Final report of the phase I trial of single-dose WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid].

Two hundred one patients were entered in a single-dose phase I trial of WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid]. Major toxic effects included emesis and hypotension. The observed minor toxic effects were somnolence and sneezing. Two infusion schedules were tested: long-infusion time, which fixed the rate, but varied total time; and short-infusion time, which varied the rate, but fixed the time to 15 minutes. Emesis was significantly influenced by infusion time; the long schedule caused a 57% incidence whereas the short schedule caused only a 28% incidence. Within the long-infusion group, higher-dose patients and women were more likely to vomit. Although only 15% of the entire group had hypotension, the long-infusion schedule had a hypotension incidence of 23%; the short schedule had an incidence of only 3% (P less than 0.0005). Within the long-infusion group, dose and tumor site significantly influenced the incidence of hypotension. No factors were associated with these toxic effects in the short-infusion schedule. However, certain toxic effects were too infrequent to detect significant differences. For future trials we recommend 740 mg/m2 infused in 15 minutes. With this schedule, vomiting was seen in 25% of infusions and hypotension was seen in only one of 68 infusions. To date, no delayed toxic effects have been detected in any organ system, and the trial resulted in no toxic deaths.

Response of human neuroblastoma and melanoma multicellular tumor spheroids (MTS) to single dose irradiation.

The growth characteristics of 6 human cell line derived multicellular tumor spheroids (MTS) were studied. Melanoma MTS (C32, HML-A, HML-B) were slow growing with baseline growth rates of 13.9 to 27.3 microns diameter/day. Neuroblastoma MTS (Lan-1, NB-100, NB-134) grew rapidly, with baseline growth rates of 32.1 to 40.3 microns diameter/day, that is, 1.2 to 2.9 times as fast as the melanomas. Delay constants were calculated for all six lines. The neuroblastomas were more sensitive to radiation than melanomas, as reflected in a greater value for the radiation-induced growth delay constant. One neuroblastoma line, Lan-1, was highly radioresponsive; that is, after a subcurative dose of radiation, the MTS diameter decreased beyond the original diameter, which was followed by recovery and regrowth. Irrespective of these initial changes in diameter, growth delay sensitivity (value of delay constant) was the same for Lan-1 and NB-100, an MTS line that did not show the responsive pattern.

Potential application of perfluorochemicals in cancer therapy.

PFC emulsions, because of their excellent oxygen-transporting properties and possibly other properties as well, have broad and promising potential in cancer therapy. Although this potential is readily apparent to both laboratory and clinical investigators who are concerned with tumor hypoxia, it should be equally apparent that the formulations that are currently available are wholly inappropriate for such an application. The sublethal toxicities of these emulsions may appear to indicate a poor prognosis for this approach, but they may be a blessing in disguise. All known approaches to tumor hypoxia are limited by our inability to administer enough of the agents to be effective. In many cases, the limiting factor was not uncovered until the approach had been introduced into clinical trials. Had we relied on such parameters as lethality in the case of PFC emulsions, the same mistake might have been made here. Instead, the death of a single rat has caused us to focus our attention on what would surely have produced major problems in the initial clinical trials. Recognition of the sublethal toxic effects of PFC emulsions at this early stage should allow us to optimize our formulations at the preclinical stage, thereby increasing the likelihood that PFC emulsions will someday live up to their full potential.

Quantitation of the response of human tumor spheroids to daily radiation exposures.

Two human tumor lines, NB-100 neuroblastoma and C-32 melanoma, were grown as multicellular tumor spheroids (MTS) and exposed to daily doses of gamma rays, 5 days per week. It required daily doses of 200 rad to arrest the growth of the NB-100 MTS, while 350 rad per day was required to arrest the growth of the C-32 MTS. Calculation of the delay in time to grow 200 um beyond the original size yielded similar differences in radiation resistance. When the volume of the treated MTS was expressed as a fraction of their potential volume and plotted as a function of cumulative dose, there appeared to be little fraction size dependence over the range studied. This prediction was tested experimentally and confirmed: daily administration of two 100 rad doses separated by 4 hours to NB-100 MTS was only marginally less effective than a daily single dose of 200 rad. These data suggest that MTS may prove to be valuable tools in studying the response of human tumors to clinical exposure protocols, including hyperfractionation.

Variation in normal tissue responsiveness to WR-2721.

