Comparative studies of the cytostatic action and metabolism of 5-azacytidine and 5,6-dihydro-5-azacytidine.

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.

Therapeutic and pharmacological studies of tetrachloro(d,l-trans)1,2-diaminocyclohexane platinum (IV) (tetraplatin), a new platinum analogue.

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.

Application of a human tumor colony-forming assay to new drug screening.

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.

Quantitative structure--activity correlations of rifamycins as inhibitors of viral RNA-directed DNA polymerase and mammalian alpha and beta DNA polymerases.

Twenty-two 3-substituted rifamycins were tested for inhibition of mammalian alpha and beta DNA polymerase and viral RNA-dependent DNA polymerase ("reverse transcriptase"). Quantitative structure--activity relationships (QSAR) were formulated for the three systems. Inhibition is linearly dependent on the partition coefficient and is highly favored by the presence of bulky hydrazones or oximes. None of these agents proved to be a selective or specific inhibitor of reverse transcriptase. A correlation in terms of log P and (log P)2 was obtained from data on a more closely related set of analogues from a published study. For murine reverse transcriptase, log P0 = 5.1.

A strategy for the development of two clinically active cisplatin analogs: CBDCA and CHIP.

The antitumor agent cisplatin has a broad antitumor spectrum and has been incorporated into regimens that are curative for some malignant diseases. However, one of the major limitations to its clinical usefulness is the incidence of severe toxicities involving several major organ systems. Therefore, much enthusiasm has been generated for the development of cisplatin analogs that demonstrate an improved therapeutic index in some preclinical models. The two most promising analogs are CBDCA (carboplatin) and CHIP (iproplatin). The preclinical and early clinical trial results have demonstrated that these two compounds show activity in cisplatin-responsive tumors. The preclinical background providing the rationale for the clinical development of these two analogs is described. We suggest a means of screening for each analog's clinical antitumor activity and determining the analogs' utility against specific malignant diseases compared with that of the parent compound or standard treatment.

Nude mouse models as predictors of chemotherapy in man: thymidine and pyrimidines.

The National Cancer Institute cancer treatment screening program has been reorganized incorporating, as an important feature, a panel of human tumors growing as xenografts in congenitally athymic mice. The new screening program is a prospective experiment in the search for new and more effective agents for the treatment of clinical neoplasia. The new program is described and questions that are being asked prospectively are presented. Data are summarized on the activity against human tumor xenografts for a number of clinically established antitumor drugs and examples are presented in which there is interest in compounds for the clinic on the basis of activity in the new screen. Studies are outlined in which high dose thymidine inhibited the growth of human melanoma and teratocarcinoma transplanted in athymic mice. Studies are discussed employing murine tumors in which marked augmentation of the in vivo antitumor activity of 5-fluorouracil was obtained by combination therapy with the pyrimidine nucleosides thymidine, uridine and cytidine. The desirability of investigating combination chemotherapy with pyrimidine nucleosides and 5-fluorouracil and other pyrimidine antagonists in the treatment of human tumor xenografts is stressed. There is a broad range of investigations that can be conducted in nude mouse models and it is important to conduct such programs in relation to the clinic.

Antitumor activity of cis-dichlorodiammineplatinum(II).

cis-Dichlorodiammineplatinum(II) has shown good broad-spectrum activity in the current Division of Cancer Treatment (National Cancer Institute) tumor panel. It meets Decision Network criteria for activity in at least five of ten tumor systems (the ip B16 melanoma, the sc CD8F1 mammary carcinoma, the ip colon tumor 26, the ip L1210 leukemia, and the ip P388 leukemia). Activity against the sc colon tumor 38, the iv Lewis lung tumor, the ic ependymoblastoma, and the human breast tumor xenograft (MX-1) was marginal. No activity was detected against the human lung tumor xenograft (LX-1), but good, reproducible activity was observed against the murine M5076 ovarian carcinoma. No schedule-dependency was observed after ip administration against the ip L1210 leukemia. The recently developed subrenal capsule assay offers promise as a rapid way (less than or equal to 11 days) of ranking the sensitivity of a variety of human tumors to cis-dichlorodiammineplatinum(II) and other chemotherapeutic agents.

Binding of maytansine to tubulin: competition with other mitotic inhibitors.

The effect of maytansine on the binding of [3H]vinblastine and [3H]colchicine to tubulin was examined by Sephadex gel filtration column chromatography. When varying concentrations of maytansine were employed, competition between vinblastine and maytansine was observed at a vinblastine to maytansine ratio of 1:10 and 1:100, while a colchicine to maytansine ratio of 1:100 did not affect the binding of colchicine to tubulin. These results confirm earlier findings with a DEAE-cellulose disc paper assay and a tubulin polymerization assay that maytansine shares a common binding site with vinblastine and support the view that tubulin has at least two drug binding sites.

Potentials and drawbacks of the human tumor stem cell assay.

The human tumor stem cell assay (HTSCA) provides a means of performing drug sensitivity measurements on human tumor cells in primary culture. Results from such assays offer potential for improving cancer chemotherapy by identifying drugs useful for treatment of individual patients' tumors and through application to screening new compounds for antitumor activity. While existing data supports the potential of the assay in both areas, the assay also poses significant drawbacks. Many of these drawbacks relate to technical aspects of the assay and can be eliminated or reduced by further assay development. In this paper, we describe some of the technical drawbacks in detail and some approaches which have been successful in minimizing them. Continued advances in this area should make it possible to more fully realize the potential of the human tumor stem cell assay.

Synthesis, physical properties, and antitumor activity of tetraplatin and related tetrachloroplatinum(IV) stereoisomers of 1,2-diaminocyclohexane.

The synthesis, physical properties, and antitumor activity of the cis-, d,l-trans-, d-trans-, and l-trans- stereoisomers of 1,2-diaminocyclohexane tetrachloroplatinum(IV) are described. The objective of the study was to produce a platinum complex with activity against cisplatin-resistant tumor cells and with suitable pharmaceutical properties for formulation development. The isomers had the following solubilities in saline: cis-, 2 mg/ml; d,l-trans-, 6.5 mg/ml; and d-trans- and l-trans-, 15-16 mg/ml. The four complexes showed slightly better activity than cisplatin against the ip implanted murine L1210 leukemia. In contrast to cisplatin, all complexes produced significant increases in life span against L1210/cisplatin, a subline of L1210 with acquired resistance to cisplatin. However, the cis- isomer was less active against L1210/cisplatin. The d,l-trans- isomer (tetraplatin) was selected for further studies based on greater ease for large-scale synthesis. It showed superior activity to cisplatin against P388/cisplatin and like cisplatin showed significant and reproducible activity against the ip implanted B16 melanoma, ip implanted M5076 sarcoma, ip implanted P388 leukemia, and MX-1 human breast xenograft implanted under the renal capsule. Purity and stability (greater than 24 hours in saline) were evaluated by high-performance liquid chromatography and found to be suitable for development of a parenteral dosage form. Preliminary studies in a rat model (to be reported elsewhere) showed it to be less nephrotoxic than cisplatin on a molar basis and worthy of further study.