Efficacy of Neulasta or Neupogen on H-ARS and GI-ARS Mortality and Hematopoietic Recovery in Nonhuman Primates After 10-Gy Irradiation With 2.5% Bone Marrow Sparing.

A nonhuman primate model of acute, partial-body, high-dose irradiation with minimal (2.5%) bone marrow sparing was used to assess endogenous gastrointestinal and hematopoietic recovery and the ability of Neulasta (pegylated granulocyte colony-stimulating factor) or Neupogen (granulocyte colony-stimulating factor) to enhance recovery from myelosuppression when administered at an increased interval between exposure and initiation of treatment. A secondary objective was to assess the effect of Neulasta or Neupogen on mortality and morbidity due to the hematopoietic acute radiation syndrome and concomitant gastrointestinal acute radiation syndrome. Nonhuman primates were exposed to 10.0 Gy, 6 MV, linear accelerator-derived photons delivered at 0.80 Gy min. All nonhuman primates received subject-based medical management. Nonhuman primates were dosed daily with control article (5% dextrose in water), initiated on day 1 postexposure; Neulasta (300 µg kg), administered on days 1, 8, and 15 or days 3, 10, and 17 postexposure; or Neupogen (10 µg kg), administered daily postexposure following its initiation on day 1 or day 3 until neutrophil recovery (absolute neutrophil count ≥1,000 cells µL for 3 consecutive days). Mortality in the irradiated cohorts suggested that administration of Neulasta or Neupogen on either schedule did not affect mortality due to gastrointestinal acute radiation syndrome or mitigate mortality due to hematopoietic acute radiation syndrome (plus gastrointestinal damage). Following 10.0 Gy partial-body irradiation with 2.5% bone marrow sparing, the mean duration of neutropenia (absolute neutrophil count <500 cells µL) was 22.4 d in the control cohort vs. 13.0 and 15.3 d in the Neulasta day 1, 8, 15 and day 3, 10, 17 cohorts, relative to 16.2 and 17.4 d in the Neupogen cohorts initiated on day 1 and day 3, respectively. The absolute neutrophil count nadirs were 48 cells µL in the controls; 117 cells µL and 40 cells µL in the Neulasta days 1, 8, and 15 or days 3, 10, and 17 cohorts, respectively; and 75 cells µL and 37 cells µL in the Neupogen day 1 and day 3 cohorts, respectively. Therefore, the earlier administration of Neulasta or Neupogen was more effective in this model of marginal 2.5% bone marrow sparing. The approximate 2.5% bone marrow sparing may approach the threshold for efficacy of the lineage-specific medical countermeasure. The partial-body irradiation with 2.5% bone marrow sparing model can be used to assess medical countermeasure efficacy in the context of the concomitant gastrointestinal and hematopoietic acute radiation syndrome sequelae.

A non-human primate model of radiation-induced cachexia.

Cachexia, or muscle wasting, is a serious health threat to victims of radiological accidents or patients receiving radiotherapy. Here, we propose a non-human primate (NHP) radiation-induced cachexia model based on clinical and molecular pathology findings. NHP exposed to potentially lethal partial-body irradiation developed symptoms of cachexia such as body weight loss in a time- and dose-dependent manner. Severe body weight loss as high as 20-25% was observed which was refractory to nutritional intervention. Radiographic imaging indicated that cachectic NHP lost as much as 50% of skeletal muscle. Histological analysis of muscle tissues showed abnormalities such as presence of central nuclei, inflammation, fatty replacement of skeletal muscle, and muscle fiber degeneration. Biochemical parameters such as hemoglobin and albumin levels decreased after radiation exposure. Levels of FBXO32 (Atrogin-1), ActRIIB and myostatin were significantly changed in the irradiated cachectic NHP compared to the non-irradiated NHP. Our data suggest NHP that have been exposed to high dose radiation manifest cachexia-like symptoms in a time- and dose-dependent manner. This model provides a unique opportunity to study the mechanism of radiation-induced cachexia and will aid in efficacy studies of mitigators of this disease.

