mesna and carboxyphosphamide

31P NMR and cell perfusion techniques were used to investigate the conversion of the individual enantiomers of aldophosphamide (AP) to carboxyphosphamide (CBP) as catalyzed by aldehyde dehydrogenase in human erythroleukemia K562 cells. R- and S-cyclophosphamides (CPs) were treated with ozone and hydrogen peroxide to yield Rp- and Sp-cis-4-hydroperoxycyclophosphamides (Rp- and Sp-cis-4-HO2-CP); reduction of each hydroperoxide gave the corresponding enantiomer of AP [along with its tautomer 4-hydroxycyclophosphamide (4-HO-CP)]. In separate experiments, K562 cells embedded in agarose gel threads were perfused at pH 7.4, 21 +/- 1 degrees, with solutions of 1.4 mM Rp- and Sp-4-HO-CP/AP, both with and without added mesna (an acrolein scavenger). A comparison of the 31P NMR spectral data derived from the experiments revealed little statistical difference (+/- 10-20% error limits) in the normalized intensities of the CBP peaks arising from the individual AP enantiomers [with added mesna, the ratio Rp-CBP:Sp-CBP was 1.00:1.24 +/- 0.13 (average deviation); without mesna, the same ratio was 1.00:1.35]. Using conventional methods for evaluating the in vitro drug toxicities, CP-resistant L1210 cells were treated in separate experiments with Rp- and Sp-cis-4-HO2-CP; there were no significant differences between the toxicities exhibited by the stereoisomers.

Topics: Aldehyde Dehydrogenase; Animals; Cyclophosphamide; Humans; Magnetic Resonance Spectroscopy; Mesna; Mice; Oxidation-Reduction; Phosphoramide Mustards; Stereoisomerism; Tumor Cells, Cultured

31P nuclear magnetic resonance (NMR) spectroscopy was used in conjunction with cell perfusion techniques to monitor the intracellular chemistry of the cyclophosphamide (CP, CAS 6055-19-2) metabolites 4-hydroxy-cyclophosphamide (4-HO-CP) and aldophosphamide (AP) in U937 human histiocytic (CP-sensitive) and K562 human erythroleukemia (CP-resistant) cells. Similar experiments were carried out using the ifosfamide (IF, CAS3778-73-2) metabolites 4-hydroxyifosfamide (4-HO-IF) and aldoifosfamide (AIF). The hydroxy and aldehydic metabolites were generated by the triphenylphosphine reduction of 4-hydroperoxycyclophosphamide (4-HO2-CP) or 4-hydroperoxyifosfamide (4-HO2-IF) or by a spontaneous elimination/addition reaction involving water and 4-thiocyclophosphamide analogs 4-(2-hydroxyethyl) thiocyclophosphamide (4-ESCP) or mafosfamide. Cell death resulting from 4-HO-CP/AP perfusions was mimicked by perfusion with acrolein or an acrolein producing but non-alkylating, dechloro-CP analog. Acrolein toxicity was minimized by the presence of 2-mercaptoethanol or mesna (sodium 2-mercaptoethanesulfonate) in perfusion solutions as well as by fractional dose drug perfusions (sequential 2.5-3.0 h perfusions separated by cell washes with drug-free medium). The intracellular half-life for phosphoramide mustard (PM) at an intracellular pH value of 7.1 +/- 0.1 and an ambient probe temperature of 23 +/- 1 degree C in U937 cells was 2.1 h [k = (5.4 +/- 0.3) x 10(-3) min-1] and in K562 cells was 3.1 h [k = (3.7 +/- 0.4) x 10(-3) min-1]. Similar half-lives (2-4 h) were determined for intracellular isophosphoramide mustard (IPM). Fractional dose perfusion of U937 or K562 cells with 1.5 mmol/l 4-HO-CP/AP (generated from 4-HO2-CP) and 0.3 mmol/l mesna allowed for the observation of intracellular carboxyphosphamide (CBP); CBP was formed in higher concentrations in the CP-resistant K562 cells. Similar results were obtained using 4-ESCP and mafosfamide as sources of 4-HO-CP/AP. Identification of CBP was based on chemical shift, chemical stability, and membrane permeability studies of synthetic CBP. Concentrations of carboxyifosfamide (CBIF) formed in K562 cells were also greater than that in U937 cells.

Topics: Acrolein; Cell Death; Cyclophosphamide; Free Radical Scavengers; Half-Life; Humans; Ifosfamide; Magnetic Resonance Spectroscopy; Mesna; Perfusion; Phosphoramide Mustards; Phosphorus Isotopes; Sulfhydryl Compounds; Tumor Cells, Cultured