The radioprotection afforded normal tissues by WR-2721 or S-2-[3-aminopropylamino]ethylphosphorothioic acid varies widely, with some of the most responsive tissues showing low levels of absorbed drug and vice versa. While the oxygen tension of the respective tissues may be contributing to this variation, it can be shown that the drug is not homogeneously distributed within each tissue, and that its location within the sub-cellular compartments can vary widely. Recognition of this variability, when combined with the newly developed HPLC assays for drug quality, should provide new insights into the sources of variation in normal tissue responsiveness to these agents.

Toxicity of WR-2721 administered in single and multiple doses.

Two hundred forty-three patients have received WR-2721 in Phase I-II studies. Separate studies were conducted in which patients with advanced malignancies received WR-2721 before single or multiple doses of radiotherapy or in single doses prior to cyclophosphamide, nitrogen mustard or cis-platinum. Single doses were escalated from 25 to 1330 mg/m2. An Acceptable Tolerated Dose (ATD) of 740 mg/m2 infused in 15 minutes has been established and is currently used in Phase II studies. Significant persistent hypotension (greater than 20 torr systolic) as a dose-limiting toxicity has occurred in 5% of patients in the single dose study. Fifty-five patients have been entered in the multiple dose trial. Dose levels of 340 mg/m2, four times a week for three weeks, and 250 mg/m2, four times a week for six weeks have been reached. There were five idiosynchratic reactions (fever, chills, rash, hypotension), one of which was severe. Some patients withdrew from the multiple dose study because of vomiting after each injection, or fear. No deaths nor any long-term untoward effects were observed. There is no suggestion of tumor protection.

Differential radioprotection of normal tissues by hydrophilic chemical protectors.

Analogous to certain radiosensitizers which are too hydrophilic to enter tumor cells, certain radioprotectors, because of their hydrophilicity, may also be hindered from entering tumor cells and thus protect only normal tissues. In testing this hypothesis, we utilized thin layer chromatography as convenient means to measure radioprotector hydrophilicity. Dose reduction factors (DRF's) for hematopoietic radioprotection were determined in BALB/c mice given half maximum tolerated doses (MTD/2) of 11 radioprotectors 30 min prior to graded doses of gamma rays. DRF's for tumor protection were determined in MCa-11 tumor-bearing mice using a regrowth delay assay. Differential radioprotection was found to be significantly correlated (r = 0.86) with hydrophilicity. Thus, radioprotector hydrophilicity appears to be a significant factor in the differential radioprotection observed and should be useful in designing or selecting better differential radioprotectors.

Perfluorocarbon emulsions in cancer therapy: preliminary observations on presently available formulations.

Perfluorocarbon (PFC) emulsions, due to their favorable oxygen transporting properties, have been proposed as tumor sensitizers for application in both radiotherapy and chemotherapy. While this application is a very promising one it is by no means simple, and presently available formulations are inadequate. Intravenous administration of these emulsions can produce a severe hemodilution which tends to offset the desired effect; these emulsions can alter the pharmacokinetics of simultaneously administered drugs. Unless these variables are taken into account the risk of false negative and false positive results will be excessive. Of more serious concern are the profound disturbances produced by these emulsions in the reticuloendothelial system. Using two of the more popular PFC, Fluosol-DA and DMA/NONANE, we have shown that daily administration of these emulsions can produce 9-fold increases in liver size and 27-fold increases in spleen size. This problem appears to involve the surfactant used in both emulsions, pluronic F-68. It will be necessary to circumvent this problem before further study of their potential application can proceed.

Liquid-overlay culture of cellular spheroids.

The capacity of different types of human cells to form and grow as spheroids was tested by using suspension culture over a bottom surface, which prevented cell attachment (liquid-overlay culture). The best and most convenient bottom surface so far tested was the bottom of a normal culture dish covered with a thin film of agarose. The agarose film was allowed to dry prior to the addition of medium. Several different human cell lines were tested, and it was found that many types of tumor cells formed and grew as spheroids, Large variations in growth rate, cell morphology, thickness of the viable cell layer and PO2 profiles were found among the tested spheroids. This variability parallels the variability seen in solid tumors. The morphology and the growth rate of solid tumors vary a great deal, depending on the type and position of the tumors (Ackerman and Rosai 1975; Steel 1977). Some tumors have a slow growth rate, with a few proliferating cells, while others grow faster and have higher proportions of proliferative cells (Charbit et al. 1971; Malaise et al. 1973; Friedman 1974; Lightdale and Lipkin 1975; Steel 1977). Local variations in both morphology and proliferation are usually found even within single tumors (Denekamp and Kallman 1973; Ackerman and Rosai 1974). Considering the variability in morphology, growth rate, and radiosensitivity between different types of spheroids, it is not difficult to imagine that wide variations may exist in these variables between different types of tumors or even between different regions within single tumors. The use of a group of different spheroids, showing a spectrum of growth rates, radiosensitivities, etc., will, it is hoped, be helpful in elucidating factors of importance in cancer therapy.

