Tumor dependence of observed thymidine index as a function of emulsion exposure.

The graphs of observed pulse thymidine index against duration of emulsion exposure for seven experimental tumor systems have been observed to exhibit significant differences; either they were parallel and displaced, or they were nonparallel. The immediate practical consequence of this phenomenon is that identical emulsion exposure times, other experimental conditions being equal, may not be sufficient to provide valid comparison of thymidine indices among tumors. A mathematical model is described which relates observed autoradiographic labeling index to the distribution of radioactive atoms among individual cells that initially incorporate labeled material. Two distributions for uptake are considered, and the results are compared. One model assumes uniform uptake, and the other assumes that the number of radioactive atoms among cells acquiring label follows a gamma distribution. The gamma-distributed uptake model fits the observed thymidine index data for the seven tumors and may be expressed as Lj(t) = a[1 - Qj(1 + bt) -u] where Lj(t) is the labeling index for emulsion exposure duration of t, Qj is a polynomial, and a, b, and u are adjustable parameters. The counting threshold for scoring a cell as labeled is j and for j = 1, Q1 = 1. The model indicates that the labeling index should increase with the duration of emulsion exposure and that this increase is related to the proportion of "lightly" labeled cells. The parameter a is the maximum expected labeling index, and an estimate of this parameter should theoretically be independent of emulsion exposure time.

Disposition of indicine N-oxide in mice and monkeys.

The disposition of tritium-labeled indicine N-oxide (INO) was evaluated in mice and rhesus monkeys. Disappearance of INO from the serum of BDF1 mice given iv doses of 100 or 500 mg/kg occurred with an initial half-life of about 11 mins followed by a second phase greater than 100 mins. At 2 hrs after iv injection of mice, the highest concentrations of INO were present in kidney, liver, and intestine. In CDF1 mice bearing P388 leukemia cells and injected ip with a dose of 500 mg/kg, INO was found, in equal concentrations, in cells of the parent line, which is resistant to INO, and in cells of a line resistant to cyclophosphamide but sensitive to INO. Serum levels of INO in these mice decreased with an initial half-life of about 20 mins. For monkeys given iv doses of 24, 2.4 of 0.24 mg/kg, INO disappeared from the serum in three phases, with average half-lives of 3, 32, and 180 mins, respectively. Half-lives for the two observed phases of urinary excretion were 40 and 240 mins. In 24 hrs, both mice and monkeys excreted greater than 80% of the doses unchanged.

Adequacies and inadequacies in assessing murine toxicity data with antineoplastic agents.

Previous retrospective analyses have suggested a very positive correlation in toxic doses of antineoplastic agents between mice and humans. Additional toxicological information has now been accumulated and reveals a noticeable variability in the existing data base. Nevertheless, it is likely that mouse toxicological studies will become a principal determinant for estimating initial doses to be used in humans. Recognition of the factors responsible for differences in determinations of toxic dose levels in mice will enhance the proper utilization of this approach.

Effect of adriamycin on the reproductive integrity of cultured leukemia L1210 and P388 cells.

Proliferating cultured P388 cells exhibited a greater degree of sensitivity to adriamycin than did proliferating cultured L1210 cells, although both leukemia cell populations had approximately the same doubling time. The rate of reduction in viability when cultured L1210 cell populations were exposed to adriamycin (0.0625-2.0 microgram/ml, concentrations that are comparable to tissue drug levels during therapy) was concentration-dependent. Therefore, the results indicated a possible therapeutic advantage to be gained by an increase in drug concentrations (within the limits of acceptable host toxicity) at the target cell site.

Kinetics of reduction in viability of cultured leukemia L1210 cells exposed to purine analogs.

Rates of reduction in viability and degree of cell killing were relatively independent of concentrations when replicating cultured L1210 cells were exposed to increasing concentrations of 6-thioguanine, 6-methylthiopurine ribonucleoside, 9-beta-D-arabinofuranosyl-9H-purine-6-thiol, or 9-ethyl-6-thiopurine. The relative lack of dependence of cell killing rate on cytotoxic concentrations suggest (a) that these agents may be only effective aganinst proliferating cells, and (b) that only limited therapeutic advantage can be gainged by increasing their concentration beyond a minimum effective level. In contrast, the rate and degree of cell killing were dependent upon concentrations when cells were exposed to increasing amounts of 4-aminopyrrolo(2,3-d)pyrimidine-beta-D-ribofuranoside or to 2-fluoroadenosine, indicating that these analogs are not cell-cycle-stage-specific and that nonreplicating cell populations may be sensitive to them.

