THE IMPORTANCE OF DURATION OF THERAPY IN ACUTE LYMPHATIC LEUKEMIA (ALL)

In 1968-1969, previously untreated patients with ALL were given high dose combination chemotherapy (POMP). This included on days 1-5, i.v., prednisolone 1000 mg/m2; methotrexate (MTX) 7.5 mg/m2; 6-mercaptopurine (6MP) 125 mg/m2; and, on day 1, vincristine 2 mg/m2. Once remission was achieved POMP was given every 2 weeks. The doses of 6-MP and MTX were increased to maximally tolerated levels in all pts. After 6 months, bischloroethylnitrosourea (BCNU) was given for 3 days at 50 mg/ m2 and pts. were randomized to receive POMP monthly for 6 months or no further therapy. Complete remission was achieved in 61/66 pts. (91%). Median remission duration for all 61 pts. was 13 months; 48 pts. completed the initial 6 months of therapy without relapse; 24 were randomized to receive further POMP therapy and 24 to no further therapy. The further therapy group had a median remission of 20 months with 7 pts. still in initial remission. The no further therapy group had a median remission of 11 months with 2 still in initial remission. These differences are significant at the p <.05 level by the generalized Wilcoxon test. A comparable group of 35 pts. had an intermediate remission duration of 17 months. These data indicate that even with intensive therapy in the first 6 months, cure of leukemia will not be possible in the majority of pts. Prolonged therapy is required for improved remission duration.

Sensitivity of Human and Murine Hemopoietic Precursor Cells to Chemotherapeutic Agents Assessed in Cell Culture

Abstract The sensitivity of human and murine hemopoietic precursor cells to 1,3 bis (2 chloroethyl)-1-nitrosourea (BCNU), nitrogen mustard, 5-fluorouracil, and melphalan was assessed in culture using colony assay techniques. The results indicate that human and murine hemopoietic cells may differ in their intrinsic sensitivity to drugs. The hematologic toxicity of some chemotherapeutic agents cannot be extrapolated directly from mouse to man.

Studies on the mechanism of action of the tumour inhibitory triazenes

A series of imidazole and phenyl dialkyi triazenes were synthesized and investigated for their anti-cancer activity in experimental animals. Active triazenes had a broad spectrum of anti-tumour action and like bischloroethylnitrosourea (BCNU) were active against tumours not sensitive to conventional alkylating agents. It was confirmed that at least one N-methyl group was necessary for anti-cancer activity but there was no correlation between dealkylation by liver microsomes and activity since a diethyl triazene was readily dealkylated but not active. A factor appears to be present in normal cell cytoplasm which can detoxify triazenes but which is absent from tumours sensitive to these agents.

Blood: Chemotherapy of Mouse Myeloma: Quantitative Cell Cultures Predictive of Response In Vivo

Abstract The effects of chemotherapeutic agents on mouse myeloma and bone marrow precursor cells were studied using cell culture and spleen colony assay techniques, and the results were assessed for their values in predicting therapeutic effectiveness. For cyclophosphamide, nitrogen mustard, and 1,3 bis (2 chloroethyl)-1-nitrosourea (BCNU), selective toxic effects on mouse myeloma (Adj. PC-5) cells were seen while no such differential effect was observed for 5-fluorouracil. The differential effects of these agents could not be explained by the cell cycle effects but appeared to be dependent on the intrinsic properties of the two cell classes. Marked differences were noted in the sensitivity of three mouse myelomas to melphalan. The ratio of the melphalan dose required to reduce marrow CFU to 37% (D37) to the dose required to reduce myeloma CFU equally was 75 for Adj. PC-5, 18 for MOPC 46B, and 7 for MOPC 460D. These results predict that melphalan would be more effective than 5-fluorouracil for Adj. PC-5 and that melphalan would be relatively ineffective for MOPC 460D. These predictions were confirmed by survival tests.