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the effect of VPA on irradiated cells, hippocampal neurons were treated with 0.6 mM VPA for 7 days prior
to irradiation. Radioprotection, as measured by clonogenic survival, was observed in VPA-treated
neuronal cells compared to cells treated with radiation alone. In contrast, VPA acted as a radiosensitizer
in GL261, D54 and Daoy brain cancer cells. To elucidate the mechanism of this selective increased survival
hippocampal neurons we monitored for apoptosis by Annexin V-APC or DAPI staining. Irradiated
hippocampal neurons pretreated with VPA demonstrated a twofold decrease in apoptosis than irradiated
cells. Similar results were obtained using irradiated mice. Seven-day old C57BL6 pups were treated with
VPA (300mg/Kg) or PBS for 7 days and were irradiated with the single dose of 7 Gy. VPA pretreatment
protected hippocampal neurons from radiation-induced apoptosis as demonstrated by TUNEL staining.
Cell cycle analysis showed that VPA induced cell cycle arrest in cancer cells (GL261), but not in normal HT-
22 cells. Immunoblot analysis showed that VPA inhibited histone deacetylase (HDAC) and glycogen
synthase kinase-3β (GSK-3β) in hippocampal neurons. Furthermore, the level of Bax was reduced and Bcl-
2 was increased in VPA treated cells. The GL261 intracranial glioma model was used to evaluate tumor
growth using MRI and measure survival following treatment with VPA and radiation. VPA, in combination
with radiation, significantly reduced tumor growth and improved mouse survival. In conclusion, VPA
protects hippocampal neurons, but not cancer cell lines, from radiation-induced cytotoxicity in vitro and
in vivo. Inhibition of the enzymatic activity of both HDAC and GSK-3β appear to be involved. VPA could be
a useful in attenuating neurocognitive deficits resulting from cranial irradiation and also to enhance the
efficiency and survival outcomes of glioma radiotherapy.
(PS2-84) Dose optimization study of AEOL 10150 in mitigation of radiation induced lung injury in CBA/J
mice. Francis N. Murigi; Chiwei Hung; Shabnam Salimi; Wilfried Goetz; Carolyn M.S. Buck; Radmila
Pavlovic; Isabel L. Jackson; and Zeljko Vujaskovic, University of Maryland School of Medicine, Baltimore,
MD
Objective: AEOL 10150 is a catalytic metalloporphyrin antioxidant being developed by Aeolus
Pharmaceuticals, Inc as a countermeasure for pulmonary radiation injury. The goal of the study was to
determine the most effective AEOL 10150 dose in reducing functional lung damage and improving 180
day survival in CBA/J mice after whole thorax lung irradiation (WTLI). Methods: Mice received 14.6 Gy of
X-irradiation in a single fraction delivered to the whole thorax at a dose rate of 125.42 cGy min-1 (320
kVp, 12mA, Filter = 2.00 mm Al, HVL ~ 1 mm Cu). Mice were randomized into the following groups: WTLI
alone, WTLI + saline, or WTLI + 5, 10, 25, or 40 mg/kg/day of AEOL 10150. Sham-irradiated mice were
included as controls. AEOL 10150 or saline was delivered by subcutaneous injection starting 24 hours post
WTLI and continued daily for 28 days. Animals were monitored daily for morbidity/mortality for up to 180
days post-WTLI. Lung function and body weights were assessed bi-weekly. Lung weights (a marker of
pulmonary edema) were determined at the time of necropsy. Histopathology (Masson’s trichrome for
fibrosis) was performed to assess lung damage. Results: Survival data demonstrated AEOL 10150 at a
dose of 25 mg/kg/day was the most effective in improving survival by >30% compared to WTLI alone.