Increased Expression of Connective Tissue Growth Factor (CTGF) in Multiple Organs After Exposure of Non-Human Primates (NHP) to Lethal Doses of Radiation.

Exposure to sufficiently high doses of ionizing radiation is known to cause fibrosis in many different organs and tissues. Connective tissue growth factor (CTGF/CCN2), a member of the CCN family of matricellular proteins, plays an important role in the development of fibrosis in multiple organs. The aim of the present study was to quantify the gene and protein expression of CTGF in a variety of organs from non-human primates (NHP) that were previously exposed to potentially lethal doses of radiation. Tissues from non-irradiated NHP and NHP exposed to whole thoracic lung irradiation (WTLI) or partial-body irradiation with 5% bone marrow sparing (PBI/BM5) were examined by real-time quantitative reverse transcription PCR, western blot, and immunohistochemistry. Expression of CTGF was elevated in the lung tissues of NHP exposed to WTLI relative to the lung tissues of the non-irradiated NHP. Increased expression of CTGF was also observed in multiple organs from NHP exposed to PBI/BM5 compared to non-irradiated NHP; these included the lung, kidney, spleen, thymus, and liver. These irradiated organs also exhibited histological evidence of increased collagen deposition compared to the control tissues. There was significant correlation of CTGF expression with collagen deposition in the lung and spleen of NHP exposed to PBI/BM5. Significant correlations were observed between spleen and multiple organs on CTGF expression and collagen deposition, respectively, suggesting possible crosstalk between spleen and other organs. These data suggest that CTGF levels are increased in multiple organs after radiation exposure and that inflammatory cell infiltration may contribute to the elevated levels of CTGF in multiple organs.

The Effect of Radiation Dose and Variation in Neupogen® Initiation Schedule on the Mitigation of Myelosuppression during the Concomitant GI-ARS and H-ARS in a Nonhuman Primate Model of High-dose Exposure with Marrow Sparing.

A nonhuman primate (NHP) model of acute high-dose, partial-body irradiation with 5% bone marrow (PBI/BM5) sparing was used to assess the effect of Neupogen® [granulocyte colony stimulating factor (G-CSF)] to mitigate the associated myelosuppression when administered at an increasing interval between exposure and initiation of treatment. A secondary objective was to assess the effect of Neupogen® on the mortality or morbidity of the hematopoietic (H)- acute radiation syndrome (ARS) and concurrent acute gastrointestinal radiation syndrome (GI-ARS). NHP were exposed to 10.0 or 11.0 Gy with 6 MV LINAC-derived photons at approximately 0.80 Gy min. All NHP received medical management. NHP were dosed daily with control article (5% dextrose in water) initiated on day 1 post-exposure or Neupogen® (10 µg kg) initiated on day 1, day 3, or day 5 until recovery [absolute neutrophil count (ANC) ≥ 1,000 cells µL for three consecutive days]. Mortality in both the 10.0 Gy and 11.0 Gy cohorts suggested that early administration of Neupogen® at day 1 post exposure may affect acute GI-ARS mortality, while Neupogen® appeared to mitigate mortality due to the H-ARS. However, the study was not powered to detect statistically significant differences in survival. The ability of Neupogen® to stimulate granulopoiesis was assessed by evaluating key parameters for ANC recovery: the depth of nadir, duration of neutropenia (ANC < 500 cells µL) and recovery time to ANC ≥ 1,000 cells µL. Following 10.0 Gy PBI/BM5, the mean duration of neutropenia was 11.6 d in the control cohort vs. 3.5 d and 4.6 d in the day 1 and day 3 Neupogen® cohorts, respectively. The respective ANC nadirs were 94 cells µL, 220 cells µL, and 243 cells µL for the control and day 1 and day 3 Neupogen® cohorts. Following 11.0 Gy PBI/BM5, the duration of neutropenia was 10.9 d in the control cohort vs. 2.8 d, 3.8 d, and 4.5 d in the day 1, day 3, and day 5 Neupogen® cohorts, respectively. The respective ANC nadirs for the control and day 1, day 3, and day 5 Neupogen® cohorts were 131 cells µL, 292 cells µL, 236 cells µL, and 217 cells µL, respectively. Therefore, the acceleration of granulopoiesis by Neupogen® in this model is independent of the time interval between radiation exposure and treatment initiation up to 5 d post-exposure. The PBI/BM5 model can be used to assess medical countermeasure efficacy in the context of the concurrent GI- and H-ARS.