Cytophotometric measurement of the cellular DNA content of [3H]thymidine-labelled spheroids. Demonstration that some non-labelled cells have S and G2 DNA content.

Spheroids from the V279-171b and MCa-11 cell lines were incubated continuously for 24 hr in [3H]thymidine for labelling of the outer cells of the viable rim. The spheroids were dispersed into single cells, and the DNA content of photomapped cells was measured by absorption cytophotometry. Autoradiographs were then prepared from which we ascertained cellular labelling. For spheroids of both cell lines, we found a larger proportion of cells with a G0/G1 DNA content among the non-labelled inner spheroid cells than among the labelled outer cells (P less than 0.001). This block of non-labelled spheroid cells in G0/G1 was not a cell cycle perturbation caused by the isotope for the MCa-11 spheroids. Approximately 8% of non labelled MCa-11 spheroid cells had S/G2 DNA content, suggesting that non-cycling cells in spheroids may be blocked in S and G2 as well as in the G0/G1 phase of the cell cycle.

Computer-assisted morphometric analysis of renal radiation response.

A single x-ray dose of 1,200 to 1,600 rads to the mouse kidney is associated with definite morphologic alteration but minimal functional impairment at six months; this progresses to profound structural and functional impairment by one year after irradiation. Subjective morphologic assessment of renal damage at six months correlates well with total radiation dose, fractionation schedule and energy characteristics of the radiation beam but does not provide adequate quantitative numerical data for sophisticated statistical tests of significance or for comparisons of effect variability at given dose levels. This investigation assessed the applicability of computer-assisted morphometric analysis (CAMA) for quantitation of effects and in making statistical comparisons of significance between kidneys subjectively classified as to degree of histologic alterations. Images of renal cortex tubular nuclei from the various histologic grades were digitized, recorded and analyzed with the CAMA system. Results indicate that the reliability of specific grade assignment by CAMA for individual nuclei was inadequate but that separation of irradiated and unirradiated renal tissue (bivariate group means) was quite distinct and of high reliability. Differences were present among the four irradiated histologic grades, but they were not marked, especially among the three highest grades. More accurate quantitation of nuclear size variations was achieved, and chromatin textural differences were detected that were not apparent to the eye. Computer-assisted morphometric analysis appears to have a valuable application in the quantification and analysis of chronic radiation effects.

Circumvention of the tumor membrane barrier to WR-2721 absorption by reduction of drug hydrophilicity.

In attempting to account for the ability of most solid tumors to restrict the absorption of WR-2721, aminopropyl-aminoethylphosphorothioate, we examined a number of drug characteristics which might allow for this restriction, and observed that drug hydrophilicity was a major contributing factor. When the highly hydrophilic WR-2721 was dephosphorylated, the drug became less hydrophilic and could readily cross tumor cell membranes. In addition, conventional radioprotectants, such as cysteine and mercaptoethylamine, were shown to be less hydrophilic than WR-2721 and also to cross tumor membranes readily. Therefore, drug hydrophilicity would appear to be the factor underlying the ability of WR-2721 to selectively protect normal tissues while most other protectors alter the radiation resistance of normal and tumor tissue alike. A red blood cell model for studying this problem in greater detail is described.

Clinical trials of WR-2721 with radiation therapy.

The radioprotector with clinical potential, S-2-(3 aminopropylamino)-ethylphosphorothioic acid (WR-2721) is undergoing two Phase I trials. The objectives of these trials are 1) to determine the maximum tolerated dose (MTD) of WR-2721 in a single dose and 2) to determine the highest dose of WR-2721 that can be tolerated daily in the greatest number of fractions per week. A total of 65 patients have been treated. The single maximum tolerated dose has not yet been reached, though 740 mg/m2 is well tolerated. A single dose of 910 mg/m2 has been successfully administered to one patient. The multiple dose MTD is at an early stage with patients currently receiving 170 mg/m2 four times a week. Among the toxicities noted in both trials are hypotension, hypertension, emesis and somnolence. In addition, in the multiple dose trial there have been three patients who have had allergic reactions including one which was life-threatening. Phase II studies are planned and will begin when the maximum tolerated dose is established from each Phase I trial.