AEOL 10150 at 25 mg/kg/day shifted the response 30% from an LD90/180 to an LD60/180. 40 mg/kg/day
was less effective, improving survival by 20% vs. WTLI alone. AEOL 10150 at 25 mg/kg/day significantly
decreased respiratory distress between 14 and 18 weeks post WTLI. Pulmonary edema / congestion were
also significantly reduced at the time of necropsy among mice treated with 25 or 40 mg/kg AEOL 10150
compared to WTLI alone (p < 0.02). The lungs of AEOL 10150 (25 and 40 mg/kg/day) treated mice
displayed a reduction in alveolar walls thickness, congestion and inflammation. Doses of 5 and 10
183 | P a g e
to irradiation. Radioprotection, as measured by clonogenic survival, was observed in VPA-treated
neuronal cells compared to cells treated with radiation alone. In contrast, VPA acted as a radiosensitizer
in GL261, D54 and Daoy brain cancer cells. To elucidate the mechanism of this selective increased survival
hippocampal neurons we monitored for apoptosis by Annexin V-APC or DAPI staining. Irradiated
hippocampal neurons pretreated with VPA demonstrated a twofold decrease in apoptosis than irradiated
cells. Similar results were obtained using irradiated mice. Seven-day old C57BL6 pups were treated with
VPA (300mg/Kg) or PBS for 7 days and were irradiated with the single dose of 7 Gy. VPA pretreatment
protected hippocampal neurons from radiation-induced apoptosis as demonstrated by TUNEL staining.
Cell cycle analysis showed that VPA induced cell cycle arrest in cancer cells (GL261), but not in normal HT-
22 cells. Immunoblot analysis showed that VPA inhibited histone deacetylase (HDAC) and glycogen
synthase kinase-3β (GSK-3β) in hippocampal neurons. Furthermore, the level of Bax was reduced and Bcl-
2 was increased in VPA treated cells. The GL261 intracranial glioma model was used to evaluate tumor
growth using MRI and measure survival following treatment with VPA and radiation. VPA, in combination
with radiation, significantly reduced tumor growth and improved mouse survival. In conclusion, VPA
protects hippocampal neurons, but not cancer cell lines, from radiation-induced cytotoxicity in vitro and
in vivo. Inhibition of the enzymatic activity of both HDAC and GSK-3β appear to be involved. VPA could be
a useful in attenuating neurocognitive deficits resulting from cranial irradiation and also to enhance the
efficiency and survival outcomes of glioma radiotherapy.
(PS2-84) Dose optimization study of AEOL 10150 in mitigation of radiation induced lung injury in CBA/J
mice. Francis N. Murigi; Chiwei Hung; Shabnam Salimi; Wilfried Goetz; Carolyn M.S. Buck; Radmila
Pavlovic; Isabel L. Jackson; and Zeljko Vujaskovic, University of Maryland School of Medicine, Baltimore,
MD
Objective: AEOL 10150 is a catalytic metalloporphyrin antioxidant being developed by Aeolus
Pharmaceuticals, Inc as a countermeasure for pulmonary radiation injury. The goal of the study was to
determine the most effective AEOL 10150 dose in reducing functional lung damage and improving 180
day survival in CBA/J mice after whole thorax lung irradiation (WTLI). Methods: Mice received 14.6 Gy of
X-irradiation in a single fraction delivered to the whole thorax at a dose rate of 125.42 cGy min-1 (320
kVp, 12mA, Filter = 2.00 mm Al, HVL ~ 1 mm Cu). Mice were randomized into the following groups: WTLI
alone, WTLI + saline, or WTLI + 5, 10, 25, or 40 mg/kg/day of AEOL 10150. Sham-irradiated mice were
included as controls. AEOL 10150 or saline was delivered by subcutaneous injection starting 24 hours post
WTLI and continued daily for 28 days. Animals were monitored daily for morbidity/mortality for up to 180
days post-WTLI. Lung function and body weights were assessed bi-weekly. Lung weights (a marker of
pulmonary edema) were determined at the time of necropsy. Histopathology (Masson’s trichrome for
fibrosis) was performed to assess lung damage. Results: Survival data demonstrated AEOL 10150 at a
dose of 25 mg/kg/day was the most effective in improving survival by >30% compared to WTLI alone.
AEOL 10150 at 25 mg/kg/day shifted the response 30% from an LD90/180 to an LD60/180. 40 mg/kg/day
was less effective, improving survival by 20% vs. WTLI alone. AEOL 10150 at 25 mg/kg/day significantly
decreased respiratory distress between 14 and 18 weeks post WTLI. Pulmonary edema / congestion were
also significantly reduced at the time of necropsy among mice treated with 25 or 40 mg/kg AEOL 10150
compared to WTLI alone (p < 0.02). The lungs of AEOL 10150 (25 and 40 mg/kg/day) treated mice
displayed a reduction in alveolar walls thickness, congestion and inflammation. Doses of 5 and 10
183 | P a g e
