Doses Of CCNU Research Articles

Correlation between in vitro DNA synthesis, DNA strand separation and in vivo multiplication of cancer cells.

The chemicals 9, 10-dimethylbenzanthracene (DMBA), ethionine, daunorubicin, actinomycin D, 1-(2-chloroethyl-1)-nitrosourea (CCNU), steroids, croton oil and dimethyl-sulfoxide (DMSO) were used in order to correlate their effect on the in vitro synthesis of normal and cancer DNA, on DNA strand separation and on accelerated in vivo multiplication of cancer cells. All of the compounds tested strongly stimulate the synthesis of cancer DNA in vitro catalyzed by DNA-dependent DNA polymerase I and measured as an acid-precipitable labeled product. Under the same conditions, the synthesis of DNA originating from healthy tissues is only slightly enhanced, except in the case of croton oil and DMSO. These substances are almost equally active on cancer and normal DNA. Although both cancer and normal DNA contain a large amount of double-stranded regions, the extent of DNA strand separation measured by the increase in UV absorbance (hyperchromicity) in the presence of each compound tested is much higher for all cancer DNA than for corresponding normal DNA. In contrast, DMSO and croton oil do not appear to distinguish cancer DNA from normal DNA. Additive and differential effects of various compounds on cancer DNA strand separation can be observed. Small doses of DMBA and CCNU stimulate the multiplication of Ehrlich ascites tumor cells in vivo in mice. There is thus a possible correlation between DNA strand separation, DNA synthesis, multiplication and differentiation of cancer cells in the presence of the above compounds, which is different from the response of normal cells to these compounds.

The effect of phenobarbital on the toxicity and tumoricidal activity of CCNU in a murine brain tumor model.

✓ The nitrosoureas are known to be substrates for hepatic microsomal enzymes. Since phenobarbital (PB) is a potent inducer of hepatic microsomal enzymes, and since PB and other enzyme-inducing drugs are commonly used in patients with brain tumors, the authors have assessed the effect of PB pretreatment on the toxic and tumoricidal activity of CCNU in a murine model. In C57B1/6J mice, PB pretreatment markedly reduced the lethal toxicity of high doses of CCNU. The LD99 dose of CCNU was found to be 80 mg/kg given intraperitoneally. Pretreatment with PB reduced the toxic death rate of CCNU at 40, 60, 90, or 120 mg/kg to less than 10% (p < 0.01). Pretreatment with PB also reduced the tumoricidal activity of CCNU. In an intracerebral murine ependymoblastoma, intraperitoneal CCNU alone, at 30 mg/kg given on the 5th day after tumor transplantation, produced a percent increased life span (%ILS) of > 300, and 18 of 25 were long-term survivors (LTS). In contrast, after four daily doses of PB prior to the same dose of CCNU, the %ILS was reduced to 85 with no LTS among 25 mice (p < 0.01). When CCNU was given alone at 30 mg/kg intraperitoneally on Day 10, a %ILS of > 300 with 22 of 25 LTS resulted; whereas, PB pretreatment reduced the %ILS to 15, with two of 25 LTS (p < 0.01). When CCNU was administered directly into the tumors intracerebrally at 30 mg/kg on Day 10, the %ILS was > 300, with 16 of 20 LTS; whereas, PB pretreatment reduced the %ILS to 50 with five of 20 LTS (p < 0.01). Furthermore, PB pretreatment significantly reduced the ability of CCNU to retard tumor growth in a subcutaneous murine ependymoblastoma. Thus, PB pretreatment significantly altered the activity of CCNU. Since enzyme-inducing drugs are so commonly used in patients with brain tumors, it is possible that the clinical failure of nitrosoureas in some cases may be due to an unsuspected drug antagonism.

