Significant Reduction of Radiation-Induced Death in Mice Treated with PrC-210 and G-CSF after Irradiation

Abstract

The search for single or combined radiation countermeasures that mitigate the development of Acute Radiation Syndrome (ARS) after radiation exposure remains a prominent goal of the U.S. government. This study was undertaken to determine whether PrC-210 and G-CSF, when administered 24–48 h postirradiation, would confer an additive or synergistic survival benefit and mitigate ARS in mice that had received an otherwise 96% lethal radiation dose. Our results show that optimum systemic doses of PrC-210 and G-CSF, when administered 24 h or later after a 96% lethal dose of whole-body irradiation, conferred: 1. strong individual survival benefits (PrC-210 44%, P = 0.003), (G-CSF 48%, P = 0.0002), 2. a profound combined 85% survival benefit (P < 0.0001) when administered together, and on day 14 postirradiation, 3. peripheral white blood cell/lymphocyte counts equal to unirradiated controls, 4. dense bone marrow cell density (>65% of unirradiated controls), 5. jejunal villi density that equaled 90% of unirradiated controls, and 6. spleen weights that equaled 93% of unirradiated controls. Our results show that PrC-210 and G-CSF given together 24 h after irradiation confer strong additive efficacy by protecting the immune system, and enabling recovery of the bone marrow, and they work synergistically to enable recovery of peripheral white blood cells in circulating blood.

INTRODUCTION

The risk of human radiation exposure, possibly catastrophic in proportion, has risen in today’s world (1, 2). Whether from intentional or unintentional use of nuclear weapons, terror-based use of nuclear “dirty bombs” in urban settings, or natural disasters or unintended disasters at nuclear reactor facilities like Fukushima or Chernobyl (3, 4), such events would yield thousands to millions of victims who would benefit from significant protection and mitigation of the human pathologies associated with postirradiation survival.

In this study, we explore the single and combined benefits of two known radiomitigator molecules, PrC-210 (5) and G-CSF [Neupogen^® (6, 7)], to determine whether their known, individual, conferred survival benefits after irradiation are additive or synergistic when administered together after lethal, whole-body irradiation. Such combined efficacy would support the use of a “radiomitigator cocktail” in the field after human radiation exposures.

PrC-210 is a new, direct-acting aminothiol radioprotector that can be administered orally, subcutaneously, intraperitoneally or intravenously (8, 9). PrC-210’s positive-charged backbone “hovers” above negative-charged chemical structures, such as DNA, or proteins, or lipids and thereby physically extends its thiol group several bond-lengths away to capture free radicals before they modify the cell biomolecules. When it is administered as a radiation protector minutes before irradiation, it confers 100% survival to rodents who have received an otherwise 100% whole-body lethal dose of ionizing radiation (10, 11), and when PrC-210 is administered 24 h postirradiation as a radiomitigator, it confers a 45% survival benefit to mice given an otherwise 100% lethal radiation dose (5). PrC-210 has been established as a safe compound in numerous animal studies with no measurable nausea/emesis nor hypotension side effects (9).

Hematopoietic acute radiation syndrome is characterized by dose- and time-dependent loss in circulating white blood cells, lymphocytes, neutrophils and platelets resulting in severe myelo- and immunosuppression with subsequent hemorrhage and sepsis. Granulocyte colony-stimulating factor (G-CSF; Neupogen) enhances recovery of white blood cells and reduces the duration of neutropenia in patients undergoing myelosuppressive chemo- or radiotherapy (12). Neupogen has been approved by the U.S. Food and Drug Administration (FDA) for treatment of the neutropenia associated with acute radiation syndrome (ARS) after a radiological nuclear accident. A new, longer plasma half-life, pegylated form of recombinant G-CSF, Neulasta, has now also been FDA approved for treatment of ARS. The scheduling of G-CSF administration is important in achieving a favorable hematological recovery. The consensus is that G-CSF treatment should be initiated as soon as possible after TBI (total body irradiation) and that treatment should continue until the peripheral blood absolute neutrophil count normalizes (13). In a previous study (6), tests of several G-CSF doses and dose regimens identified significant survival benefit (57%) with three G-CSF doses of 0.17 mg/kg administered to irradiated mice over three days.