Pegfilgrastim Improves Survival of Lethally Irradiated Nonhuman Primates.

Leukocyte growth factors (LGF), such as filgrastim, pegfilgrastim and sargramostim, have been used to mitigate the hematologic symptoms of acute radiation syndrome (ARS) after radiation accidents. Although these pharmaceuticals are currently approved for treatment of chemotherapy-induced myelosuppression, such approval has not been granted for myelosuppression resulting from acute radiation exposure. Regulatory approval of drugs used to treat radiological or nuclear exposure injuries requires their development and testing in accordance with the Animal Efficacy Rule, set forth by the U.S. Food and Drug Administration. To date, filgrastim is the only LGF that has undergone efficacy assessment conducted under the Animal Efficacy Rule. To confirm the efficacy of another LGF with a shorter dosing regimen compared to filgrastim, we evaluated the use of pegfilgrastim (Neulasta(®)) in a lethal nonhuman primate (NHP) model of hematopoietic acute radiation syndrome (H-ARS). Rhesus macaques were exposed to 7.50 Gy total-body irradiation (the LD(50/60)), delivered at 0.80 Gy/min using linear accelerator 6 MV photons. Pegfilgrastim (300 µg/kg, n = 23) or 5% dextrose in water (n = 23) was administered on day 1 and 8 postirradiation and all animals received medical management. Hematologic and physiologic parameters were evaluated for 60 days postirradiation. The primary, clinically relevant end point was survival to day 60; secondary end points included hematologic-related parameters. Pegfilgrastim significantly (P = 0.0014) increased 60 day survival to 91.3% (21/23) from 47.8% (11/23) in the control. Relative to the controls, pegfilgrastim also significantly: 1. decreased the median duration of neutropenia and thrombocytopenia; 2. improved the median time to recovery of absolute neutrophil count (ANC) ≥500/µL, ANC ≥1,000/µL and platelet (PLT) count ≥20,000/µL; 3. increased the mean ANC at nadir; and 4. decreased the incidence of Gram-negative bacteremia. These data demonstrate that pegfilgrastim is an additional medical countermeasure capable of increasing survival and neutrophil-related parameters when administered in an abbreviated schedule to a NHP model of lethal H-ARS.

The ability of filgrastim to mitigate mortality following LD50/60 total-body irradiation is administration time-dependent.

The identification of the optimal administration schedule for an effective medical countermeasure is critical for the effective treatment of individuals exposed to potentially lethal doses of radiation. The efficacy of filgrastim (Neupogen®), a potential medical countermeasure, to improve survival when initiated at 48 h following total body irradiation in a non-human primate model of the hematopoietic syndrome of the acute radiation syndrome was investigated. Animals were exposed to total body irradiation, antero-posterior exposure, total midline tissue dose of 7.5 Gy, (target lethal dose 50/60) delivered at 0.80 Gy min, using linear accelerator-derived 6 MV photons. All animals were administered medical management. Following irradiation on day 0, filgrastim (10 µg kg d) or the control (5% dextrose in water) was administered subcutaneously daily through effect (absolute neutrophil count ≥ 1,000 cells µL for three consecutive days). The study (n = 80) was powered to demonstrate a 25% improvement in survival following the administration of filgrastim or control beginning at 48 ± 4 h post-irradiation. Survival analysis was conducted on the intention-to-treat population using a two-tailed null hypothesis at a 5% significance level. Filgrastim, initiated 48 h after irradiation, did not improve survival (2.5% increase, p = 0.8230). These data demonstrate that efficacy of a countermeasure to mitigate lethality in the hematopoietic syndrome of the acute radiation syndrome can be dependent on the interval between irradiation and administration of the medical countermeasure.