Chlorozotocin

Chlorozotocin is a chloroethyl nitrosourea with a glucose carrier that has curative activity for the murine L1210 leukemia, but is nonmyelosuppressive in mice. To determine the mechanism for this unique property of reduced bone marrow toxicity, comparative studies were conducted with chlorozotocin and CCNU, a myelotoxic chloroethyl nitrosourea. Suspensions of L1210 leukemia and murine bone marrow cells were incubated for 2 h with 0.1 mM [(14)C]-chloroethyl chlorozotocin or CCNU. Chlorozotocin demonstrated a fourfold increased covalent binding of the chloroethyl group to L1210 nuclei when compared to equimolar CCNU. Chlorozotocin alkylation of L1210 cells resulted in the binding of 57 pmol of [(14)C]ethyl group/mg of DNA, which represented a 2.3-fold increased alkylation when compared to CCNU. In marked contrast, the binding of the chloroethyl group to bone marrow nuclei was equivalent for both drugs. In addition, chlorozotocin alkylation of murine bone marrow DNA, 45 pmol of [(14)C]ethyl group/mg of DNA, was equivalent to that of CCNU. The ratio of L1210:bone marrow DNA alkylation was 1.3 for chlorozotocin compared to 0.6 for CCNU. The intracellular carbamoylation of L1210 and bone marrow protein by CCNU was 400- to 600-fold greater than that produced by chlorozotocin. After a 2-h exposure to 0.1, 0.05, or 0.01 mM drug, both chlorozotocin and CCNU produced a reduction in the cloning efficiency of L1210 cells that was dose dependent. However, chlorozotocin was significantly more cytotoxic than CCNU at all three molar concentrations (P < 0.01). Chlorozotocin, 0.1 mM, reduced L1210 DNA synthesis to 1% of control by 48 h, in contrast to 16% with equimolar CCNU (P < 0.01). In mice bearing 10(5) L1210 cells, chlorozotocin produced its optimal antitumor activity (332% increased life span [ILS]) at doses of 48-64 mumol/kg, with >50% indefinite survivors. In contrast, CCNU at the same molar doses resulted in only a 191% ILS; a CCNU dose of 128 mumol/kg was required for comparable optimal L1210 antitumor activity, 413% ILS. On a molar basis, the dose of chlorozotocin that produced optimal in vivo L1210 antitumor activity was one-third to one-half that of CCNU. Chlorozotocin, unlike CCNU, produced no murine bone marrow toxicity at its optimal therapeutic dose. This unique combination of antitumor activity without myelosuppression can be correlated with the advantageous ratio of L1210:bone marrow in vitro DNA alkylation by chlorozotocin (1.3) as compared to equimolar CCNU (0.6).

Cytochrome P-450 interactions with the 2-chloroethylnitrosoureas and procarbazine

Summary Monooxygenation of 1-(2-chloroethyl)-3-( trans -α - methylcyclohexyl) - 1 - nitrosourea (Methyl CCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the presence of NADPH and O 2 by liver microsomes prepared from normal and phenobarbital-induced rats and mice were compared. The rate of Methyl CCNU hydroxylation was about 20 to 30 per cent of the rate of CCNU hydroxylation. A NADPH cytochrome P-450 dependent denitrosation of Methyl CCNU to the parent urea was observed. Single doses of CCNU (7.5, 15 and 30 mg/kg body wt) administered to rats two weeks prior to sacrifice and preparation of liver microsomes resulted in a dose-related decrease in cytochrome P-450 content, ethylmorphine N -demethylase and nitroreductase activities. These decreases gave values of 57, 33 and 60 per cent respectively relative to controls at the 30 mg/kg body wt dose level. Similar experiments are reported for the effects of CCNU treatment in vivo on CCNU monooxygenation by microsomes. These results are discussed in relation to previous studies with procarbazine.

The effect of amphotericin B on the survival of brain-tumor-bearing mice treated with CCNU.

The membrane-active polyene, amphotericin B (AMB) has been shown to enhance or potentiate the effects of various chemotherapeutic agents against tumor cells in tissue culture. Because of the need to increase the efficacy of the nitrosoureas in brain-tumor chemotherapy, we have studied the effect of the nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), with or without AMB pretreatment. The intraperitoneal administration of 25 mg/kg of AMB 24 hours before 5, 10, 20 or 30 mg/kg of intraperitoneal CCNU did not result in a significant improvement in survival. The failure of intraperitoneal AMB to potentiate the effect of CCNU was likely a failure in drug delivery, since AMB crosses the blood-brain barrier very poorly. To circumvent the drug-delivery problem, AMB was administered intracerebrally directly into the tumor-bearing hemisphere. With 0.5 mg/kg of intracerebral AMB administered 24 hours before 10 mg/kg CCNU was given intraperitoneally, the life-span was significantly increased over the control group. The same dose of CCNU alone did not significantly increase survival. When 0.2, 0.5, or 1.0 mg/kg AMB was administered intracerebrally 24 hours before 20 mg/kg of interaperitoneal CCNU, survival was significantly increased over those groups receiving the same dose of CCNU alone. It is concluded that direct intracerebral administration of AMB enhances or potentiates the therapeutic effect of CCNU in this brain-tumor model.