In this study, we hypothesized that the small molecule aminothiol (PrC-210), which works as a radioprotector, and a hematopoietic growth factor (G-CSF), working via a presumably independent pathway, would provide complementary radiomitigation effects, and by so doing provide a substantial survival benefit to the otherwise lethally irradiated mice. The results of this study clearly demonstrate the efficacy of a multidrug treatment with one approved drug, Neupogen, and one drug in development, PrC-210, for mitigation of ARS.

MATERIALS AND METHODS

Materials and PrC-210

PrC-210 HCl (MW: 220) was synthesized for these studies as previously described (14, 15). ICR (CD-1) mice (female, 25–30 gm) were purchased from Envigo (Madison, WI). Solvents and chemicals were purchased from Sigma-Aldrich (St Louis, MO).

Animal Studies

This research was approved by the School of Medicine and Public Health Institutional Animal Care and Use Committee at the University of Wisconsin (Protocol M006610). All procedures were performed in accordance with the Animal Care and Use Policies at the University of Wisconsin. Mice were maintained on a 12 h light/dark cycle and were provided ad libitum water and lab chow (Harlan Teklad 8604). Unanesthetized mice in a plexiglass mouse pie cage (Braintree Scientific, MPC-2) were irradiated in an AP orientation with 8.25 Gy (a previously demonstrated LD₉₅ dose) in an XStrahl CIX3 Irradiator (see https://xstrahl.com/cix3/for beam and filtration details) with 300 keV of X rays at 1.37 Gy/min. Dose was standardized using thermoluminescent dosimeters (TLDs). Twenty-four or 48 h after irradiation, mice received a single intraperitoneal (IP) injection, either 100 μl saline or PrC-210 [0.3 X Maximum Tolerated Dose (MTD); the mouse IP MTD = 504 μg/gm body weight (bw); see ref. (8)] dissolved in approximately 100 ml of saline adjusted to pH 6.5. In some experiments, mice received a single IP injection of 0.5 MTD PrC-210 30 min before the same irradiation, or control mice received a 100 ul injection of saline.

At times ranging from 12 h to 72 h after irradiation (see Fig. 2), some mice received four subcutaneous injections of 0.17 mg/kg of G-CSF (Neupogen, Amgen, Thousand Oaks, CA).

FIG. 2.

Mouse organ histology postirradiation. Representative sternal bone marrow (panel A) and jejunal small intestine (panel B) histology in mice from the indicated treatment groups. Mice were euthanized on day 14 after 8.25 Gy irradiation. Jejunum cross sections (taken 6 cm from stomach) or sterna were fixed in 10% formalin, sterna de-calcified and then worked up to include H&E staining of sections. “10×” magnifications of slides were scanned using an Optika Optiscan10 scanner.

After drug treatments, mice were returned to cages and observed each day for the next 30 days. On day 14 postirradiation, from three randomly chosen mice per treatment group, mouse jejunum segments (taken 6 cm beyond the stomach), sterna and intact spleens were fixed in 10% formalin, sterna were decalcified, and tissues were embedded in paraffin, sections were then mounted and stained with hematoxylin-eosin. Slides were scanned using a 10× objective on an Optika Optiscan10 scanner to generate jpg images.

In mice euthanized on day 14 after irradiation, blood was collected, and complete blood counts (CBC) were performed on 50 ul aliquots of blood using an HM5 VetScan hematology analyzer.

Bone Marrow and Intestine Histology and Digital Analysis

Sections of H&E-stained sternal bone marrow and jejunal cross-section were scanned using a 10× objective on an Optika Optiscan10 scanner to generate jpg images.

To score jejunal villi density in cross sections, the 10× jpg images were opened in Image J, and using Image J subprograms, both total pixels per image and “non-white” pixels per image were determined. The total “non-white” pixels in the jejunal cross-section (highlighted in blue by Image J; see Fig. 6) were divided by the “total” pixels in the jejunal cross-section to yield a frequency (“jejunal non-white pixels/jejunal cross-section area”).

FIG. 6.

Jejunal histology in irradiated mice. Sample H&E-stained jejunal cross-section images were analyzed using Image J to identify and quantify “non-white” pixels (highlighted in blue by Image J), which were then normalized against the total pixel area of the jejunum in each JPEG image. The mean and SEM of the three frequencies in each treatment group were then plotted versus the 30-day percent survival for the same treatment group. Treatment group labels are shown for each group.

