Mechanisms of venoocclusive disease resulting from the combination of cyclophosphamide and roxithromycin

BACKGROUND: High doses (≥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 μmol/L were not toxic to endothelial cells as assessed by lactate dehydrogenase (LDH) leakage assay. However, the combination of roxithromycin (500 μmol/L) and cyclophosphamide was toxic for all the tested cyclophosphamide concentrations (0.05 to 500 μmol/L) without clear concentration dependence (LDH ratio 38.3 ± 11.0 [mean ± SEM] for the combination with cyclophosphamide 0.05 μmol/L and 50.2 ± 10.2 for the combination with cyclophosphamide 500 μmol/L; P ≤ 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 μmol/L), cyclophosphamide (500 μmol/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.