Limiting dilution analysis of a novel tripeptide anticancer agent Ambamustine (PTT-119): effect on K-562, CCRF-SB and multidrug resistant LoVo-Dk cell lines.

Cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SB or K-562 lines. After incubating the cell mixtures in vitro with different dose levels of Ambamustine (PTT-119), a quantity of 10(4) treated cells were dispensed into microculture plates, and graded cell numbers of the lines used to contaminate the normal marrow were added. Limiting dilution analysis (LDA) was used to estimate the frequency of leukemic cells persisting after treatment. Incubation with 50 micrograms/mL of PTT-119 produced a total elimination of K-562 acute myelogenous blasts, whereas nearly 0.17 and 0.27 leukemic cells were still present in the cell mixtures after treatment with 5 and 25 micrograms/mL, respectively. When normal bone marrow was contaminated with CCRF-SB lymphoblastic cells, incubation with either 50 or 25 micrograms/mL of PTT-119 produced a complete clearing of leukemic cells, whereas with 5 micrograms/mL the leukemic cells in each well were 0.18. When PTT-119 was incubated with LoVo-DX, a colon cancer cell line which expresses the pleiotropic drug resistance MDR phenotype, virtually complete inhibition of clonogenic colonies was observed with as little as 5 micrograms/mL. This suggests that PTT-119 could be used in clinical trials as a non-cross-resistant agent in multidrug protocol.

Effect of two cyclophosphamide derivatives on hemopoietic progenitor cells and pluripotential stem cells.

The studies described herein were undertaken to help define the effects of certain cyclophosphamide derivatives that have been used for selective removal of leukemic cells from marrow samples used for autologous transplantation. We have tested the effect of 4-HC and another cyclophosphamide congener, ASTA-Z 7557, on pluripotent stem cells (CFU-S) and committed progenitor cells (CFU-GM) in mice. The CFU-S were evaluated by the spleen colony assay at eight days and 12 days after transplant. The eight-day colonies are transient in nature, rapidly growing, mainly erythroid, and lack pluripotential precursors. The 12-day colonies are believed to provide a measure of hemopoietic stem cells as they slowly grow and do contain primitive precursors. Our data show that at the maximum dose levels tested, both drugs caused a 100% loss of CFU-GM and about 80%-95% inhibition of early transient CFU-S. In contrast, about 70% of the pluripotent 12-day CFU-S were spared. These data appear to explain the hemopoietic recovery seen in man after transplantation with marrow cells treated with 4-HC despite their relative absence of hemopoietic progenitor cells.

Limiting-dilution analysis for the determination of leukemic cell frequencies after bone marrow decontamination with mafosfamide or merocyanine 540.

To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.