Impact of Abbreviated Filgrastim Schedule on Survival and Hematopoietic Recovery after Irradiation in Four Mouse Strains with Different Radiosensitivity

Abstract

Filgrastim (Neupogen^®, granulocyte-colony stimulating factor) is among the few countermeasures recommended for management of patients in the event of lethal total-body irradiation. Despite the plethora of studies using filgrastim as a radiation countermeasure, relatively little is known about the optimal dose schedule of filgrastim to mitigate radiation lethality. We evaluated the efficacy of filgrastim in improving 30-day survival of CD2F1 mice irradiated with a lethal dose (LD_70/30) in the AFRRI cobalt-60 facility. We tested different schedules of 1, 3, 5, 10 or 16 once-daily injections of filgrastim initiated one day after irradiation. Time optimization studies with filgrastim treatment were also performed, beginning 6-48 h postirradiation. Maximum survival was observed with 3 daily doses of 0.17 mg/kg filgrastim. Survival efficacy of the 3-day treatment was compared against the conventional 16-day filgrastim treatment after irradiation in four mouse strains with varying radiation sensitivities: C3H/HeN, C57BL/6, B6C3F1 and CD2F1. Blood indices, bone marrow histopathology and colony forming unit assays were also evaluated. Filgrastim significantly increased 30-day survival (P < 0.001) with a 3-day treatment compared to 16-day treatment. Filgrastim did not prevent cytopenia nadirs, but facilitated faster recovery of white blood cells, neutrophils, red blood cells, platelets, lymphocytes and hematocrits in all four strains. Accelerated hematopoietic recovery was also reflected in faster bone marrow reconstitution and significant increase in hematopoietic progenitors (P < 0.001) in all four mouse strains. These data indicate that prompt and abbreviated filgrastim treatment has potential benefit for triage in the event of a radiological incident for treating acute hematopoietic syndrome.

FIG. 1

Survival of CD2F1 mice after 9.25 Gy TBI and therapy with different schedules of filgrastim (G-CSF). Panel A: A single dose of filgrastim [0.17–0.68 mg/kg, 1 day after TBI (single arrow)] compared to 5% dextrose. Panel B: Three daily doses (days 1–3) postirradiation (indicated by arrows), of 0.17 or 0.34 mg/kg filgrastim compared to 5% dextrose. Panel C: Five doses (cross-hatch) of either 5% dextrose or filgrastim, 0.17 or 0.34 mg/kg. Panel D: Ten doses (dashed bar) of either 5% dextrose or filgrastim, 0.12 or 0.17 mg/kg. Kaplan-Meier survival curves were plotted using GraphPad software; n = 24 mice/group and trend in survival is compared between vehicle and drug-treated groups.

FIG. 2

Optimization of filgrastim (G-CSF) dose to improve survival of lethally irradiated mice. Panel A. Effect of increasing filgrastim dose administered s.c. on days 1, 2 and 3 after 9.25 Gy TBI on 30-day mouse survival (n = 24/group). Three doses of 0.17 mg/kg filgrastim showed optimal mitigation, followed by 0.34 mg/kg and 0.51 mg/kg, while survival in the 0.68 mg/kg filgrastim-treated group was similar to the 5% dextrose-treated group (control). Panel B. Optimization of time of filgrastim administration in relationship to irradiation. Mice were irradiated and injected with 3 daily doses of either 0.17 or 0.34 mg/kg filgrastim beginning at 6 and 12 h, days 1, 2 or 3 after TBI. *P < 0.001 for 30-day survival of 0.17 mg/kg filgrastim-treated group compared to survival of 0.34 mg/kg group.

FIG. 3

Comparison of the radiation sensitivities of four mouse strains exposed to different radiation doses. All mice were irradiated on the same day. There were 16 mice per group per dose per strain. Probit survival curves were generated for each strain exposed to LD_5/30 to LD_95/30 TBI. Mortality at 30 days postirradiation is plotted against dose for C3H/HeN mice (irradiated with 6–8.5 Gy), C57BL/6 mice (7–9 Gy), B6C3F1 mice (7.5–9 Gy) and CD2F1 mice (8.5–10.5 Gy).

FIG. 4

Mitigation of radiation lethality in four different mouse strains with 3 vs. 16 doses of filgrastim starting 24 h postirradiation. Male mice (12–14 weeks old) of each strain were exposed to LD_70/30 TBI and treated with either 5% dextrose (3 or 16 doses) or filgrastim (0.17 mg/kg on days 1, 2 and 3 or 0.12 mg/kg filgrastim at days 1–16 postirradiation). Panel A: 8 Gy irradiated C3H/HeN mice. Panel B: 8.2 Gy irradiated C57BL/6 mice. Panel C: 8.5 Gy irradiated B6C3F1 mice. Panel D: 9.25 Gy irradiated CD2F1 mice. Log-rank test, P < 0.0001–0.009 (n = 24 mice per group).

FIG. 5

Photomicrographs show regeneration in representative sections of sternal bone marrow after filgrastim treatment in C3H/HeN, C57BL/6, B6C3F1 and CD2F1 mice exposed to a sublethal dose of radiation. Panels A–D: Nonirradiated mice injected with 3 doses of 5% dextrose. Panels E–H: Irradiated mice 14 days after exposure injected with 3 doses of 5% dextrose (vehicle) on days 1, 2 and 3 after TBI, showing bone marrow atrophy, infiltration by adipocytes and rare megakaryocytes. Panels I–L: Mice at 14 days after TBI, s.c. injected with 0.17 mg/kg filgrastim on days 1, 2 and 3 postirradiation, show increased cellularity and myeloid cells. All sections were routinely H&E stained (200× magnification).

FIG. 6

Filgrastim accelerates hematopoietic progenitor recovery after a nonlethal radiation dose in all four mouse strains. CD2F1, C57BL/6, C3H/HeN and B6C3F1 mice (n = 6 per group) were irradiated with a sublethal dose and treated with filgrastim on days 1, 2 and 3 after TBI. Filgrastim treatment after irradiation enhanced survival of bone marrow hematopoietic stem and progenitor cells. Clonogenic potential of bone marrow cells was assessed by a CFU assay on days 4, 7, 10 and 14 postirradiation. Cells from two femurs were pooled, counted and each sample plated in triplicate to be scored 12 days after plating. Data are expressed as mean ± SEM for day 14 only, since earlier time points did not yield statistically quantifiable data. Statistical significance was determined between irradiated control and filgrastim-treated groups as described. *P < 0.001 compared to irradiated control. When compared with the vehicle-treated group, filgrastim-treated animals showed significant increase (P ≤ 0.002) in BFU-E and CFU-GEMM on day 14 in all four mouse strains. Similarly, for CFU-GM, significant increase was obtained (P < 0.05–0.00) on day 14 after TBI in all strains. However, significant CFU-E increase (P < 0.0001) was observed only in B6C3F1 mice on day 14 postirradiation.