Sensitivity of bone marrow hematopoietic colony-forming cells from mice, dogs, and humans to carminomycin, marcellomycin, aclacinomycin A, and N,N-dibenzyldaunorubicin and its relationship to clinical toxicity.

The sensitivity of bone marrow granulocyte-macrophage colony-forming cells to 4 anthracyclines, carminomycin, marcellomycin, aclacinomycin A, and N,N-dibenzyldaunorubicin, was studied using the agar diffusion chamber technique which allows exposure of target cells to drug metabolized by the chamber-bearing host after i.v. injection. Colony-forming cells from mice, dogs, and humans were all found to have exponential dose-response curves for the agents studied, with variation of the slopes between species and agents. Species sensitivities as determined by the assay related well to the available toxicological and clinical data for specific drugs. The rank order of sensitivity of human marrow colony-forming cells to five anthracyclines tested in this and a previous study related very closely to doses producing moderate leukopenia in Phase I and II clinical studies. A dose of 200 mg/sq m of N,N-dibenzyldaunorubicin would be expected to produce moderate leukopenia in future clinical trials. This assay may be useful in predicting human bone marrow toxicity of new agents before actual clinical trial because of the ability to study the survival of human colony-forming cells directly.

Evaluation of some anthracycline antibiotics in an in vivo model for studying drug-induced human leukemia cell differentiation.

Human acute promyelocytic leukemia cells (HL-60) were induced to undergo terminal differentiation by treatment in vivo with marcellomycin. This was accomplished by cloning HL-60 cells in 0.3% agar in diffusion chambers, which were subsequently implanted in the peritoneal cavities of mice. These animals were given injections i.v. with anthracycline antibiotics, and the chambers were transferred to a second recipient 24 hr later. After an additional 8 days, the chambers were removed, the cloning efficiency was determined, and colonies were scored for the presence of differentiated cells based on their ability to reduce nitro blue tetrazolium (NBT). Marcellomycin produced dose-dependent decreases in cloning efficiency and increases in the number of differentiated cells present in chambers. At all dose levels of marcellomycin tested, three types of colonies were observed; these were colonies consisting entirely of undifferentiated cells and approximately equal numbers of colonies consisting of solely differentiated cells and those with mixtures of both differentiated and undifferentiated cells. An 8-fold increase in colonies containing differentiated cells (both pure and mixed) was observed after a single injection of marcellomycin (10 mg/kg), a dose which reduced cloning efficiency by 40%. At that dosage level, aclacinomycin A also induced differentiation, while doxorubicin was ineffective, a finding consistent with the effects of these anthracyclines on HL-60 cells in suspension culture. In addition to the functional changes accompanying differentiation, commitment was characterized by a limitation in proliferative potential. Thus, the average size of uniform NBT-positive colonies was approximately 16 cells, and few clones of NBT-positive cells greater than 32 cells in number were observed; this contrasted with NBT-negative clones which contained up to 100 cells. This finding suggests that HL-60 cells undergo an average of four and a maximum of five divisions upon commitment to granulocytic differentiation. The in vivo system described may be useful in further evaluation of differentiation-inducing agents for therapeutic potential after an initial in vitro screen to identify active compounds.

Effect of a perfluorochemical emulsion on the development of artificial lung metastases in mice.

Intravenous infusions of perfluorochemical emulsions, combined with administration of inspired oxygen or carbogen have been found to improve tumor oxygenation and increase the response of solid tumors in animals to radiotherapy. Fluosol-DA 20 per cent, the only perfluorochemical emulsion currently approved for testing in humans in the United States, has recently entered clinical trials as an adjunct to radiotherapy in the treatment of head and neck carcinoma. The studies reported here were undertaken as part of our laboratory evaluation of the safety and clinical potential of this oxygen-transport fluid as an adjunct to cancer therapy; they asked whether single or multiple treatments with Fluosol and an oxygen-enriched atmosphere produced immunologic perturbations, pulmonary damage, or other effects which altered the development of artificial lung metastases in experimental animals. Neither single nor multiple treatments with clinically relevant regimens of Fluosol and carbogen (95 per cent O2/5 per cent CO2) had any effect on the development of lung nodules from intravenously injected EMT6 tumor cells.

Enhancement of artificial lung metastases by misonidazole.

The effect of treatment with the hypoxic cell radiosensitizer misonidazole on the formation of artificial lung metastases was examined. Both single treatments with large doses of misonidazole and fractionated treatments with smaller doses of misonidazole were found to increase the number of lung tumors developing after intravenous injection of EMT6 mouse mammary carcinoma cells. The immunologic and physiologic effects of the nitroimidazole were postulated to be responsible for the enhancement of lung tumor formation.

