A review of radiation countermeasure work ongoing at the Armed Forces Radiobiology Research Institute.

PURPOSE: The hazard of exposure to ionizing radiation is a serious public and military health concern that has justified substantial efforts to develop medically effective radiation countermeasure approaches, including radiation protectors, mitigators, and therapeutics. Although such efforts were initiated more than half a century ago, no safe and effective radiation countermeasure has been approved by the United States Food and Drug Administration (FDA) for the acute radiation syndrome. This situation has prompted intensified research among government laboratories, academic institutions, and pharmaceutical companies to identify a new generation of countermeasures. In this communication we discuss selected promising radiation countermeasures at advanced stages of development. CONCLUSION: Other than granulocyte colony-stimulating factor, which has an Emergency Use Investigational New Drug (IND) status, four countermeasures have FDA IND status and other promising countermeasures are in development. Here we review primarily the in vivo efficacy of selected countermeasures in animal models and clinical studies.

CBLB613: a TLR 2/6 agonist, natural lipopeptide of Mycoplasma arginini , as a novel radiation countermeasure.

To date, there are no safe and effective drugs available for protection against ionizing radiation damage. Therefore, a great need exists to identify and develop non-toxic agents that will be useful as radioprotectors or postirradiation therapies under a variety of operational scenarios. We have developed a new pharmacological agent, CBLB613 (a naturally occurring Mycoplasma-derived lipopeptide ligand for Toll-like receptor 2/6), as a novel radiation countermeasure. Using CD2F1 mice, we investigated CBLB613 for toxicity, immunogenicity, radioprotection, radiomitigation and pharmacokinetics. We also evaluated CBLB613 for its effects on cytokine induction and radiation-induced cytopenia in unirradiated and irradiated mice. The no-observable-adverse-effect level of CBLB613 was 1.79 mg/kg and 1 mg/kg for single and repeated doses, respectively. CBLB613 significantly protected mice against a lethal dose of (60)Co γ radiation. The dose reduction factor of CBLB613 as a radioprotector was 1.25. CBLB613 also mitigated the effects of (60)Co γ radiation on survival in mice. In both irradiated and unirradiated mice, the drug stimulated induction of interleukin-1ß (IL-1ß), IL-6, IL-10, IL-12, keratinocyte-derived chemokine, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-1α. CBLB613 also reduced radiation-induced cytopenia and increased bone marrow cellularity in irradiated mice. Our immunogenicity study demonstrated that CBLB613 is not immunogenic in mice, indicating that it could be developed as a radioprotector and radiomitigator for humans against the potentially lethal effects of radiation exposure.

Radioprotective efficacy of tocopherol succinate is mediated through granulocyte-colony stimulating factor.

The purpose of this study was to elucidate the role of granulocyte colony-stimulating factor (G-CSF) induced by α-tocopherol succinate (TS) in protecting mice from total-body irradiation. CD2F1 mice were injected with a radioprotective dose of TS and the levels of cytokine in serum induced by TS were determined by multiplex Luminex. Neutralization of G-CSF was accomplished by administration of a G-CSF antibody and confirmed by cytokine analysis. The role of G-CSF on gastrointestinal tissue protection afforded by TS after irradiation (11 Gy, 0.6 Gy/min of 60Co γ-radiation) was determined by analysis of jejunum histopathology for crypt, villi, mitotic figures, apoptosis, and cell proliferation. Our results demonstrate that TS protected mice against high doses of radiation-induced gastrointestinal damage and TS also induced very high levels of G-CSF and keratinocyte-derived chemokine (KC) production in peripheral blood 24 h after subcutaneous administration. When TS-injected mice were administered a neutralizing antibody to G-CSF, there was complete neutralization of G-CSF in circulating blood, and the protective effect of TS was significantly abrogated by G-CSF antibody. Histopathology of jejunum from TS-injected and irradiated mice demonstrated protection of gastrointestinal tissue, yet the protection was abrogated by administration of a G-CSF antibody. In conclusion, our current study suggests that induction of G-CSF resulting from TS administration is responsible for protection from 60Co γ-radiation injury.

Tocopherol succinate: modulation of antioxidant enzymes and oncogene expression, and hematopoietic recovery.

PURPOSE: A class of naturally occurring isoforms of tocopherol (tocols) was shown to have varying degrees of protection when administered before radiation exposure. We recently demonstrated that α-tocopherol succinate (TS) is a potential radiation prophylactic agent. Our objective in this study was to further investigate the mechanism of action of TS in mice exposed to (60)Co γ-radiation. METHODS AND MATERIALS: We evaluated the effects of TS on expression of antioxidant enzymes and oncogenes by quantitative RT-PCR in bone marrow cells of (60)Co γ-irradiated mice. Further, we tested the ability of TS to rescue and repopulate hematopoietic stem cells by analyzing bone marrow cellularity and spleen colony forming unit in spleen of TS-injected and irradiated mice. RESULTS: Our results demonstrate that TS modulated the expression of antioxidant enzymes and inhibited expression of oncogenes in irradiated mice at different time points. TS also increased colony forming unit-spleen numbers and bone marrow cellularity in irradiated mice. CONCLUSIONS: Results provide additional support for the observed radioprotective efficacy of TS and insight into mechanisms.

