title: Mechanisms of venoocclusive disease resulting from the combination of cyclophosphamide and roxithromycin.
abstract: BACKGROUND
  High doses (>or=500 mg/m) of cyclophosphamide are known to cause venoocclusive disease (VOD). The authors recently observed a patient treated with immunosuppressive cyclophosphamide doses (100 mg/day) and roxithromycin who developed VOD. Because roxithromycin inhibits cytochrome P450 (CYP) 3A4 and P-glycoprotein, the patient may have been exposed to higher cyclophosphamide and/or cyclophosphamide metabolite concentrations.
  
  
  METHODS
  The effect of roxithromycin on the metabolism and toxicity of cyclophosphamide was studied using human hepatic microsomes and a human endothelial cell line.
  
  
  RESULTS
  Cyclophosphamide or roxithromycin at concentrations from 0.05 to 500 micromol/L were not toxic to endothelial cells as assessed by lactate dehydrogenase (LDH) leakage assay. However, the combination of roxithromycin (500 micromol/L) and cyclophosphamide was toxic for all the tested cyclophosphamide concentrations (0.05 to 500 micromol/L) without clear concentration dependence (LDH ratio 38.3 +/- 11.0 [mean +/- SEM] for the combination with cyclophosphamide 0.05 micromol/L and 50.2 +/- 10.2 for the combination with cyclophosphamide 500 micromol/L; P <or= 0.005 for all tested combinations vs. control). Although roxithromycin did not favor the generation of toxic metabolites from cyclophosphamide, it led to cyclophosphamide accumulation due to inhibition of both CYP3A4 and CYP2B6. Although roxithromycin inhibited P-glycoprotein, this was not the mechanism by which cyclophosphamide toxicity was increased because cyclophosphamide in combination with other P-glycoprotein inhibitors was not toxic to endothelial cells. In the presence of roxithromycin (500 micromol/L), cyclophosphamide (500 micromol/L) induced apoptosis in endothelial cells (34.3 +/- 10.4% apoptotic cells [in % of total cells] for the combination of cyclophosphamide and roxithromycin, 0.7 +/- 0.25% for cyclophosphamide alone, 0% for roxithromycin alone; P < 0.0001) most probably by mitochondrial membrane permeability transition and release of cytochrome c.
  
  
  CONCLUSIONS
  The combination cyclophosphamide and roxithromycin, but not the individual compounds, is toxic to endothelial cells by inducing apoptosis. Inhibition of P-glycoprotein and formation of toxic metabolites are unlikely causes.
authors.affiliation: Division of Clinical Pharmacology and Toxicology, University Hospital Basel, Basel, Switzerland.
authors.forename: Priska
authors.initials: P
authors.lastname: Kaufmann
authors.forename: Manuel
authors.initials: M
authors.lastname: Haschke
authors.forename: Michael
authors.initials: M
authors.lastname: Török
authors.forename: Johannes
authors.initials: J
authors.lastname: Beltinger
authors.forename: Katrijn
authors.initials: K
authors.lastname: Bogman
authors.forename: Markus
authors.initials: M
authors.lastname: Wenk
authors.forename: Luigi
authors.initials: L
authors.lastname: Terracciano
authors.forename: Stephan
authors.initials: S
authors.lastname: Krähenbühl
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D003520:Cyclophosphamide / Q000008:administration & dosage* / Q000009:adverse effects*; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003601:Cytotoxicity Tests, Immunologic; D004359:Drug Therapy, Combination; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006504:Hepatic Veno-Occlusive Disease / Q000139:chemically induced*; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D015575:Roxithromycin / Q000008:administration & dosage* / Q000009:adverse effects*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D065607:Cytochrome P-450 Enzyme Inhibitors; D015575:Roxithromycin; D003520:Cyclophosphamide; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D007654:Ketoconazole