To score white blood cell density in sternal bone marrow cross-sections, scoring “non-white” pixels using Image J was inadequate. In bone marrows that were vacant of cells after LD₉₆ irradiation, there was still a high background of “non-white” pixels, basically stained marrow space support structure. Thus, in an extensive effort using code-writing expertise accessed through CodeMentor (https://www.codementor.io/) new code was written that complemented existing image analysis capability in existing software (Python, Jupiter Notebook, and Image J). This yielded a software code [named “Image(-ine)”] in which a scanned jpg image was i) opened, ii) total pixels determined, iii) “blue pixel areas” determined (i.e., they matched the blue color chosen from a color matrix on a separate screen), and iv) “total blue pixels” were determined within the aggregate “blue pixel areas.” Printouts of the data are shown in Fig. 3.

FIG. 3.

Sternal bone marrow density after irradiation. H&E-stained sternal bone marrow spaces (white boxes in panel A) were copy/pasted as new JPEGS into an image analysis program [“Image(ine)”] written in our lab which enabled selection and quantification in this case of blue pixels which were then normalized against the total number of pixels within the same JPEG image (panels B and C are examples).

Caspase 3,7 Assay

Activated caspase 3 and 7 activity in mouse plasma was determined using the Apo-ONE fluorescent substrate (Promega, Madison, WI) (5). The activated caspase assay was performed as follows: 20 μl of mouse plasma (stored at −80°C) was mixed with 30 ul buffer and 50 μL of the undiluted Apo-ONE substrate in the well of a black, opaque, 96 well plate to initiate the 60 min reaction. Plates were shaken at 200 RPM at 37°C for 60 min. The DEVD caspase substrate peptide cleavage was measured using a BMG Clariostar fluorescent plate reader at an excitation wavelength of 499 nm and an emission wavelength of 521 nm. A caspase standard was included in each experiment.

Caspase 1 Assay

Activated caspase 1 activity in mouse plasma was determined using the Caspase-Glo 1 Inflammasome Assay (G9951, Promega, Madison, WI). The activated caspase 1 assay was performed as follows: 20 μl of mouse plasma (stored at −80°C) was mixed with 30 ul buffer and 50 μL of the Z-WEHD caspase 1 substrate in the well of a black, opaque, 96 well plate. The plate was shaken at 200 rpm for 60 sec and then allowed to sit at room temperature for 30 min. Chemiluminescence in the wells was measured using a BMG Clariostar fluorescent plate reader. A caspase 1 standard was included in each experiment.

Statistical Analyses

For statistical analysis, either the Student’s T test was used for simple comparisons between groups, or the Mantel-Cox test was used to compare differences in survival between groups. For correlation analyses, Pearson correlation coefficients (“R”) were calculated using Graphpad Prism.

RESULTS

Survival in Mice Administered PrC-210, G-CSF, or Combination after Irradiation

Earlier studies from our lab (5) and AFRRI (6) demonstrated sizable survival advantages when either PrC-210 was administered once 24 h after irradiation, or G-CSF was administered three times after irradiation. Although a few different doses of G-CSF worked, the 0.17 mg/kg × 3 worked the best. Based upon these earlier studies we sought to determine whether administration of both molecules together after irradiation would confer any additive or synergistic effect.

In the radiation survival experiments of this study, 25 ICR mice were enrolled into each of ten treatment groups, where at 24–72 h after irradiation, the groups received either: 1. Nothing; 2. a single IP dose of PrC-210 (0.3 MTD; 151 ug/gm bw); 3. three doses of G-CSF (0.17 mg/kg), or 4. various combinations of PrC-210 and G-CSF over 24–72 h postirradiation (see Fig. 1). Twenty-two mice per group were scored for survival, and three mice per group were euthanized on day 14 for histology and complete blood count.

FIG. 1.

Drug-enhanced postirradiation mouse survival. Survival (panel A) in ten groups (25 mice per group) of ICR female mice given either i) a single IP (intraperitoneal) injection of either saline or PrC-210 (0.3 MTD, 151 mg/kg) 24 or 48 h after irradiation with 8.25 Gy, or ii) three single SC (subcutaneous) injections of G-CSF (0.17 mg/kg) at the indicated times (panel B) after irradiation, or iii) a combination of both PrC-210 and G-CSF injections at the indicated times after irradiation. Mice were observed each day through day 30. Mantel-Cox statistical analyses of survival curves provided the indicated P values between treatment groups in panel C.

The combined administration of PrC-210 at +24 h and G-CSF at +24, +36, +48 provided a profound survival advantage of 85% (i.e., 89–4%, P < 0.0001, Fig. 1A and B). The 85% survival, i.e., essentially additive, is consistent with the combined survival of the PrC-210 alone (Group B, 40%, 44–4%) and the G-CSF alone (Group D, 49%, 53–4%).

