Effects of a prospective antitumor agent, 1,4-bis(2''-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate, on cultured mammalian cells

The effects of 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate (CBH; NSC 57198) on cell viability, growth, progression through the cell cycle, survival, and differentiation were investigated in suspension cultures of murine lymphocytic leukemia (L1210) and erythroleukemic (FL) cells and normal human lymphocytes stimulated with phytohemagglutinin (PHA) and in adherent cultures of Chinese hamster ovary (CHO) cells. CBH was equally cytotoxic toward stationary and exponentially growing CHO cells. Cell viability was diminished by 50% following 24 hr exposure to approximately 50 μg CBH per ml. Treatment of quiescent human lymphocytes for 24 hr with up to 100 μg CBH per ml did not appreciably diminish cell viability though the subsequent stimulation of such lymphocytes with PHA was inhibited in a dose dependent fashion. L1210, FL cells, and PHA stimulated human lymphocytes were equally sensitive to CBH, 50% inhibition of growth was obtained following 24 hr treatment with 25 μg CBH per ml. Incubation for up to 48 hr with CBH did not result in differentiation of FL cells to mature hemoglobin containing cells. Constant exposure of L1210 cells and PHA-stimulated human lymphocytes to 10-50 μg CBH per ml resulted in accumulation of cells in G₂ + M phase; higher drug concentrations resulted in cell arrest in mid to late S phase and G₂ phase. A short 1-hr pulse of the drug resulted in a transient accumulation of L1210 cells in S and G₂ phases. However, cells recovered from a short pulse of drug and by 48 hr, both cell proliferation and the cell cycle distribution appeared normal. A detailed analysis of cell cycle progression of L1210 cells in the presence of the drug indicated that the duration of G₂ phase was extended at low concentrations (10 μg/ml) while the transit of cells through S was retarded with subsequent accumulation in late S and G₂ phase at higher (50 μg/ml) concentrations. Concomitant with cell arrest in S and G₂ phase an increase in cellular RNA content indicating unbalanced growth was observed. This state of unbalanced growth was reversible in cultures exposed to a 1-hr pulse of up to 100 μg CBH per ml; cellular RNA content returned to control values by 48 hr. No effect on nuclear chromatin as assayed by acid denaturation was observed. Though the exact mechanism of drug action is not known, the data are not incompatible with the drug acting as an alkylating agent.     

Effects of ellipticine on cell survival and cell cycle progression in cultured mammalian cells

The effects of ellipticine [5,11-dimethyl-6H-pyrido(4,3-b)carbazole; NSC 71795] on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent [Chinese hamster ovary (CHO)]tumor cell systems as well as in mitogen-stimulated human peripheral blood lymphocyte cultures. Cell cycle progression and the terminal point of action of the drug were monitored by flow cytometry. Ellipticine was cytostatic for all cell lines tested, blocking cells in G2 phase following 24 hr constant exposure at concentrations in the range of 1.0 microgram/ml. A 10 times higher drug concentration was required to block cells in G2 if the cells were exposed for only 30 min to the drug followed by 23.5 hr culture in drug-free medium. Formation of CHO cell colonies was inhibited by 50% following exposure to ellipticine for 2 hr at 6.0 microgram/ml or for 24 hr at 0.3 microgram/ml. Fifty % cell kill in asynchronously growing Friend leukemia and L1210 cells was obtained following exposure to ellipticine for 24 hr at 2.0 microgram/ml and 1.15 microgram/ml, respectively, whereas human peripheral blood lymphocytes required 66 hr exposure to 1.0 microgram/ml to kill 50% of the cells. Phytohemagglutinin-stimulated lymphocytes were remarkably resistant to the cytotoxic effect of ellipticine but did display a dose-dependent inhibition of stimulation and accumulation in G2 whether the drug was added prior to our during active cell proliferation. Ellipticine, at cytostatic concentrations, had a marked effect on cellular RNA content. Friend leukemia cells, blocked in G2 by the drug, doubled their RNA content compared to control cells. L1210 and CHO cells, but not lymphocytes, also increased in RNA content following ellipticine treatment. Drug concentrations which blocked cells in G2 also led in the case of Friend leukemia and L1210 but not CHO cells to an increase in the proportion of cells with greater than 4C amounts of DNA.     

Effects of aclacinomycin on cell survival and cell cycle progression of cultured mammalian cells

The effects of aclacinomycin (ACM; NSC 208734) on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent (Chinese hamster ovary) cell systems. Cell cycle progression and the effect of the drug on various transition points in the cell cycle (i.e. G1 to S phase, through a window in early S phase and G2 phase to mitosis) were monitored by flow cytometry. Formation of Chinese hamster ovary cell colonies was inhibited by 50% following 24 hr of exposure to 0.05 micrograms ACM per ml whereas 1 hr of exposure to 1.0 micrograms ACM per ml reduced colony formation by only 30%. Stationary cultures required a drug concentration more than 5 times higher to reduce colony formation by an equivalent amount when present for 24 hr. Short-term (1-hr) exposure to drug concentrations up to 1.0 micrograms/ml had no effect on colony formation of stationary-phase Chinese hamster ovary cells. Cell growth was inhibited by 50% in suspension cultures of Friend leukemia and L1210 cells when exposed for 24 hr to 0.024 and 0.053 micrograms ACM per ml, respectively. Continuous drug exposure of Friend leukemia and L1210 cells to ACM concentrations of 0.05 to 0.1 micrograms/ml led to a slow down in cell progression manifested as an accumulation of cells in G2 + M phase by 24-hr and then in G1 phase by 48-hr culture. However, brief (1-hr) exposure of L1210 cells to 0.5 micrograms/ml resulted in an irreversible accumulation of cells in G2 + M phase. A more detailed examination of drug effects on the cell cycle determined that 0.1 micrograms ACM per ml resulted in a slow down in L1210 cells leaving G1 phase and entering mitosis and an accumulation of cells in G2 phase, although early S-phase cells appeared unaffected. At a 5 times higher drug concentration, exit of cells from G1 was almost completely halted, passage of cells through early S was slowed, and the entrance of cells into mitosis plateaued 3.5 hr after addition of the drug; G2-phase cells were only mildly affected. The RNA content of all cells examined was reduced by 35 to 50% depending upon dose and time of exposure. These findings are discussed in terms of the known biochemical effects of ACM on RNA and protein synthesis.     

Effects of new antitumor bifunctional intercalators derived from 7H-pyridocarbazole on sensitive and resistant L 1210 cells

The antitumor properties of 7H-pyridocarbazole dimers, a new series of bifunctional intercalators, have recently been described (Pelaprat, D. Delbarre, A., Le Guen, I., Roques, B. P., and Le Pecq, J. B. J. Med. Chem., 23: 1336-1343, 1980; and Roques, B. P., Pelaprat, D., Le Guen, I., Porcher, G., Gosse, C., and Le Pecq, J. B. Biochem. Pharmacol., 28: 1811-1815, 1979). In order to study the mechanism of action of these compounds, an L1210 subline was made resistant to one dimer (NSC 335153; ditercalinium). Selection of resistant cells was based on an in vitro-in vivo procedure as follows. Ascitic cells were taken from a leukemic mouse and incubated in vitro with the dimer for 1 hr. They were then injected into mice. After the development of the ascites, L1210 cells were collected and the process was repeated 13 times, until establishment of the resistance. Cloned resistant cells have maintained their resistance for 18 months of in vitro culture. The effects of two dimers (NSC 335153 and NSC 335154) on cell viability, growth, colony formation, and cell cycle progression were investigated on parental and resistant L1210 cells. The cross-resistance of these two L1210 cell lines to several cytotoxic agents was estimated. Several observations indicate that the mechanism of action of these dimers might be different from that of monointercalating agents: (a) these drugs induce a delayed toxicity (growth arrest occurring five generations after drug exposure) in sensitive but not in resistant cells; (b) cells exposed to the dimers arrested almost randomly in all phases of the cell cycle, whereas the corresponding monomer provokes a block in the G2 + M phase. Resistant cells were cross-resistant to 7H-pyridocarbazole monomer, Adriamycin, and vincristine but not to 6H-pyridocarbazole monomer derivatives, actinomycin D, and methotrexate.     