Alpha-tocopherol succinate protects mice from gamma-radiation by induction of granulocyte-colony stimulating factor.

PURPOSE: The purpose of this study was to further elucidate the role of granulocyte-colony stimulating factor (G-CSF)-induced in response to alpha-tocopherol succinate (TS) administration in protecting mice from total body irradiation (TBI). MATERIAL AND METHODS: The dose, route, and schedule of TS administration for optimal G-CSF induction were determined by giving TS through subcutaneous (sc) and oral routes to male CD2F1 mice. The level of cytokine in serum was determined by multiplex Luminex. The role of G-CSF on survival after TBI was determined by first treating mice with a protective dose (400 mg/kg) of TS 24 h before exposure to a lethal dose (9.2 Gy, 0.6 Gy/min) of cobalt-60 gamma-irradiation. The treated mice were then given neutralising antibody to G-CSF 16 h before TBI to abrogate the radioprotective efficacy of TS. The efficacy of whole blood samples obtained from TS-treated mice was evaluated to protect naïve lethally irradiated mice. The hematopoietic stem cells in blood from TS-treated mice were analysed by fluorescence-activated cell sorting (FACS). RESULTS: Maximal levels of G-CSF were observed in peripheral blood 24 h after sc administration of TS. When TS-treated mice were given neutralising antibody to G-CSF, TS failed to protect against TBI. After being challenged with an LD90/30 (lethal dose causing 90% mortality over 30 days) dose of gamma-radiation, mice infused with whole blood from TS- and AMD3100 (1,1'-{1,4-phenylenebis(methylene)}bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride)-treated mice exhibited significantly higher survival compared with those infused with whole blood from vehicle-injected mice. FACS data revealed that hematopoietic stem cells were mobilised into the peripheral blood. CONCLUSIONS: The results indicate that G-CSF-induced by the administration of TS, mobilises hematopoietic stem cells and is responsible for the protection from ionising radiation.

Preclinical development of a bridging therapy for radiation casualties.

OBJECTIVE: Victims of a terrorist attack presenting with the hematopoietic syndrome resulting from exposure to excessive levels of ionizing radiation will succumb to sepsis if not adequately treated. The probability of survival is increased substantially if the victim's immune system is allowed to recover before sepsis sets in. We report here preclinical development of a new bridging therapy that will allow the victim's immune system to recover from damage caused by ionizing radiation. MATERIALS AND METHODS: The hematopoietic progenitor cells in blood from tocopherol succinate (TS)-injected mice were analyzed quantitatively by standard in vitro soft matrix colony procedures. CD2F1 mice were irradiated with lethal, whole-body doses (9.2 Gy) of (60)Co gamma-rays and then transfused intravenously (periorbital sinus, venous plexus behind the eye) with whole blood, peripheral blood mononuclear cells, or plasma from TS-injected mice 2 and 24 hours postirradiation. Survival was monitored for 30 days after transfusion of whole blood, peripheral blood mononuclear cells, or plasma. RESULTS: Progenitor cell analyses revealed that hematopoietic progenitors were mobilized into the peripheral blood of TS-injected mice. Our results demonstrated that infusions of whole blood or peripheral blood mononuclear cells from TS-injected mice greatly improved chances of extended survival of lethally irradiated mice. CONCLUSION: TS-stimulated granulocyte colony-stimulating factor mobilizes high numbers of progenitors into the peripheral circulation; in turn, this blood-these progenitors-can be used upon subsequent transfusion to effectively mitigate and repair primary acute radiation injury. The transfused cells act secondarily as a bridging therapy for irradiated mice while their own immune system recovers from the radiation-induced damage.

Tocopherol succinate: a promising radiation countermeasure.

Vitamin E is composed of a family of eight isomers known as tocols; consisting of four tocopherols and four tocotrienols that exist in four isomeric forms: alpha (alpha), beta (beta), gamma (gamma), and delta (delta). Earlier we have demonstrated the radioprotective efficacy of the succinate ester of alpha-tocopherol (alpha-tocopherol succinate, TS). CD2F1 mice were injected subcutaneously with 400 mg/kg of TS and irradiated with different doses of 60Co gamma-radiation to determine its radioprotective efficacy. The dose reduction factor (DRF) for TS was also determined. We also investigated effects of TS on cytokine production by multiplex Luminex and message by quantitative RT-PCR. Peripheral blood cells were enumerated from irradiated (3 and 7 Gy) and non-irradiated mice. TS significantly protected mice against lethal doses of 60Co gamma-radiation and the DRF was 1.28. TS stimulated granulocyte-colony stimulating factor (G-CSF) with a peak at 24 h after drug injection, and also stimulated G-CSF message as judged by RT-PCR in bone marrow cells at 12 and 24 h after injection. Further, pancytopenia studies revealed that TS significantly reduced thrombocytopenia, neutropenia, and monocytopenia. TS had no significant effect on lymphocytes indicating that it may be helpful only for the myeloid cell compartments. The stimulation of G-CSF by TS supports its effects on myeloid cell compartments. Our studies indicate that TS may be developed as a radioprotectant for humans against the potentially lethal effects of radiation exposure.