Starting both PrC-210 and G-CSF at +48 h (Fig. 1A and B) was also highly significant in the conferred survival advantage when compared to mice that were only irradiated.

Complete Blood Counts in Irradiated Mice

An 8.25 Gy whole-body radiation exposure profoundly reduces all the peripheral blood cell counts in mice 14 days later (Table 1). This is particularly apparent when comparing CBCs in non-irradiated mice to those in irradiated mice (reductions show P values of 0.0001 to 0.03). Several red blood cell parameters, particularly oxygen-carrying elements (i.e. red blood cells, hemoglobin; Table 1) are significantly suppressed (Table 1), and PrC-210 or G-CSF had little effect on their restoration by +14 days. Completely opposite to this, after 14 days, two white blood cell counts (i.e., white blood cells: P = 0.408, lymphocytes: P = 0.440) were restored to unirradiated control levels by PrC-210 + G-CSF, and one count (Neutrophils) was nearly restored to the unirradiated control level (Table 1). Whereas PrC-210 alone had a suppressive effect on white blood cell counts, the combination of PrC-210 + G-CSF restored white blood cell counts to unirradiated control levels.

TABLE 1.

Blood Cell Counts 14 Days Postirradiation ± PrC-210 Dose at +24 Hours

	Day 14 postirradiation
	Treatment Group					P value
	Nonirradiated	A	B	D	F
Blood parameter	Ctrl	XRT	XRT + PrC-210	XRT + G-CSF	XRT + PrC-210 + GCSF	Nonirradiated vs. A	F vs. A	F vs. nonirradiated
Hemoglobin (gm/deciliter)	16.4 ± 0.4	6.3 ± 0.3	9.5 ± 1.6	8.3 ± 1.4	9.8 ± 1.6	0.0001	0.0014	—
Platelets (109/liter)	294.7 ± 24.0	17.2 ± 7.2	29.3 ± 15.7	8.7 ± 5.6	21.3 ± 3.2	0.0004	—	—
Hematocrit (%)	50.6 ± 0.9	29.3 ± 5.7	33.8 ± 6.2	28.6 ± 4.7	27.0 ± 3.4	0.0108	—	—
Red blood cells (1012/liter)	10.31 ± 0.47	5.98 ± 1.46	6.81 ± 1.09	5.86 ± 0.94	5.52 ± 0.97	0.0240	—	—
White blood cells (109/liter)	1.55 ± 0.14	0.82 ± 0.02	0.46 ± 0.08	0.81 ± 0.16	1.51 ± 0.35	0.0044	0.0020	0.408
Neutrophils (109/liter)	0.37 ± 0.03	0.29 ± 0.09	0.05 ± 0.03	0.32 ± 0.12	0.31 ± 0.14	0.0405	—	—
Lymphocytes (109/liter)	1.12 ± 0.12	0.81 ± 0.02	0.40 ± 0.04	0.51 ± 0.02	1.10 ± 0.12	0.0329	0.0090	0.440

Bone Marrow White Blood Cell Density

Irradiation of mice with 8.25 Gy caused depletion or suppression of sternum bone marrow spaces in all mouse groups at 14 days after irradiation (see examples in Fig. 2A). Sternal bone marrow white blood cell densities (i.e., blue pixels/total pixels) were quantitatively analyzed using a new image analysis program written in our lab [“Image(ine)”]. Figure 3B and C show representative analyses of two marrow spaces. Figure 3A shows a JPEG image of a 10× scan of an H&E-stained sternal cross section. Image-selected samples within the bone marrow space were copy-pasted as new JPEGs (such as Fig. 3B panel), and the total number of blue pixels and total number of pixels in each image were then recorded in a spreadsheet; this generally yielded an N of nine for the three mice per treatment group harvested on day +14 for histology. Calculated “blue pixels per 100,000 pixels” values (such as Fig. 3B and C) were then plotted versus each of the 10 treatment group survival rates at +30 days (Fig. 4). Next to the “No XRT” control mice, the highest marrow blue pixel densities were seen in two of the PrC-210 + G-CSF groups (i.e., Groups I and F). There was a very strong correlation (R = 0.790; P = 0.004) between the marrow blue pixel densities and “% Survival” over the 10 treatment groups.

FIG. 4.