Antitumor activity of a novel antitumor antibiotic, quinocarmycin citrate (KW2152)

A novel antitumor antibiotic, 2a,3,4,5,6,6a,7,11b-octahydro-11-methoxy-12-methyl-3,6-imino-1H-2-oxa-11 c- azanaphth(1,2,3-cd)azulene-5-carboxylic acid monocitrate (quinocarmycin citrate; KW2152) was selected for investigation in a number of experimental tumor systems because of its efficacy against P388 leukemia. In the initial studies with P388 leukemia (i.p.-i.p.), KW2152 gave an increase in life span of greater than 80%. The activity was schedule dependent and daily administration was the most effective. KW2152 caused marginal activity against L1210 leukemia, B16 melanoma, and M5076 sarcoma. The effect on cultured cells suggested that KW2152 was not cross-resistant to Adriamycin (ADM) but was cross-resistant to mitomycin C (MMC); however, KW2152 caused prolongation of life span against mice bearing P388/ADM or P388/MMC. In tests against human tumors xenografted s.c. in nude mice, KW2152 significantly inhibited the growth of MX-1 mammary carcinoma with all tumors cured at i.v. doses of 4.4 mg/kg/day and p.o. doses of 26.2 mg/kg/day given daily for 7 days. KW2152 also inhibited distinct human gastric carcinomas, St-4 and St-15 tumors, and colon carcinoma Co-3 by daily administration for 7 days. Against St-4, KW2152 gave a treated versus control percentage of 27, compared to 52 for cis-diamminedichloroplatinum. Against Co-3, KW2152 was at least as effective as MMC, ADM, cis-diamminedichloroplatinum, and bleomycin, giving a treated versus control percentage of 18 at a dose of 8.6 mg/kg/day given daily for 7 days. KW2152 showed growth inhibitory activity against cultured murine tumors and human cells. The order of in vitro efficacy of KW2152 against murine tumors, P388 leukemia greater than L1210 leukemia, B16 melanoma, correlated with the order of the sensitivity on the i.p.-i.p. systems of these tumors. The 50% inhibitory concentrations against P388 leukemia cells were 5.3 X 10(-6) and 1.1 X 10(-7) M after 1 and 72 h exposure, respectively. KW2152 caused significant inhibition of RNA synthesis after a short time exposure. In P388 leukemia cells exposed for 1 h with KW2152, the 50% inhibitory concentration for RNA synthesis was 10(-5) M, 30-fold less than that for DNA synthesis. White blood cell depression or platelet depression was not significant after administration of the i.v. 10% lethal dose given daily for 7 days. Because of its good activity against human mammary tumor MX-1 and some effectiveness against other gastric and colon carcinomas and its water solubility, a novel antitumor antibiotic, KW2152, is being developed as a Phase I anticancer agent.     

Antitumor activity and biochemical effects of cyclopentenyl cytosine in mice

Cyclopentenyl cytosine, a recently synthesized inhibitor of cytidine 5'-triphosphate synthesis, has marked antitumor activity. Treatment with 1 mg/kg i.p. on days 1-9 following inoculation with tumor produced 111-122% increased median life span in mice bearing L1210 leukemia, 73-129% increased median life span in mice bearing P388 leukemia, and 58-62% increased median life span in mice with B16 melanoma. A subline of L1210 selected for resistance to 1-beta-D-arabinofuranosylcytosine was more sensitive to cyclopentenyl cytosine than the parent tumor line. L1210 cell growth in cultures was greatly inhibited (greater than 90%) by 0.1 microM cyclopentenyl cytosine, but cells were protected from the growth inhibitory effects by cytidine (20 microM) and to a lesser extent by uridine or deoxycytidine. Exposure of cultured L1210 cells to 1 microM cyclopentenyl cytosine inhibited formation of [3H]cytidine nucleotides from [3H]uridine by 30% during the first 15 min of exposure to drug and by greater than 95% after 2 h of exposure. Treatment of mice bearing L1210 ascites with cyclopentenyl cytosine (1 mg/kg) produced rapid depletion of cytidine nucleotide pools in the tumor cells; these pools fell to 35% of control within 30 min. The effects of cyclopentenyl cytosine on nucleotide pools were tissue selective; the cytidine nucleotide pools of spleen, liver, kidney, and intestine were less sensitive than that of the L1210 ascites tumor. Cytidine nucleotide pools of spleen and liver were depleted by higher doses (10 mg/kg) of cyclopentenyl cytosine.     

