No Evidence for Cell Activation or Vaso-Occlusion with Plerixafor Treatment of Sickle Cell Mice

Introduction: Gene therapy for sickle cell disease (SCD) is currently in active trials. Finding a safe and effective method for collection of hematopoietic stem cells (HSC) in SCD remains a challenge. Granulocyte colony stimulating factor (G-CSF), used most commonly for collecting HSC, can cause life-threatening vaso-occlusion in SCD. Bone marrow harvest requires general anesthesia and multiple punctures. Plerixafor is an inhibitor of the CXCR4 chemokine receptor on HSC and interferes with binding to SDF-1 on bone marrow stroma. As pre-clinical data in support of a clinical trial in SCD patients studying plerixafor mobilization (NCT02193191), we administered plerixafor to SCD mice to assess the risk of cell activation and vaso-occlusion.

Methods: 3-6 month old SS Berkeley (n=12) or SS Townes mice (n= 18) were used. Littermate mice were randomized to subcutaneous treatment with plerixafor (Genzyme-Sanofi) 10 mg/kg once, G-CSF (Amgen) 250 ug/kg daily for 5 days, or equivalent volume (5 uL/g) normal saline daily for 5 days. Peripheral blood was harvested at 1-2 hr (plerixafor) or 4-5 hr (G-CSF and normal saline) after the last dose for the following studies: CBC (Advia), enumeration of HSC mobilization (Lin-Sca-1⁺ c-kit⁺ Flt3^- (LSKF) cells by flow cytometry), neutrophil activation (L-selectin shedding by flow cytometry), and endothelial activation (soluble P-selectin by ELISA). Berkeley mice underwent MRI imaging before and after completion of treatment.

Results: CBC showed the mean WBC and platelet counts of both plerixafor and G-CSF groups to be significantly different from saline, but the WBC differential was only significantly different (in % neutrophils and lymphocytes only) from saline in the G-CSF group (Table 1). The percentages of HSC subsets were significantly higher in both plerixafor and G-CSF groups compared to saline, with no significant differences between plerixafor and G-CSF (Table 2). L-selectin was low and soluble P-selectin high only in the G-CSF group, with both markers significantly different from both plerixafor and saline (Table 3). MRI imaging showed no significant differences in cerebral blood flow (measures oxygen delivery), mean diffusivity (measures vasogenic swelling), or fractional anisotropy (measures axonal integrity) pre- compared to post-treatment in any group. Tables show mean ± SD and significant p-values compared to saline.

Discussion: Plerixafor and G-CSF were effective as evidenced by expected changes in WBC and platelet counts with treatment compared to saline. Both plerixafor and G-CSF significantly mobilized HSC subsets. There was a trend towards higher mobilization with G-CSF of the more primitive LSKF subset, but clinical data indicate that addition of plerixafor to G-CSF mobilizes a higher number of more primitive CD34+CD38- than G-CSF alone (Fruehauf S, Cytotherapy 2009). In support of potential safety of plerixafor in SCD patients, there was no evidence of neutrophil or endothelial activation with plerixafor, in contrast to G-CSF. Despite the evidence of neutrophil and endothelial activation with G-CSF, there was no evidence of perfusion-related organ damage as measured by MRI parameters. These findings suggest that plerixafor canbe safely and effectively used for HSC mobilization from SCD patients for use in gene therapy.

Acknowledgments: We are grateful to Farid Boulad, MD and Tsiporah Shore, MD for providing plerixafor.