Reactions of tumors and normal tissues in mice to irradiation in the presence and absence of a perfluorochemical emulsion.

The effect of pre-treatment with the perfluorochemical emulsion, Fluosol-DA, on the radiation response of normal tissues and EMT6 mammary tumors in BALB/c mice was examined. Pre-treating tumor-bearing mice with .015 ml/g of Fluosol and 30 min of carbogen (95% O2/5% CO2) increased the number of tumor cells killed by irradiation with doses of 2.5-20 Gy; the change in the radiation dose-response curve was consistent with a reduction in the hypoxic fraction. Fluosol did not alter the response of tumors in air-breathing or N2-asphyxiated mice and carbogen alone did not alter the radiation response of this tumor significantly. Carbogen treatments 5-60 min in duration produced similar enhancements of tumor radiosensitivity in Fluosol-treated animals. Pre-treatment with Fluosol plus carbogen also increased the number of tumor cells killed by a fractionated regimen of four 2.5 Gy fractions given over 2 days. Pre-treatment with Fluosol-DA plus carbogen, therefore, increased the antineoplastic effects of radiotherapy in both single-dose and multi-fraction radiation regimens. In contrast, Fluosol did not increase the effect of radiation on the partially committed (CFU-GM) or pluripotent (CFU-S) stem cells of the bone marrow or on the CFU-GM of the spleen. The radiation response of the skin was only slightly enhanced by pre-treatment with Fluosol plus carbogen. These data show that treatment of mice with perfluorochemical emulsions plus carbogen can produce therapeutic gain by improving the radiation response of solid tumors, without producing an equivalent increase in the radiation response of potentially dose-limiting normal tissues. These findings encourage further evaluations of these agents as adjuncts to clinical radiotherapy.

Effect of 1-methyl-5-sulfonamide-4-nitroimidazole and 1-methyl-5-bromide-4-nitroimidazole on the radiosensitivity of EMT6 tumor cells.

The compounds 1-methyl-5-sulfonamide-4-nitroimidazole and 1-methyl-5-bromide-4-nitroimidazole are extremely effective radiosensitizers of hypoxic cells in vitro. Preincubation of hypoxic cells with these and related drugs produces greater sensitization than is obtained with short incubations or is expected from the electron affinities of the compounds. The experiments reported here were undertaken to determine whether these agents were effective in sensitizing the naturally occurring hypoxic cells of solid tumors in mice. Neither of the 4-nitroimidazoles produced significant radiosensitization of the hypoxic cells of EMT6 mouse mammary tumors in vivo with any of the single- or multiple-injection regimens tested.

Effects of the mode of administration of mitomycin on tumor and marrow response and on the therapeutic ratio.

The antineoplastic and toxic effects of mitomycin (MC) administered as a single pulse injection were compared with the effects of MC given as infusions over 2, 5, or 24 hours and with the effects of the drug administered in fractionated regimens incorporating 2-20 injections. The antineoplastic effects of MC were assessed using EMT6 mouse mammary tumors implanted in BALB/c mice; tumor response was assessed using both cell survival and tumor growth assays. The host toxicity of MC was assessed by measuring the survival of bone marrow stem cells (CFU-S) and by determining LD50/30's (which reflect primarily marrow injury). The survival curve for cells from tumors treated with varying doses of MC was exponential, with no shoulder which might suggest accumulation of sublethal lesions. No evidence was found that the tumor cells repaired MC damage when left in situ for up to 24 hours after treatment (repair of potentially lethal damage) or that the tumor repaired sublethal damage during times of up to 24 hours between drug treatments or during drug infusions. The responses of tumors treated with single injections of MC, with infusions over 2, 5, and 24 hours, with two injections separated by 5 or 24 hours, and with five daily injections were all similar. The survival curve for CFU-S from mice treated with varying doses of MC was exponential, with no shoulder which might suggest accumulation of sublethal lesions. LD50/30's were the same whether MC was administered as single injections, as infusions over 2, 5 or 24 hours, or as five daily fractions. Longer fractionated regimens, in which mice received daily injections, 5 days per week, for up to 4 weeks, produced less host toxicity but also less antineoplastic effect; the therapeutic ratio for the most protracted regimen tested was lower than that for single injections. The therapeutic ratios achieved with infusions and fractionated regimens were never greater than that obtained with a large single dose, confirming clinical impressions that infrequent, large doses of MC are an optimal method of treatment with this drug.