Effects of 5-aza-2'-deoxycytidine on survival and cell cycle progression of L1210 leukemia cells

The in-vitro effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-dCyd), on DNA synthesis, growth, cloning in agar, and cell cycle traverse of L1210 leukemia cells were studied. 5-Aza-dCyd at 0.1 microgram/ml for 10 hr (cytotoxic concentration) did not inhibit DNA synthesis but produced a very potent growth inhibition, and changed markedly the DNA flow cytometric histograms. A 5-h continuous exposure to the drug at concentrations ranging from 0.1 to 10 micrograms/ml caused an accumulation of cells in the S portion of the DNA histograms indicating a slowing of the progression of cells in the S phase. A longer exposure time (10 h) at the same concentrations led to a bimodal DNA distribution (peaks at G1 and G2-M) and a depletion of the S phase. When the exposure time to 5-aza-dCyd (0.1 microgram/ml) was extended to 15 and 20 h, there was a decrease in the G2-M peak and an augmentation of the G1 peak. To determine if 5-aza-dCyd produced a block in cell cycle progression, L1210 cells were treated for 10 h with colcemid and 5-aza-dCyd simultaneously for 10 h. Colcemid alone, or colcemid in combination with 5-aza-dCyd produced an accumulation of cells under a single G2-M peak. This indicates that 5-aza-dCyd did not block the progression of L1210 cells through S phase, but only produced a slowing down of this event. These results, indicating that 5-aza-dCyd does not block cell cycle progression and that its cytotoxic action is not self-limiting, are of importance for designing future clinical trials.     

Effects of the alpha-D-anomer of 5-aza-2'-deoxycytidine on L1210 mouse leukemic cells in vitro and in vivo

The alpha-D-anomer of 5-aza-2'-deoxycytidine inhibited cell growth in vitro in L1210 mouse leukemic cells (concentration causing 50% inhibition about 1 X 10(-6) M) and was also active in vivo in increasing the life span of mice with L1210 leukemia by 100% after a single i.p. injection (800 to 1000 mg/kg). This effect could be reversed by 2'-deoxycytidine. The alpha-D-anomer produced approximately 100-fold less host toxicity than did 5-aza-2'-deoxycytidine. It was observed that alpha-D-anomer, when kept at 23 degrees for 72 hr, inhibited the uptake of radioactive 2'-deoxycytidine by L1210 cells in vitro as well as the phosphorylation of 2'-deoxycytidine by a cell-free extract from L1210 cells. These effects could not be obtained with the fresh solution of the drug. It is proposed that the action of alpha-D-anomer is due to its conversion to 5-aza-2'-deoxycytidine as shown by high-performance liquid chromatography performed on water solutions of both anomers.     

Cellular pharmacology of DUP-785, a new anticancer agent

DUP-785, a new inhibitor of dihydroorotate dehydrogenase, is currently undergoing clinical evaluation for anticancer activity. We developed a GC/MS method to quantitate dihydroorotate that accumulates in cultures of L1210 cells exposed to growth inhibitory concentrations of DUP-785. This method was used to follow the onset, extent, and duration of inhibition of de novo pyrimidine synthesis in intact L1210 cells and to compare this inhibition with cell proliferation and cellular concentrations of pyrimidine nucleotides. There were direct relations between inhibition of de novo pyrimidine synthesis, changes in pyrimidine nucleotide concentrations, and cell proliferation following short (less than 24 hr) drug exposures; with prolonged exposures (greater than 24 hr), however, there was a departure from these relationships in that restoration of pyrimidine nucleotide pools and de novo pyrimidine pathway activity did not restore cell proliferation. Exposure of L1210 cells to 15 microM DUP-785 produced a maximum cell kill (99.9% as determined by cloning efficiency) at 24 hr, and no increase in cell kill was observed with drug exposure up to 96 hr.     