Ambiguity of the thymidine index.

The observed thymidine indices of seven experimental tumor lines are compared as a function of duration of emulsion exposure. The effects of dose level of tritiated thymidine and background threshold are also evaluated. The results indicate that an arbitrary high background threshold discriminates against "lightly" labeled cells at short periods of exposure but that the chosen threshold becomes less critical with longer exposure. The observed thymidine index increases with increasing duration of emulsion exposure but appears to approach a plateau for all tumor systems. The "thymidine index curves" are significantly different for each tumor. There is an inverse relationship between the dose of tritiated thymidine and the duration of exposure required to recognize the same fraction of cells as labeled in a given tumor. Similar experimental conditions do not necessarily guarantee a valid basis for comparison of observed thymidine indices among tumors.

Estimation of tumor cell kill from Gompertz growth curves.

The Gompertz function has been found to mathematically describe several types of biologic growth, including the growth of transplantable animal tumors. If it is assumed that treatment (chemotherapy, surgery, x-irradiation) of tumors causes cell death in a relatively short interval and that the surviving cells begin to repopulate immediately, producing tumor growth according to the Gompertz function before treatment, then the displacement in time between the two growth curves may be quantitatively related to tumor cell kill. This theory is developed and compared to bioassay values. It is also shown that tumor cell kill may be determined graphically without the necessity of computer-generated estimates of the Gompertz parameters.

Basic and therapeutic trial results obtained in the spontaneous AK leukemia (lymphoma) model-end of 1971.

Basic and therapeutic trial results obtained in the spontaneous AK leukemia (lymphoma) model have been brought together for comparison with available information on the much used transplanted murine leukemia models and human leukemias and lymphomas. The etiologic agent for "spontaneous" AK lymphoma is an RNA virus present at birth in AKR mice. Lymphoma cells first appear in the thymuses of animals at 5-->12 months of age. The time lapse between the first appearance of viable lymphoma cells in the thymus and clinical diagnosis (eg, with about 10(9) widely disseminated viable plus nonviable lymphoma cells in the host) is about 1 month. Thus, the overall doubling time of lymphoma cells before diagnosis is about 1 day. This estimate is compatible with the doubling time of relatively small numbers of first-passage lymphoma cells, assay data on the rate of repopulation of viable lymphoma cells after therapeutic reduction, and the median intermitotic time of dividing lymphoma cells (ie, 0.6 day). In general, the cytokinetic parameters of advanced spontaneous AK lymphoma cell populations are more like those observed in advanced human leukemias than are those of early L1210 leukemia. This paper presents assay data on the reduction in viable spontaneous AK lymphoma cells after treatment with a variety of agents, and the rate of cell repopulation after cessation of treatment. Extensive therapeutic trial data indicate that cyclophosphamide is presently the most effective single agent against spontaneous AK lymphoma, with arabinosylcytosine or palmO-ara-C a close second. Daunomycin, 5-fluorouracil, the nitrosoureas, vincristine, methotrexate, and dexamethasone provided moderate increases in host survival time. The combination of vincristine plus prednisone was a good remission inducer but the median survival time after cessation of treatment was shorter than that observed for cyclophosphamide or palmO-araC. The best responses observed to date with two-drug combinations appear better on several scores than the best that have been observed with single drugs. The best overall responses observed to date with two-drug combinations were with palmO-ara-C plus methyl-CCNU, cyclophosphamide plus methyl-CCNU, and palmO-ara-C plus cyclophosphamide. Some three- and four-drug combinations have provided better therapeutic responses than have been observed with single agents but not significantly better than those obtained with two-drug combinations. Splenomegaly assays carried out immediately after cessation of treatment and 60 days and longer after cessation of treatment, suggest that eradication of all viable lymphoma cells is being achieved in some animals by combination chemotherapy; however, such animals eventually die of lymphoma, presumably as a result of the reinduction of a second lymphoma cell population. The requirements for permanent "cure" of spontaneous lymphoma in AKR mice include eradication of all viable lymphoma cells and prevention of reinduction. Two major differences between early L1210 leukemia and clinically diagnosed spontaneous AK lymphoma are the degree of disease advancement at the time therapy is usually started (and associated cytokinetic differences) and the reinduction problem in AKR mice. Spontaneous AK lymphoma is relatively more advanced at diagnosis than is acute leukemia in man (ie, with respect to nearness of the host to death), and it is presumed that the reinduction problem in AKR mice is more acute and more prevalent than in human neoplastic disease.