Comparison of In Vitro Antileukemic Activity of 4-Hydroperoxyifosfamide and 4-Hydroperoxycyclophosphamide.

BACKGROUND/AIM: The oxazaphosphorines, ifosfamide and cyclophosphamide, represent a class of alkylating agents. The aim of the present in vitro study was to compare antileukemic activity of 4-hydroperoxyifosfamide (4-OOH-IF) and 4-hydroperoxycyclophosphamide (4-OOH-CP). MATERIALS AND METHODS: The experiments were performed on MOLT-4 and ML-1 cells. The research was conducted using flow cytometry fluorescein diacetate/propidium iodide (PI), fluorescein-conjugated annexin V/PI, CaspGLOW Red Active Caspase-8 and -9, CellEvent™ Caspase-3/7 Green assays, and tetramethylrhodamine ethyl ester test. RESULTS: 4-OOH-IF and 4-OOH-CP distinctly reduced cell viability and triggered apoptosis and necrosis, causing changes in intracellular esterase activity, plasma membrane structure and integrity, caspase activation, and mitochondrial membrane potential. The oxazaphosphorines were responsible for the different antileukemic activities. 4-Hydroperoxyifosfamide appeared to be less cytotoxic against the leukemia cells than 4-hydroperoxycyclophosphamide. MOLT-4 cells were more sensitive to the action of the oxazaphosphorines than ML-1 cells. CONCLUSION: The findings provide a new insight on the mechanisms of cytotoxic action of 4-OOH-IF and 4-OOH-CP on the human acute lymphoblastic and myeloblastic leukemia cells.

In vitro effects of new generation oxazaphosphorines on human promyelocytic leukemia cells.

Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864) and glufosfamide (D-19575, beta-D-glucose-isophosphoramide mustard) are new generation oxazaphosphorine agents. The present investigation was undertaken to determine the activity of these three oxazaphosphorines in human promyelocytic leukemia HL-60 cells. The research was conducted using the spectrophotometric MTT assay and the electronic Beckman Coulter and microscopy methods. Functional and morphological changes were observed after exposure of HL-60 cells to the oxazaphosphorine agents. The various patterns of temporary alterations in cell viability, size and count, and also in the frequency of leukemic cells undergoing mitotic catastrophe, apoptosis and necrosis, were shown. Different leukemic cell responses to the action of the three oxazaphosphorines were evaluated. These are the first data comparing the in vitro activity of D-17272, D-18864 and D-19575 against human promyelocytic leukemia cells.

Comparative effects of new generation oxazaphosphorines on the size and viability of human acute myeloblastic leukemia cells.

Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864), and beta-D-glucose-isophosphoramide mustard (D-19575, glufosfamide) are three new generation oxazaphosphorine agents. The aim of the present study was to compare the cell response to the action of these three oxazaphosphorines. The experiments were performed in vitro on human acute myeloblastic leukemia ML-1 cells. After exposure of ML-1 cells to the oxazaphosphorines, the size, viability and count of these cells were determined. The research was conducted using the spectrophotometric MTT assay and the electronic Beckman Coulter method. The temporary changes in the ML-1 cell size, viability and count, were dependent on the oxazaphosphorine agent tested, its dose, and the time intervals after its application. Among the three oxazaphosphorine agents, D-18864 proved to be the most cytotoxic, and D-19575 was characterized by the lowest cytotoxicity. The results suggest the possibility of using the electronic sizing and counting method and the MTT assay as a rapid in vitro test for assessing leukemic cell sensitivity to the action of new potential chemotherapeutic agents.

Induction of DNA breakage in U937 cells by oxazaphosphorines.

Oxazaphosphorines are a class of DNA alkylating agents. The aim of the present study was to compare the possible influence of three new generation oxazaphosphorines, D-17272 (mafosfamide cyclohexylamine salt), D-18864 (4-hydro-peroxy-cyclophosphamide), and D-19575 (glufosfamide, beta-D-glucose-isophosphoramide mustard) on DNA damage induction in the human histiocytic lymphoma U937 cells. The flow cytometry APO-BRDU assay, based on the TUNEL method, was used for the in situ detection of DNA strand breaks. After exposure of U937 cells to the oxazaphosphorines, the patterns of temporary changes in the frequency of TUNEL positive U937 cells, expressing DNA breakage, were determined. The effects of the oxazaphosphorines on U937 cells were dependent on the agent tested and its dose, and the time intervals after the drug application. The different potential of D-17272, D-18864 and D-19575 to induce DNA strand breakage in the human histiocytic lymphoma U937 cells was shown.

Investigations on the pharmacokinetics of trofosfamide and its metabolites-first report of 4-hydroxy-trofosfamide kinetics in humans.

Trofosfamide (TRO), like cyclophosphamide (CYCLO) and ifosfamide (IFO), is a prodrug oxazaphosphorine derivative that requires hepatic biotransformation to form the cytotoxically active 4-hydroxy derivative (4-hydroxy-TRO). Individual 4-hydroxyoxazaphosphorines and 4-hydroxy-TRO itself have not been demonstrated in humans up to now. For investigation of the principal pharmacokinetics of TRO and its metabolites, six tumour patients (49-65 years of age, Karnofsky index >70%) with normal liver and renal function were given a single oral dose of 600 mg/m(2) TRO. Plasma was sampled using a bedside technique. Individual 4-hydroxyoxazaphosphorines and TRO together with further metabolites were determined by a specially developed HPLC-UV method and a HPLC-MS method, respectively. With a short apparent half-life (1.2 h) and high apparent clearance (Cl/F 4.0 l/min), TRO was very quickly eliminated from plasma and highly converted to its metabolites, mainly 4-hydroxy-TRO and IFO. In relation to the AUC values of TRO (1.0) the following molar quotients were calculated: 1.59 (4-hydroxy-TRO), 0.40 (4-hydroxy-IFO), 6.90 (IFO) and 0.74 (CYCLO). C(max) values were in the range 10-13 micromol/l for TRO, 4-hydroxy-TRO and IFO and in the range 1.5-4.0 micromol/l for CYCLO, 2- and 3-dechloroethyl-IFO and 4-hydroxy-IFO. Kinetic data indicate that 4-hydroxy-IFO is formed by both hydroxylation of TRO and exocyclic N-dechloroethylation of 4-hydroxy-TRO. 4-hydroxy-CYCLO was not detected above the quantification limit of the method. Only mild haemodepressive side effects were observed after oral administration of 600 mg/m(2) TRO. In relation to known data for IFO, TRO is much more 4-hydroxylated than IFO. The high 4-hydroxy-TRO/TRO ratio found suggests that TRO is a promising tumourstatic agent.