Antitumor activities and mechanisms of action of harringtonine and homoharringtonine

The antitumor activities and the mechanisms of action of harringtonine and homoharringtonine, alkaloids isolated from cephalotaxus hainanensis Li, were compared to those of vincristine. The results obtained were as follows: Harringtonine and homoharringtonine Significantly inhibited the growth of L1210 cells in culture. The IC50 values were similar to that of vincristine. Harringtonine and homoharringtonine had little effects on changes in the DNA histograms of FL cells at any concentrations, which suggesting that these drugs prolong the duration of each phase of the cell cycle evenly. Harringtonine and homoharringtonine had only a minor effect in arresting P388 cells in mitosis. Harringtonine significantly inhibited the DNA synthesis of P388 leukemia cells in culture, while vincristine weakly inhibited RNA and DNA synthesis. The successive treatment with harringtonine and homoharringtonine were as effective as the successive treatment with vincristine against P388 and L1210 leukemia, while both drugs were ineffective against Lewis lung carcinoma.     

Comparison of the mechanism of action of busulfan with hepsulfam, a new antileukemic agent, in the L1210 cell line

1,7-Heptanediol disulfamate (hepsulfam, NSC 329680) is a new antileukemic agent with close structural similarity to busulfan. The mechanism of action of hepsulfam is not known and it has recently been entered into Phase I clinical trials by the National Cancer Institute. Waud et al. have recently shown that hepsulfam has good antitumor activity against mouse L1210 leukemia in vivo (Waud et al., Proc. Am. Assoc. Cancer Res., 29:333, 1988). In contrast, busulfan was inactive against this model tumor system. In the present study, we have compared the in vitro cytotoxicity of hepsulfam with that of busulfan and we also examined the ability of these compounds to induce DNA damage in the L1210 leukemia cell line. Our results show that L1210 leukemia cells were 7-fold more sensitive to hepsulfam than busulfan. Only hepsulfam produced an appreciable quantity of DNA interstrand cross-linking in L1210 cells, with the peak of cross-link formation being delayed 12 h following a 2-h drug treatment. In contrast, both compounds also produced DNA-protein cross-linking, again with the formation of peak levels being delayed 6-12 h after drug treatment. At equimolar concentrations, hepsulfam produced a greater quantity of DNA interstrand cross-links and DNA-protein cross-links than busulfan. In contrast, busulfan produced a greater quantity of DNA-protein cross-links, when compared to hepsulfam at equitoxic concentrations.     

Chlorozotocin, 2-(3-(2-chloroethyl)-3-nitrosoureido)-D-glucopyranose, an antitumor agent with modified bone marrow toxicity.

Chlorozotocin, 2-(3-(2-chloroethyl)-3-nitrosoureido)-D-glucopyranose, is a newly synthesized, water-soluble nitrosourea antitumor agent that is active against L1210 leukemia in mice. A 701% and a 401% increase in life-span were attained with a dose that was lethal to 10% of the animals (15 to 20 mg/kg, i.p.) in mice treated on Day 2 or Day 6 of L1210 tumor growth, respectivley. Sixity % of Day 2-treated mice and 30% of Day 6-treated mice survived for 90 days. At the maximally effective dose against L1210, chlorozotocin produced no significant depression in normal bone marrow DNA synthesis nor in peripheral neutrophil count, in contrast to a sustained greater than 90% inhibition in L1210 ascites cell DNA synthesis. If the antitumor activity and reduced bone marrow toxicity of chlorozotocin are confirmed in man the use of this compound would facilitate treatment of patients with neoplastic disease who have preexisting abnormal bone marrow function or would allow for the more effective use of a nitrosourea agent in combination with anticancer agents possessing more potent myelosuppressive properties.     

