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 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.