Sternal bone marrow densities. Sample H&E-stained sternal bone marrow spaces (as shown in Fig. 3) from each of the 10 treatment groups obtained from three marrow spaces per mouse × three mice per group (i.e., an N of 9 per treatment group analyzed), were then plotted vs. the 30-day percent survival for the same treatment group.

Spleen Weights

Spleens were removed from three euthanized mice from each treatment group 14 days after irradiation. Histology of the spleens revealed nothing remarkable between groups, but weights of the intact spleens (Fig. 5A) were strongly correlated (Fig. 5B; R = 0.766; P = 0.0049) with “% Survival” between the 10 treatment groups. As seen in Fig. 5A, postirradiation treatment of mice with PrC-210 alone at 24 h (Group B, P = 0.005), G-CSF alone at 24 or 48 h (Groups D, E, P = 0.009), or their combined treatment either at 24 h or 48 h (Groups H, I, P = 0.012), provided similar significant increases in the weight of the spleens in mice in these respective treatment groups. PrC-210 given alone at 48 h postirradiation did not show a significant benefit in spleen weight. Like the Group H PrC-210 + G-CSF white blood cell and lymphocyte peripheral blood counts that equaled unirradiated controls (Table 1), the Group H spleen weights equaled that seen in unirradiated controls (Fig. 5A). Thus, the PrC-210 + G-CSF conferred repopulation of peripheral white blood cells after irradiation, and its associated spleen weight, is 1. highly correlated with mouse post-radiation survival, and 2. this protection is achieved with a PrC-210 + G-CSF administration window through +48 h.

FIG. 5.

Spleen weights in irradiated mice. Spleen weights (panel A) in mouse treatment groups 14 days after 8.25 Gy irradiation. Three mice from each group were euthanized on day 14 and entire spleens were removed and transferred to 10% formalin. Subsequently, fixed spleens were blotted on tissue and weighed. Treatment groups are indicated in panel B. The mean spleen weight of the three spleens in each treatment group was then plotted versus the 30-day percent survival for the same treatment group. Treatment group labels are shown for each group.

Jejunum Villus Density

Jejunum cross-section tissue density (i.e., non-white pixel densities) were quantitatively analyzed using Image J. Fig. 6 shows JPEG images of H&E-stained jejunal cross-sections, as well as Image J selected “non-white” pixels (colored blue by Image J). In a given image, the total number of non-white pixels was divided by the total cross-sectional area of the jejunal slice to yield non-white tissue pixels/jejunum cross-section area densities in each jejunum cross-section. These density values were then recorded in a spreadsheet; this yielded an N of three for the three mice per treatment group harvested on day +14 for histology. Calculated jejunal “densities” (Fig. 6) were then plotted versus the treatment group survival at +30 days. For treatment groups with ≥50% survival, there was a very strong correlation (R = 0.883; P = 0.0037) between jejunal villus density and survival of the mice from an otherwise 96% lethal radiation dose (Fig. 6).

Plasma Caspase Activities

As a measure of radiation-induced organ damage (Caspase 3,7) and radiation-induced damage and activation of the inflammasome (Caspase 1), levels of these activated caspases were measured in blood plasma of mice euthanized at the postirradiation time points indicated in Fig. 7A and B. In both cases, caspase activation in irradiated organs, and its spillover into the plasma compartment, was profound in the 24 h after irradiation. With both caspases, a single IP dose (0.3 MTD) of PrC-210 at +24 h postirradiation conferred significant suppression (P values of 0.004 to 0.026) of their plasma levels.

FIG. 7.

Plasma caspase levels in irradiated mice. Levels of Caspase 3/7 or Caspase 1 in mouse plasma in the six days after irradiation; intraperitoneal PrC-210 was administered 24 h after irradiation (see arrows). Mice (3–4 per timepoint) were euthanized, and blood collected at the indicated timepoints after irradiation, plasma was frozen on dry ice and then −80C until caspase analyses. P values were calculated using the Student T test.