The effect of vinca alkaloids in enhancing the sensitivity of a methotrexate-resistant (L1210/R7A) line,studied by flow cytometric and chromosome number analysis

Summary Two L1210 murine lymphoma cell lines sensitive and resistant to methotrexate (L1210 and L1210/R7A, respectively) and previously shown to exhibit collateral sensitivity to the vinca alkaloids have been studied by flow cytofluorimetric techniques following propidium iodide staining of the DNA. Following treatment with a range of concentrations of vincristine, both cell lines showed a build-up of fluorescence in the 4n position. However, the methotrexate-resistant cell line exhibited this effect at lower doses of vincristine. On an equimolar basis, the vinca alkaloids ranked for intensity of this effect in the order vinblastine>vindesine>vincristine. DNA fluorescent histograms following various times of continuous exposure to vincristine showed an accumulation of material at the 8n position, which was shown by chromosome analysis to be due to polyploidy. It was concluded that the methotrexate-resistant cells (L1210/R7A) experience difficulty in traversing mitosis and this difficulty is enhanced by the vinca alkaloids.     

Differential activity of vincristine and vinblastine against cultured cells

Vincristine and vinblastine exhibit differential activity against tumors and normal tissues. In this work, a number of cultured cell lines were assayed for their sensitivity to the antiproliferative and cytotoxic effects of the two drugs following short-term (4 hr) or during continuous exposures. Differential activity was not seen when cells were subjected to continuous exposures. The concentrations of vincristine and vinblastine, respectively, that inhibited growth rates by 50% were: mouse leukemia L1210 cells, 4.4 and 4.0 nM; mouse lymphoma S49 cells, 5 and 3.5 nM; mouse neuroblastoma cells, 33 and 15 nM; HeLa cells, 1.4 and 2.6 nM; and human leukemia HL-60 cells, 4.1 and 5.3 nM. In contrast, differential toxicity was seen when cells were subjected to 4-hr exposures and transferred to drug-free medium: the 50% growth-inhibitory concentrations for vincristine and vinblastine, respectively, for inhibition (a) of proliferation of L1210 cells were 100 and 380 nM and of HL-60 cells were 23 and 900 nM and (b) of colony formation of L1210 cells were 6 and greater than 600 nM and of HeLa cells were 33 and 62 nM. Uptake and release of [3H]-vincristine and [3H]vinblastine were examined in L1210 cells under the conditions of growth experiments. Uptake of both drugs was dependent on the pH of culture media, and significantly greater amounts of [3H]vinblastine than of [3H]vincristine were associated with cells after 4-hr exposures to equal concentrations of either drug. When cells were transferred to drug-free medium after 4-hr exposures, vinblastine was released much more rapidly from cells than was vincristine, and by 0.5 hr after resuspension of cells, the amount of vincristine associated with the cells was greater than the amount of vinblastine and remained so for up to at least 6 hr.     

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M): a novel sulfonylhydrazine prodrug with broad-spectrum antineoplastic activity

