Low-dose parenteral busulfan provides an extended window for the infusion of hematopoietic stem cells in murine hosts

Abstract

Objective: Myeloablative total body irradiation (TBI) in the setting of autologous transplantation of genetically modified hematopoietic stem cells (HSC) is associated with substantial toxicity. Nonmyeloablative doses of TBI are less toxic, but result in low-level engraftment of genetically modified HSCs. As an alternative to TBI, escalating doses of parenteral busulfan were tested for their hematologic toxicity, their ability to promote donor leukocyte engraftment, and the time window for such engraftment.

Materials and methods: Hematologic toxicity of busulfan was assessed in C57BL6 mice after single nonmyeloablative doses of intraperitoneal busulfan ranging from 1 to 40 mg/kg by serial complete blood counts monitored up to 40 days. The level of donor engraftment attainable after nonmyeloablative busulfan was determined by infusion of 20 million congenic murine bone marrow nucleated cells (BMNC) following 5 to 40 mg/kg of busulfan. To determine the effects of delayed HSC infusions, BMNCs were infused 1, 10, 15, and 20 days after a single dose of 10 mg/kg of busulfan.

Results: Busulfan doses from 1 to 40 mg/kg produced hematologic toxicity that was most pronounced in the 2nd to 3rd week. In transplantation experiments, dose-dependent donor leukocyte engraftment was attained with levels >70% after only 20 mg/kg of busulfan. Similar levels of engraftment were achieved even when infusion of BMNCs was delayed up to 20 days after busulfan injection.

Conclusion: Nonmyeloablative parenteral busulfan produced transient myelosuppressive effects, clinically relevant levels of engraftment, and an extended time window for HSC infusion in murine hosts.

Figure 1

Total white blood cell (WBC) (A) and platelet (B) counts after one dose of busulfan at indicated doses (mg/kg). Each data point represents an average of five mice in the same dose cohort.

Figure 2

Percent donor leukocyte engraftment at 12 to 13 weeks post-transplantation. Single doses of 5, 10, 20, and 40 mg/kg of busulfan were used to condition the mice 1 day prior to bone marrow nucleated cell infusions. Numbers of animals were 5, 20, 20, and 5, respectively.

Figure 3

Pattern of lymphoid (A) and myeloid (B) engraftment over time. Five, 10, and 20 mg/kg of busulfan were given 1 day prior to bone marrow nucleated cell infusions. There were 5, 20, and 10 mice in each cohort, respectively.

Figure 4

Percent donor leukocyte chimerism at 13 weeks after transplantation; 10 mg/kg of busulfan were administered at 1, 10, 15, and 20 days before infusion of bone marrow nucleated cells. There were 15 mice per each time cohort. Comparing day −1 to other time points, p > 0.05.