A Preclinical Non-Human Primate Model to Test Reversible Myeloablation Following IV Busulfan Therapy without Stem Cell Rescue
Authors: Mahmud Nadim , Khanal Amit , Taioli Simona , Koca Emre , Gaitonde Sujata , Petro Benjamin , Sweiss Karen , Rondelli Damiano 
Published: October 2019

Abstract
Busulfan is a standard drug utilized in stem cell transplantation for myeloid malignancies, however it is not used in any induction or salvage chemotherapy regimen in these diseases. Non-human primates (NHP) represent a useful model for preclinical evaluation of chemotherapy-related toxicity. In order to identify what dose of IV busulfan could be tested in clinical studies for acute myeloid leukemia, we utilized NHP to determine the highest dose of IV busulfan needed to achieve reversible myelosuppression in unperturbed bone marrow (BM). Nine adult baboons (Papio anubis) were divided into three groups (n=3/group) for this study. Group A received the lowest dose of busulfan 6.4 mg/kg (1.6 mg/kg/day x 4 days), Group B received 8 mg/kg (3.2 mg/kg on day 1 and 1.6 mg/kg on day 2-4) and Group C received 9.6 mg/kg (3.2 mg/kg/day day 1-2 and 1.6 mg/kg/day day 3-4). Peripheral blood (PB) complete blood count (CBC), and BM CD34+ cells, colony forming unit (CFU) were monitored over 90 days after busulfan. Maximum suppression of WBC count, hemoglobin (Hb) and platelet counts in the PB of baboons in all 3 groups were observed around day 15. The suppression of WBC was 57 ± 4%, 58 ± 4% and 53 ±10% in group A, B and C respectively (group A: p = 0.006, group B: p = 0.03, group C: p = 0.07). The suppression of absolute neutrophil counts (ANC) in group A, B and C were 85 ± 2%, 8 4± 1% and 82± 5% respectively. Similarly the minimum post-busulfan Hb levels were 10± 0.6 g/dl, 10± 1g/dl, 10± 0.6g/dl in group A, B and C respectively. The suppression of platelet counts were 84 ± 6%, 81 ± 7% and 81 ± 9% in group A, B and C respectively. Taken together, PB CBC results indicate that at day 15, despite administering a higher dose of busulfan the maximum depletion of ANC, Hb and platelet was comparable among the three groups. At day 90, the PB WBC, Hb and platelet counts returned within normal ranges. Notably, in the group receiving the highest dose of busulfan (Group C) the PB WBC counts at day 90 displayed 76 ± 9% recovery and platelets a 79 ± 13% recovery. Extra-hematopoietic toxicity, including weight loss, was not significant in any group of animals. Then we examined CD34+ cells and CFU content of BM prior to and following busulfan therapy. Baboons receiving the lowest dose of busulfan (Group A) displayed 75 ± 19% CD34+ cell suppression, while the intermediate group B had 90 ± 9%, and the group C which received the highest dose of busulfan had 98 ± 0.6 % suppression. The CD34+ cell recovery at day 90 was 56%, 35% and 24% in group A, B and C, respectively, suggesting that the BM CD34+ cell compartment requires more time to recover despite near normal CBC numbers. Similarly, at day 15 the suppression of BM CFU in group A, B and C was 71 ± 10% , 86 ± 8% and 91 ± 5% respectively. Although the suppression in PB WBC count was not distinct the absolute number of CD34+ cells and CFU content of BM varied based on busulfan dose administered. At day 90 the recovery of BM CFU in group A, B & C were 51 ± 15% , 79 ± 15% and 53 ± 9%, respectively. Although there was no significant intergroup difference in degree of CFU plating efficiency, pre-busulfan versus day 90 CFU recovery in group C was statistically significant (p = 0.02) . These results further validate the hierarchy of CD34+ cells and CFU in contrast to more mature PB cells and the cell populations targeted by busulfan, emphasizing regulatory systems governing homeostasis are likely maintained by reserved BM precursors and progenitor cells. Despite near normalization of blood counts, the relatively primitive BM CD34+ cells and CFU content require longer time to return to pre treatment levels. BM biopsy results indicate a return of cellularity and tri-lineage hematopoiesis comparable to pre-treatment levels in all three groups by day 90. Taken together, our study showed that when busulfan is administered in a dose range of 6.4 to 9.6 mg/kg in an NHP model, it is capable of inducing reversible myeloablation with tolerable toxicity without requiring stem cell rescue or blood transfusions. Our results also indicate that about 30 to 40% BM reserve is capable of maintaining normal PB cell counts. Based on the results, we plan to design a phase 1/2 clinical trial where non-myeloablative doses of IV busulfan will be tested as salvage therapy in patients with acute myeloid leukemia.

Disclosures
No relevant conflicts of interest to declare.