Our laboratory has synthesized and evaluated the anticancer activity of a number of sulfonylhydrazine DNA modifying agents. As a class, these compounds possess broad spectrum antitumor activity, demonstrating significant activity against a variety of experimental murine tumors, including the P388 and L1210 leukemias, B16 melanoma, M109 lung carcinoma, and M5076 reticulum cell sarcoma, as well as against the human LX-1 lung carcinoma xenograft. The current report describes the activity of a more recently synthesized member of this class, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M). 101M was active in mice against the i.p. implanted L1210 leukemia over a wide range of doses and produced long-term survivors when administered as a single i.p. bolus of 10, 20, 40, 60, or 80 mg/kg, demonstrating a wider margin of safety than the nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Curative therapy was achieved with doses of 101M that did not produce depression of the bone marrow. 101M was also highly effective against the L1210 leukemia when administered by the oral route. The ability of 101M to penetrate the blood-brain barrier and eradicate leukemia cells in the brain was remarkable (>6 log kill). This agent was also curative against L1210 variants resistant to cyclophosphamide, BCNU, or melphalan. Mice implanted with the murine C26 colon carcinoma were also cured by two injections of 10 or 20 mg/kg of 101M. Administration of 101M by two different well-tolerated regimens caused complete regression of established human glioblastoma U251 xenografts in 100% of treated mice, and significant responses were also obtained with 101M against advanced murine M109 lung carcinomas in mice. The broad spectrum of anticancer activity of the sulfonylhydrazine prodrug 101M coupled with the wide range of therapeutic safety exhibited by this agent, makes 101M particularly attractive for further development and clinical evaluation.     

In vitro antitumor mechanism of a new platinum complex, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato+ ++) platinum(II)

The antitumor mechanism of (-)-(R)-2- aminomethylpyrrolidine (1.1-cyclobutanedicarboxylato)platinum(II) (DWA2114R) was examined using cultured murine L1210 leukemia cells by estimating its effects on parameters such as proliferation, macromolecular synthesis, morphology and cell cycle progression. Each parameter was estimated in cells concomitantly exposed to the drug for 24-48 hr. More than 0.1 microM of DWA2114R markedly inhibited cell proliferation as well as DNA synthesis, and it decreased in mitotic index in a concentration-dependent manner. One microM of DWA2114R decreased DNA synthesis by 80% in the cells treated for 24 hr, while the inhibition of RNA synthesis was less than 40%. A significant inhibition of protein synthesis was caused only by treatment with a high concentration (100 microM) of the drug. Under complete cytostatic conditions (10 microM of DWA2114R), cell volume markedly increased and about 40% of the total cells were polynucleate. In addition, flow cytometrical analysis revealed that most of these cells were accumulated in the G2/M phase of the cell cycle, and a new peak located in the G2/M phase of tetraploid cells emerged. On the other hand, the cells treated with 100 microM of the drug did not increase in volume and their progress in the cell cycle was almost completely blocked.     

Arrest in late G2 or prophase of cell cycle induced by 4,4-(1,2-ethanediyl) bis (1-isobutoxycarbonyloxymethyl 2, 6-piperazinedione) (MST-16) in cultured L1210 cells.

The effects of MST-16, a new antitumor agent derived from bis (2, 6-dioxopiperazine), on cell growth, cell-cycle progression and DNA synthesis, alone and in combination with other antitumor agents, were investigated in murine leukemia L1210 cells in vitro. The drug showed dose-dependent inhibition of cell growth, and this effect was cell-cycle phase-specific. Flow cytometric analysis indicated that the drug could retard-arrest the cells in late G2 phase or prophase and that it did not affect the progression from G1 to G2 phase. In the presence of MST-16, the change in 3H-thymidine incorporation was proportional to the retardation-arrest of the cells, suggesting that MST-16 has no direct action on DNA synthesis itself. MST-16 could continuously retard the cells which were arrested by etoposide (VP-16); and vincristine (VCR) could block the progression of the cells arrested by MST-16, but not vice versa. The addition of MST-16 followed by VCR was more effective than simultaneous addition of the 2 drugs on inhibition of cell growth. These results will be useful in designing a reasonable regimen of MST-16 chemotherapy for malignancies.     

Antitumor activity and mechanism of action of 6-thio-3-deazaguanine

6-Thio-3-deazaguanine (TDG), a relatively new purine antimetabolite, exhibits significant antitumor activity against a variety of experimental animal tumor models including C3H mammary adenocarcinoma, Lewis lung carcinoma, adenocarcinoma 755, and leukemias L1210 and P388. However, the drug was ineffective against 3-deazaguanine-resistant L1210 (both in vitro and in vivo) and CEM cells (in vitro). The resistant cells appear to lack HGPRTase activity because the extracts from these cell lines failed to convert hypoxanthine to IMP. These data indicate that TDG needs to be activated by hypoxanthine guanine phosphoribosyltransferase prior to its growth inhibitory effects. Cytotoxicity of TDG was completely reversed by hypoxanthine and inosine. TDG inhibited the synthesis of DNA and RNA equally and effectively, whereas the inhibition of protein synthesis required a prolonged drug exposure and appears to be a consequence of the inhibition of DNA and RNA synthesis. Data from these studies suggest that TDG is an effective antitumor agent, and its spectrum of antitumor activity and mechanism of action appears to be different from that of 3-deazaguanine.     

