Differential Toxicity Profile of Busulfan and Treosulfan on Endothelial Cells - Relevance to Hematopoietic Stem Cell Transplantation

Abstract

The success of Hematopoietic stem cell transplantation (HSCT), an established curative treatment option for hematological malignancies and non-malignant diseases, is often limited by regimen related toxicity (RRT) caused by conditioning regimen drugs. Despite their intended marrow toxicity, the conditioning drugs also potentially damage endothelium of different organs leading to RRT such as sinusoidal obstruction syndrome. Among different conditioning drugs, busulfan (Bu) and treosulfan (Treo) (a structural analog of Bu), although widely used in HSCT, exhibit differences in their toxicity profile, the mechanism of which is still unclear. Here we compared the effect of Bu and Treo in mice model to identify factors causing the differences in toxicity. BALB/c mice (6-8 weeks old) were injected with myeloablative doses of either Bu (4 × 25 mg/Kg) or Treo (3 × 1000 mg/kg) intraperitoneally. Groups of animals were euthanized on day +1 & day +7 post drug administration. WBCs, CD31+ bone marrow endothelial cells (BMECs), CD146+ Liver sinusoidal endothelial cells (LSECs) were enumerated by flow cytometry. Liver, spleen and bone marrow sections were evaluated by histology and immunofluorescence. Mice treated with Bu showed decrease in bone marrow WBCs on day +1 that persisted till day +7. Treo showed moderate decrease in WBC compared to untreated controls on day +1, the WBCs recovered on day +7. Hematoxylin & eosin (H&E) staining of bone marrow sections showed loss of bone marrow cellularity in Bu & Treo treated mice on day +1, while on day +7, mice treated with Treo alone resulted in increased marrow cellularity indicating probable recovery. BMECs were decreased by Bu on day +1 and day +7 while Treo slowly decreased BMECs from day +1 to day +7. Similarly, LSECs were highly damaged by Bu treatment on day +1 & day +7 while Treo resulted in relatively less damage on both day +1 & day +7. This was confirmed by immunofluorescence staining of liver sections with LSEC specific SE-1 antibody. Further, CD31, a marker for capillarization of LSECs were observed more in Bu compared to Treo treatment as evidenced by immunofluorescence. Treo also upregulated angiogenesis related genes [BMX, EGFL7, MMRN2, LYL1] in liver possibly explaining lesser damage. Histology of liver & spleen corroborated the findings of severe damage mediated by Bu than Treo. Bu treatment resulted in decreased bone marrow cellularity, increased damage to BMECs and capillarization of LSECs while Treo resulted in moderate decrease in bone marrow cellularity, lesser damage to BMECs & LSECs. These results indicate that although Bu & Treo had profound myeloablative effect, their toxicity profile on endothelial cells differs. Further studies are warranted to elucidate the mechanism of differential endothelial toxicity and probable hematopoietic recovery by Treo treatment.