Bone Marrow Small Molecule Radioprotectors.

Abstract Development of small molecule radioprotectors is a major national priority. Two groups of compounds have particular promise. The first group targets the mitochondria based upon previous data with transgene MnSOD which when expressed in the mitochondria prevents apoptosis and increases radioprotection. These agents contain the antioxidant tempol or nitric oxide synthetase inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) attached to a hemi-gramicidin linker which targets the mitochondria. The second group consists of the dietary agent resveratrol and acetylated variants. Mouse hematopoietic progenitor 32Dcl3 cells were incubated for 1 hr in 10 μM tempol, AMT, or gramicidin linked tempol XJB-5-125 (tempol), XJB-7-75 (tempol) or JP4-039 (AMT). In separate experiments, 32Dcl3 cells were incubated for 1 hr in resveratrol or acetylated resveratrol. The cells were then irradiated to doses ranging from 0 to 8 Gy, plated in 0.8% methylcellulose, and incubated in a 5% CO2 incubator for 7 days. Colonies of greater than 50 cells were counted with the data analyzed using linear quadratic or single-hit, multi-target models. 32Dcl3 cells incubated in 10 μm tempol before irradiation resulted in no change in radiation sensitivity while incubation in XJB-5-125 or XJB-7-75 had decreased radiosensitivity. XJB-5-125 had an increased Do of 1.91 ± 0.67 Gy compared to 1.32 ± 0.09 Gy for 32Dcl3 cells incubated in tempol and 1.35 ± 0.27 Gy for control 32Dcl3 cells (p = 0.045 or 0.040, respectively). Incubation in XJB-5-75 resulted in an increased shoulder on the survival curve with an ñ of 19.4 ± 2.6 compared to 8.7 + 1.6 for cells incubated in tempol or 6.9 +1.8 for control 32Dcl3 cells (p = 0.025 or 0.022). Incubation in JP4-039 resulted in an increased Do of 2.2 ± 0.1 Gy compared to 1.24 ± 0.15 or 1.13 ± 0.06 for cells incubated in AMT or control 32Dcl3 cells only, respectively (p = 0.0115 or 0.0098, respectively). Incubation of 32Dcl3 cells in resveratrol or acetylated resveratrol before irradiation resulted in an increased shoulder on the survival curve of 33.2 ± 5.7 or 57.5 ± 9.9, respectively, compared to 6.9 ± 1.8 for 32Dcl3 cells (p = 0.0122 or 0.0072, respectively). These compounds were tested in mice receiving an LD50/30 irradiation dose. C57BL/6NHsd mice were injected intraperitoneally with 10 mg/kg of XJB-5-125, XJB-7-75or JP4-039 or 25 mg/kg of resveratrol or acetylated resveratrol and irradiated 10 mins later along with control mice to 9.5 Gy whole body irradiation. The mice injected with XJB-5-125, XJB-7-75, JP4-039 or acetylated-resveratrol had increased survival compared to control irradiated mice (p ≤ 0.0004). Therefore, four new small molecules have been identified which demonstrate significant radioprotective properties both in vitro and in vivo.