Antitumor activity of ethyl 5-amino-1,2-dihydro-2-methyl-3-phenyl-pyrido [3,4-b]pyrazin-7-ylcarbamate, 2-hydroxyethanesulfonate, hydrate (NSC 370147) against selected tumor systems in culture and in mice

Ethyl 5-amino-1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin- 7-ylcarbamate, 2-hydroxyethanesulfonate, hydrate (NSC 370147) was evaluated for antitumor activity against a spectrum of tumor systems in culture and in mice. NSC 370147 was cytotoxic to a variety of mouse and human cell lines at nanomolar concentrations. The compound exhibited good in vivo antitumor activity against several murine tumors (P388 and L1210 leukemia, colon 11/A and 36, mammary 16/C, and M5076 sarcoma). Activity was largely independent of route of administration but favored a prolonged treatment schedule. NSC 370147 was as active against murine leukemia sublines resistant to Adriamycin, amsacrine, vincristine, melphalan, cisplatin, methotrexate, and CI-920 (a topoisomerase II inhibitor) as against the corresponding parental lines. Only the 1-beta-D-arabinofuranosylcytosine-resistant P388 subline exhibited any cross-resistance to NSC 370147. NSC 370147 has a spectrum of activity similar to that of vincristine and, unlike vincristine, is active against multidrug-resistant cell lines. Therefore, NSC 370147 is a candidate for clinical trial because of its favorable activity compared to vincristine, its effectiveness against multidrug-resistant cells, and its retention of activity for p.o. administration.     

Comparison of in vitro and in vivo effects of thymidine on L1210 leukemia in mice

Previous investigators have shown that L1210 leukemia in vitro growth can be inhibited by thymidine (dThd) by exposure for 24 hours of dThd concentrations as low as 0.1 mM. These experiments were conducted in an attempt to determine why dThd, although effective as a cytostatic agent against L1210 in vitro, is ineffective against L1210 in (BALB/c X DBA/2)F1 (CD2F1) mice. Systemic variations of dThd concentration and exposure times were examined in both a growth-inhibition assay and a soft agar cloning assay in vitro. The growth of cells exposed to 0.1 - 1.0 mM dThd for 15 hours was inhibited dramatically during exposure, but growth recovered to control rate rapidly when dThd was washed off the cells. With 96 hours of exposure to 1.0 mM dThd, growth rate did not return to control rate up to 120 hours after washing. Cloning efficiency was reduced to less than 1% of control by prior 96-hour exposure of L1210 cells to more than 0.5 mM dThd. Concentrations of deoxycytidine (dCyd) of 1.0 microM partially reversed cytotoxicity caused by dThd greater than 0.1 mM, and 10 microM dCyd completely reversed this cytotoxicity. In vivo plasma dThd concentrations during ip injections of 3,600 mg dThd/kg body weight every 8 hours for 4 days (approximately lethal dose for 10% of animals treated) ranged from 10 to 0.1 mM, but these concentrations were ineffective in prolonging survival time of L1210-bearing mice. Plasma concentrations of dCyd prior to dThd exposure were less than 1.0 microM. Following injection of dThd (3,600 mg/kg body wt), plasma dCyd was 2.8 +/- 0.5 microM (normal mice) and 6.1 +/- 0.5 microM (L1210-bearing mice). Thus the rise of dCyd in plasma following dThd administration in vivo prevented the cytotoxicity of dThd seen in vitro from being manifested in vivo in CD2F1 mice.