Abstract
Hematopoietic stem cell transplantation patients conditioned with cyclophosphamide (CY) and total body irradiation have substantially greater risk of nonrelapse mortality when plasma area under the concentration-time curve (AUC) of O-carboxyethylcyclophosphoramide mustard (CEPM) is high. The discovery was paradoxical because CEPM is a nontoxic elimination route of the protoxic CY metabolite hydroxycyclophosphamide (HCY). CY was administered to Wistar and TR- rats (a Wistar strain lacking functional ABCC2) at doses of 100 and 200 mg/kg CY, respectively. After either dose, Wistar rats excreted 4-glutathionylcyclophosphamide (GSCY) abundantly in bile; GSCY was absent from bile of TR- rats. Liver AUCGSCY was 2- to 2.5-fold greater in TR- than Wistar rats after 100 and 200 mg/kg CY, respectively. Liver AUCHCY was 24-46% greater in TR- rats than in Wistar rats after the respective CY doses. Plasma AUCCEPM of TR- rats was approximately twice that of Wistar rats after 100 mg/kg, but did not differ between the two strains after 200 mg/kg. Conversely, plasma AUCHCY was not different after 100 mg/kg CY, but was 40% greater in TR- rats after 200 mg/kg. The dose dependence of plasma AUCCEPM and AUCHCY was explained by the concentrations of HCY attained and the in vitro Km of aldehyde dehydrogenase and inhibition of aldehyde dehydrogense in TR- rats. We conclude that GSCY is a substrate of ABCC2, and plasma AUCCEPM functions as a reporter of liver exposure to HCY and toxins formed from it when HCY concentration is below the Km of aldehyde dehydrogenase and the activity is not compromised.
Footnotes
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↵1 Evaluation of the transport of GSCY was limited to in vivo studies by the complexity of the metabolism required to produce GSCY in cells. ABCC2-transfected Madin-Darby canine kidney (a common model for ABCC2 transport) is unable to form HCY from CY and have insufficient GST activity to efficiently form GSCY from HCY. (our unpublished data). Because GSCY is too polar to cross the membrane, it cannot be loaded into cells as such.
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This work is supported in part by National Institutes of Health Grant CA18029, and fellowship support to R.Q. was provided by GlaxoSmithKline.
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DOI: 10.1124/jpet.103.059105.
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ABBREVIATIONS: CY, cyclophsophamide; HSCT, hematopoietic stem cell transplantation; SOS, sinusoidal obstruction syndrome; HCY, 4-hydroxycyclophosphamide; CEPM, O-carboxyethylphosphoramide mustard; GSH, glutathione; SEC, sinusoidal endothelial cell; TBI, total body irradiation; AUC, area under the plasma concentration-time curve; GSCY, 4-glutathionylcyclophosphamide; pNPH, p-nitrophenylhydrazine; ALDH, aldehyde dehydrogenase; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; CDNB, chloro-2,4-nitrobenzene; GST, glutathione S-transferase; ANOVA, analysis of variance.
- Received August 25, 2003.
- Accepted November 11, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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