Chemotherapy of recurrent medulloblastoma with combined procarbazine, CCNU, and vincristine.

Seventeen patients with recurrent medulloblastoma were treated with a combination of three drugs: procarbazine, CCNU, and vincristine (PCV). Tumor recurrence was documented at varying periods following surgery and radiotherapy. Among 16 evaluable patients, ten showed a response to PCV on the basis of subjective neurological improvement and a decrease in tumor size by radiological criteria. Five patients were designated as having stable disease on the basis of no change in neurological status and tumor size. One patient showed uninterrupted progression of disease. The median time to progression for all patients was 45 weeks. Significnat myelotoxicity, exacerbated by prior spinal irradiation, compromised therapy. After an initial response, it was often necessary to reduce the higher doses of CCNU and procarbazine that caused concomitant bone-marrow toxicity; as a consequence of the lowered doses, tumor progression was then frequently observed. The authors conclude that PCV is an effective form of palliative therapy for recurrent medulloblastoma.

Tumour volume response, initial cell kill and cellular repopulation in B16 melanoma treated with cyclophosphamide and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

The relationship between tumour volume response and cell kill in B16 melanoma following treatment in vivo with cyclophosphamide (CY) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was investigated. Tumour volume response, expressed as growth delay, was estimated from measurements of tumour dimensions. Depression of in vitro colony-forming ability of cells from treated tumours was used as the measure of tumour cell kill. The relationship between these parameters was clearly different for the two agents studied. CY produced more growth delay (7.5 days) per decade of tumour cell kill than CCNU (2 to 3.5 days). The possibility that this was due to a technical artefact was rejected in favour of an alternative explanation that different rates of cellular repopulation in tumours treated with CY and CCNU might be responsible. Cellular repopulation was measured directly, by performing cell-survival assays at various times after treatment with doses of CY and CCNU which produced about 3 decades of cell kill. The rate of repopulation by clonogenic cells was much slower after treatment with CY than with CCNU, and this appears to account for the longer duration of the growth delay obtained with CY.

Hepatotoxicity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in dogs: The use of serial percutaneous liver biopsies

Hepatotoxicity resulting from po administration of a single dose of CCNU to dogs was studied by weekly determinations of liver function tests and performance of percutaneous liver biopsies. Histochemical stains for glycogen and alkaline phosphatase in liver sections revealed an effect on these parameters within 1–2 weeks after dosage, the earliest sign of drug toxicity. Histopathologic changes were evident 7–14 days later, when increased activity of serum alanine aminotransferase was also present. Chemical indices of liver abnormalities were reversible, whereas glycogen depletion and increased alkaline phosphatase activity in liver biopsies persisted throughout the 2- to 3-month observation period. This study demonstrates the feasibility and usefulness of serial percutaneous liver biopsies in determining both the qualitative and quantitative features of developing and recovering from drug toxicity in the dog.

A toxicologic comparison of the potency and activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in mice and rats

Toxicity resulting from po administration of single doses of BCNU and CCNU in corn oil to mice and rats was studied over a 30-day observation period. Comparison of mortality levels on a mg/kg, mg/m 2 or mmole/kg basis and evaluation of body weight changes revealed that BCNU was twice as toxic as CCNU. Species and sex mortality studies showed that both compounds were significantly more potent in mice than in rats, but neither agent exhibited a sex-related potency difference. Temporal mortality patterns indicated that the time of death was similar for equitoxic doses of the 2 compounds in either species, but mice died more quickly after treatment than did rats. Prolongation of the observation period for rats treated with BCNU affected % mortality. Consequently, all BCNU doses, except the 30-day LD1, ultimately caused 100% mortality when the observation time was extended sufficiently beyond 30 days. The peak time for mortalities was inversely related to the dose. Low doses of BCNU produced deaths as late as 100 days post treatment, though rat deaths never occurred earlier than 4 days post treatment for either compound even at doses 5 times the 30-day LD50. No consistent alterations of the mortality rate for either agent resulted from changing the vehicle, route of administration or sex of the animals. The apparently similar mechanisms of action and the significant potency differences of these compounds emphasize the importance of determining metabolic derivatives of these agents and the biologic activity of each metabolite.