Impaired Osseous Wound Healing Following Ionizing Irradiation Is Ameliorated by Mitochondrial Targeted Nitroxide JP4-039.
Authors: Rwigema Jean-Claude , Gokhale Abhay , Glowacki Julie , Dixon Tracy , Patrene Ken , Roodman G. David , Wipf Peter , Greenberger Joel 
Published: October 2019

Abstract
Abstract 4576
In the case of a nuclear explosion, combined injuries may include ionizing irradiation and bone fracture. We evaluated the potential bone wound healing stimulation capacity of a new mitochondrial targeted nitroxide - JP4-039 - that was developed and demonstrated to protect against total body irradiation. JP4-039 was synthesized by coupling Tempol (4-hydroxy-2,2,6,6-tertamethylpiperidine-N-oxyl) and a novel alkene isotere analog of a gramicidin S (GS) fragment. JP4-039 is now being tested to determine whether it can also improve osseous wound healing. A model for osseous wound healing was used in the right hind leg of C57BL/6NHsd female mice and irradiated in doses ranging from 0 to 30 Gy, then holes drilled in both tibias 24 hr later. The mice were sacrificed at 7, 14, 21, 28 or 35 days. Tibial wounding and x-rays were used for determination of wound size. The bones were fixed, sectioned, and examined for osseous wound repair. A significant irradiation- induced delay in wound repair (hole diameter) was detected at 21 days in both the 20 Gy (0.93 ± 0.11 mm, p=0.008) and 30 Gy (1.1 ± 0.1 mm, p=0.0002) groups, compared with the 0 Gy group (0.67 ± 0.23 mm). At 28 days, there were statistically significant increased hole diameters between each irradiation groups (10 Gy, 0.76 ± 0.08 mm, p=0.003; 20 Gy, 0.87 ± 0.36 mm, p=0.04; 30 Gy, 0.76 ± 0.22 mm, p=0.02) and the 0 Gy group (0.32 ± 0.32 mm). By day 35, the wounds in the 0 Gy group were healed (0.08 ± 0.17 mm), while wounds in the 20 Gy group (0.22 ± 0.24 mm, p=0.02) and 30 Gy group (0.34 ± 0.23 mm, p=0.001) remained significantly larger. To determine whether JP4-039 improved the time of osseous wound healing, mice were injected intraperitoneally with JP4-039 (10 mg/kg) 10 min before drilling holes in both rear legs. Mice injected with JP4-039 before drilling showed significant improvement in osseous wound healing in 14 days and 21 days after drilling (0.55 ± 0.3 and 0.47 ± 0.04 mm, respectively), compared with 0.84 ± 0.05 and 0.64 ± 0.05 mm, respectively for control mice (p < 0.05 according to Tukey's multiple comparison statistical test). To test whether JP4-039 can improve osseous wound healing in irradiated limbs, mice were injected with JP4-039 (10 mg/kg) 10 min before 20 Gy to the right hind leg. Wounds were made in both tibae 24 hr after irradiation. At 21 and 28 days after wounding, bones from JP4-039 treated-irradiated mice displayed improved healing with holes of 0.51 ± 0.05 and 0.37 ± 0.04 mm, respectively, compared with 1.01 ± 0.10 and 0.76 ± 0.13 mm, respectively, for control irradiated bones. This is significant at < 0.05 as calculated using a Tukey's multiple comparison statistical test. Histopathological evaluation of specimens from mice pre-treated with JP4-039 prior to irradiation confirmed radiographic evidence of improved healing. Thus, JP4-039 enhances bone repair and is effective in a combined irradiation injury setting.
Supported by NIAID/NIH grant U19AI068021 and BARDA Contract HHSO10200800062C.

Disclosures:
Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy.