Abstract

5-AED (5-androstene-3β,17β-diol) is a naturally occurring adrenal cortical steroid, which displays radioprotective effects in both rodents and non-human primates, resulting in accelerated multilineage hematopoiesis and enhanced survival after total body irradiation (TBI), including a 1-log accelerated CD34+ cell reconstitution in bone marrow of non-human primates. Pegylated granulocyte-colony stimulating factor (Peg-G-CSF) is known to stimulate lineage-specific recovery of neutrophils, whereas the effects of thrombopoietin (TPO) are broader and include protection of short-term spleen repopulating immature cells as well as platelet recovery. To gain insight into the mechanism of 5-AED on immature hematopoietic cells, the effects of 5-AED on multilineage hematopoiesis and recovery of specific repopulating stem and progenitor cell subsets after TBI was evaluated in combination with and relative to either Peg-G-CSF or TPO. For direct measurements of the radioprotective effect of 5-AED, BALB/c mice were exposed to a midlethal dose of 6 Gy TBI. Two hours after TBI, mice were injected IM with 40 mg/kg 5-AED or the carrier, with or without 0.225 mg TPO or 10 mg Peg-G-CSF IP. Radioprotective effects of 5-AED on immature repopulating cell subsets were assessed by exposing BALB/c donor mice to 3 fractions of 2 Gy TBI, separated by 24 hours, and treatment with 40 mg/kg/d 5-AED or the carrier IM, or 0.7 mg TPO IP after each fraction or a single injection of 10 mg Peg-G-CSF IP after the first fraction. Twenty four hours after the last fraction, bone marrow of donor mice was examined for immature cell content per femur using the marrow repopulating ability (MRA day 13) assay and the CFU-S day 12 after transplantation in 8 Gy irradiated mice. After 6 Gy TBI, BALB/c mice treated with 5-AED displayed an accelerated multilineage recovery with increased white blood cells (p<0.001), blood platelets (p<0.0001) and red blood cells (p<0.03), as well as increased bone marrow cellularity (p<0.0001) and elevated numbers of bone marrow colony forming cells (p<0.00001) at 14 days post-TBI in comparison to placebo-treated animals. Increasing the 5-AED dose up to 200 mg/kg did not augment this effect. Combined treatment with 5-AED and Peg-G-CSF or TPO treatment did not result in an additive effect in this setting. However, after the fractionated 3x2 Gy, a 5- and 7- fold increase in CFU-S relative to radiation controls was observed in the 5-AED and TPO groups, respectively, and a synergistic 20-fold increase in CFU-S day 12 was observed when 5-AED and TPO were used simultaneously. Consistent with earlier observations, Peg-GCSF alone did not affect CFU-S day 12 and appeared to dampen the effect of 5-AED. MRA, expressed as GM-CFU per femur at 13 days after transplantation, was found to be increased 5- to 6-fold with 1002 colonies (range 0-5785) for 5-AED versus 174 (5-360) for radiation controls. This is in contrast to TPO, which promotes CFU-S reconstitution at the expense of the more immature MRA (

Neelis et al.
1998
:
Blood
92
,
1586
). Thus, 5-AED as a single agent stimulates multilineage hematopoiesis and increases bone marrow cellularity following TBI. This effect is mediated by increased survival and/or reconstitution of immature repopulating cells in a pattern distinct from that of TPO. Consistently, 5-AED strongly synergizes with TPO at the level of immature cells from which reconstitution originates, thus revealing a novel mechanism of bone marrow protection in cytoreductive therapy.

Disclosures: Frincke:Hollis-Eden Pharmaceuticals: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Stickney:Hollis-Eden Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Reading:Hollis-Eden Pharmaceuticals Inc: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

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