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5,6-Dihydro-5-azacytidine hydrochloride, a chemically stable, soluble analog of 5-azacytidine, has cytostatic activity against mouse leukemic L1210 cells grown in culture, but concentrations on the order of 10 micronM, 10-fold higher, than the parent drug, are necessary to inhibit cell growth. The addition of either cytidine or uridine protected against growth inhibition by 5-azacytidine and 5,6-dihydro-5-azacytidine, whereas thymidine potentiated the cytostatic action of both drugs. Deoxycytidine also enhanced the action of 5-azacytidine but had no effect with the reduced analog. Cell suspensions of L1210 cells were able to phosphorylate 5-azacytidine and, to a lesser extent, 5,6-dihydro-5-azacytidine. In cell-free extracts in the presence of ATP and Mg2+, both drugs were converted to nucleotides but at less than 5% the rate of cytidine. As a substrate for mouse kidney cytidine deaminase, the apparent Km value for 5,6-dihydro-5-azacytidine (33 micronM) is of the same order of magnitude as that for cytidine (37 micronM) but less than that for 5-azacytidine (2.1 X 10(3) micronM). The Vm for deamination of the reduced analog is one-tenth that for 5-azacytidine. 3,4,5,6-Tetrahydrouridine, a potent inhibitor of cytidine deaminase, is more effective in blocking deamination of 5-azacytidine than 5,6-dihydro-5-azacytidine.

The applicability of a human tumor colony-forming assay to drug screening was investigated in terms of feasibility, validity, and potential for discovering new antitumor drugs. Feasibility was addressed in a pilot study during which basic methods, appropriate assay quality controls, and a standardized protocol for screening were developed. Considerable variability was noted in the availability and colony growth of different tumor types. The majority of the evaluable experiments utilized breast, colorectal, kidney, lung, melanoma, or ovarian tumors. For many tumor types, little evidence of growth was observed, or only rare specimens formed colonies. Colony-forming efficiencies ranged from 0.05 to 0.11% for the six most useful tumors listed above. A set of quality control measures was developed to address technical problems inherent in the assay. Testing of standard agents in the pilot study established that most of these agents could be detected as active. However, it also identified three assay limitations: compounds requiring systemic metabolic activation are inactive; medium constituents may block the activity of certain antimetabolites; and compounds without therapeutic efficacy may be positive in the assay. The assay categorized nontoxic clinically ineffective agents as true negatives with 97% accuracy. Of 79 compounds which were negative in the current National Cancer Institute prescreen (leukemia P388), 14 were active in the assay. Several demonstrated outstanding in vitro activity and are structurally unrelated to compounds already in development or in clinical trials. A subset of these active compounds were found to lack activity in a P388 in vitro colony-forming assay. This indication of differential cytotoxicity to human tumor cells makes this subset of compounds particularly interesting as antitumor drug leads. The demonstrated sensitivity to most standard agents, discrimination of nontoxic compounds, reproducibility of survival values within assays and between laboratories, and evidence of ability to identify active compounds which were negative in the in vivo prescreen suggest that the human tumor colony-forming assay may be a valuable tool for antitumor drug screening. However, because of technical limitations inherent in the current assay methodology, this must be confined to selected tumor types and limited to screening on a moderate scale.

Tetrachloro(d,l-trans)1,2-diaminocyclohexane platinum (IV) (tetraplatin), a new platinum analogue, showed greater therapeutic efficacy after i.p. administration than either cis-dichlorodiammineplatinum (II) (cisplatin) or cis-diammine-1,1-cyclobutanedicarboxylate platinum (II) (carboplatin) in mice bearing i.p. implanted L1210 leukemia. At an optimal dose of 5.7 mg/kg/injection given as a single dose on days 1, 5, and 9, tetraplatin increased the median life span over controls by more than 566% with 5 of 8 long-term (50-day) survivors. In contrast, cisplatin at the same optimal dose increased survival by 186% with 2 of 8 long-term survivors, and carboplatin at an optimal dose of 75.6 mg/kg/injection increased survival by only 120% with no long-term survivors. Tetraplatin also was more effective than cisplatin when treatment was delayed until days 3, 7, and 11 after i.p. implant. A combination of tetraplatin and Adriamycin in mice bearing i.p. implanted L1210 leukemia produced more long-term survivors over a wider range of doses than could be achieved with either drug alone. Tetraplatin at 5.7 mg/kg/injection and Adriamycin at 3 mg/kg/injection on days 1, 5, and 9 increased survival by more than 566% with 8 of 8 50-day survivors. Using the same treatment schedule, combinations of tetraplatin with either cisplatin, carboplatin, daunomycin, or 5-fluorouracil did not produce therapeutic efficacy greater than that seen with tetraplatin alone. The in vitro cellular uptake of platinum by L1210 cells at 37 degrees C was about 4-fold higher after exposure to tetraplatin compared to cisplatin following a 2-h incubation at the two concentrations examined (2.5 and 5 micrograms/ml). Comparative pharmacological studies were performed in rats at a single dose of 3 mg/kg i.v. The t1/2 beta for total platinum in plasma was 29.10 h (7.47 h for unbound platinum) after the administration of tetraplatin and 23.70 h (13.09 h for unbound platinum) after cisplatin. By 48 h the urinary excretion of platinum after tetraplatin and cisplatin was 30.1% and 41.4%, respectively. Tissue distribution of platinum was similar after either complex. Thus, tetraplatin has similar pharmacological properties to cisplatin and like cisplatin is a candidate for combination chemotherapy. However, tetraplatin may be superior to cisplatin in some therapeutic situations based on its greater efficacy against selected tumors.