Hematologic safety of chronic brain-penetrating erythropoietin dosing in APP/PS1 mice

INTRODUCTION: Low blood-brain barrier (BBB) penetration and hematopoietic side effects limit the therapeutic development of erythropoietin (EPO) for Alzheimer's disease (AD). A fusion protein of EPO and a chimeric monoclonal antibody targeting the mouse transferrin receptor (cTfRMAb) has been engineered. The latter drives EPO into the brain via receptor-mediated transcytosis across the BBB and increases its peripheral clearance to reduce hematopoietic side effects of EPO. Our previous work shows the protective effects of this BBB-penetrating EPO in AD mice but hematologic effects have not been studied. Herein, we investigate the hematologic safety and therapeutic effects of chronic cTfRMAb-EPO dosing, in comparison to recombinant human EPO (rhu-EPO), in AD mice. METHODS: Male APPswe PSEN1dE9 (APP/PS1) mice (9.5 months) were treated with saline (n = 11), and equimolar doses of cTfRMAb-EPO (3 mg/kg, n = 7), or rhu-EPO (0.6 mg/kg, n = 9) 2 days/week subcutaneously for 6 weeks, compared to saline-treated wild-type mice (n = 10). At 6 weeks, exploration and memory were assessed, and mice were sacrificed at 8 weeks. Spleens were weighed, and brains were evaluated for amyloid beta (Aβ) load and synaptophysin. Blood was collected at 4, 6 and 8 weeks for a complete blood count and white blood cells differential. RESULTS: cTfRMAb-EPO transiently increased reticulocyte counts after 4 weeks, followed by normalization of reticulocytes at 6 and 8 weeks. rhu-EPO transiently increased red blood cell count, hemoglobin and hematocrit, and significantly decreased mean corpuscular volume and reticulocytes at 4 weeks, which remained low at 6 weeks. At 8 weeks, a significant decline in red blood cell indices was observed with rhu-EPO treatment. Exploration and cognitive deficits were significantly worse in APP/PS1-rhu-EPO mice. Both cTfRMAb-EPO and rhu-EPO decreased 6E10-positive brain Aβ load; however, cTfRMAb-EPO and not rhu-EPO selectively reduced brain Aβ(1-42) and elevated synaptophysin expression. DISCUSSION: Chronic treatment with cTfRMAb-EPO results in better hematologic safety, behavioral, and therapeutic indices compared with rhu-EPO, supporting the development of this BBB-penetrable EPO analog for AD.