DISCUSSION

The search for single or combined radiation countermeasures that mitigate the development of ARS after radiation exposure remains a prominent goal of the U.S. Government (16). This study was undertaken to determine whether PrC-210 and G-CSF, when administered postirradiation, would confer an additive or synergistic survival benefit and mitigate ARS in mice that had received an otherwise 96% lethal radiation dose. Our results show that optimum systemic doses of PrC-210 and G-CSF, when administered 24 h or later after a 96% lethal dose of whole-body irradiation, conferred: 1. strong individual survival benefits (PrC-210 44%, P = 0.003), (G-CSF 49%, P = 0.0002) almost exactly replicating their earlier published single effects, 2. a profound combined survival benefit (85%, P < 0.0001) when administered together, and 14 days after irradiation, 3. fully recovered peripheral white blood cell (P = 0.0020) and lymphocyte (P = 0.0090) counts, close to normal bone marrow density (up to P = 0.001), denser jejunal villi (up to P = 0.003), and increased spleen weight (P = 0.0049) in PrC-210 + G-CSF treated mice.

In this study, the PrC-210 was administered by intraperitoneal injection, a route with somewhat limited utility in a mass casualty setting. This same study is currently being done in which the PrC-210 is instead administered by oral gavage to the irradiated mice.

While we see little recovery impact upon red cell parameters at day +14 (Table 1), we see complete restoration of white blood cell populations to untreated control levels at +14 days in PrC-210 + G-CSF treatment groups; G-CSF alone was less effective, and PrC-210 alone showed a suppressive effect upon peripheral white blood cell recovery. The peripheral blood recovery was largely mirrored by the restored bone marrow densities (Fig. 4). In bone marrows, the PrC-210 + G-CSF treatment groups (I and F) had 65% restoration of bone marrow density by 14 days postirradiation. PrC-210 alone (Group B) had a 34% marrow restoration which reflects the earlier Kumar et al. (17) observation, but this wasn’t reflected in the peripheral white blood cell counts (Table 1). It’s known in the literature (18, 19) that among its functions, G-CSF stimulates bone marrow white blood cells to move from the bone marrow space into peripheral blood.

Why do we see a suppression (Table 1) of the peripheral white blood cell recovery with PrC-210 alone? As shown in Fig. 7A and B, PrC-210 reduces apoptosis, as shown by PrC-210 suppression of the caspase 3/7 and caspase 1 inflammasome markers in plasma. With this function, PrC-210 protects peripheral immune cells postirradiation and prevents the immune system destruction associated with caspase 1 activation. This dual protective and suppressive effect leads to slow down of peripheral white blood cell recovery, but also appears to be basis for a synergistic recovery of marrow and peripheral white blood cells when combined with the hematopoietic growth factor G-CSF (Table 1).

This working hypothesis for PrC-210 + G-CSF synergy is supported by the spleen weight data. The spleen weights in the stand-alone and combination treatment groups strongly correlate (P = 0.0049) with % Survival, basically reflecting the bone marrow recovery and peripheral blood white cell counts. PrC-210 given 24 h postirradiation or in combination with G-CSF seems to be a critical part of the increased spleen weight at day 14. Only the PrC-210 given alone 48 h after irradiation doesn’t follow this scheme; this implies that the protective effect of PrC-210 wanes during the +24 h to +48 h time window, whereas the immune regulatory effect of PrC-210 (seen in Fig. 7) lasts well beyond the +48-h time point, which is in line with the long-lasting PrC-210 suppressive effect seen in the caspase experiments. Restoring those white cell populations and repopulating the spleen with white cells, is strongly associated with animal well-being, and survival from an otherwise 96% lethal radiation dose.

We also see a strong correlation between jejunal villus density and percent (%) survival (P = 0.0037). The strongest protection is in the combined PrC-210 + G-CSF group (F). Like spleen weight, PrC-210 alone when given at + 48 h is without protective effect, but, when combined with G-CSF, even at +48 h, PrC-210 significantly contributes to jejunal protection. How much this PrC-210 + G-CSF conferred jejunal protection is mechanistically connected to the like-observed PrC-210 + G-CSF white cell recovery needs to be investigated in future studies.

In summary, PrC-210 and G-CSF given as a combined therapy show additive effects in mouse survival, bone marrow density recovery, jejunal villus density, a strong synergistic effect in peripheral white blood cell recovery, and a time-dependent application effect of PrC-210 in the spleen weight gain. Based upon our findings here, the “G-CSF” and “PrC-210” effects work well together to give unparalleled survival advantages to irradiated mammals.

In developing radiomitigators, our choices are either to: 1. identify the “primary” cause of death in ARS (if there is in fact a “primary cause”) and create mitigators to suppress it (alone), or 2. identify the multifactor toxic nature of ARS and create a cocktail that suppress these multiple events; this cocktail should include PrC-210 and G-CSF to address the multiple, contributory toxicities that in aggregate constitute acute radiation syndrome.