Mitigation of total body irradiation-induced mortality and hematopoietic injury of mice by a thrombopoietin mimetic (JNJ-26366821)

The threat of a nuclear attack has increased in recent years highlighting the benefit of developing additional therapies for the treatment of victims suffering from Acute Radiation Syndrome (ARS). In this work, we evaluated the impact of a PEGylated thrombopoietin mimetic peptide, JNJ-26366821, on the mortality and hematopoietic effects associated with ARS in mice exposed to lethal doses of total body irradiation (TBI). JNJ-26366821 was efficacious as a mitigator of mortality and thrombocytopenia associated with ARS in both CD2F1 and C57BL/6 mice exposed to TBI from a cobalt-60 gamma-ray source. Single administration of doses ranging from 0.3 to 1 mg/kg, given 4, 8, 12 or 24 h post-TBI (LD70 dose) increased survival by 30–90% as compared to saline control treatment. At the conclusion of the 30-day study, significant increases in bone marrow colony forming units and megakaryocytes were observed in animals administered JNJ-26366821 compared to those administered saline. In addition, enhanced recovery of FLT3-L levels was observed in JNJ-26366821-treated animals. Probit analysis of survival in the JNJ-26366821- and saline-treated cohorts revealed a dose reduction factor of 1.113 and significant increases in survival for up to 6 months following irradiation. These results support the potential use of JNJ-26366821 as a medical countermeasure for treatment of acute TBI exposure in case of a radiological/nuclear event when administered from 4 to 24 h post-TBI.

Risk of radiation exposure due to terrorist attacks or nuclear accidents is increasing and it is critical to have multiple radiation countermeasures readily available. The extent of injury resulting from exposure to ionizing radiation is dependent on the extent of exposure, with the hematopoietic system being the most susceptible 1 . Development of radiation countermeasures for treatment of Hematopoietic Acute Radiation Syndrome (H-ARS) has focused on drugs that promote hematopoietic recovery and more specifically, agents that stimulate production of white cells and platelets following Total Body Irradiation (TBI) 2,3 .
To date, the FDA has approved 4 radiation countermeasures for the mitigation of H-ARS, each serving to stimulate white cells or platelets: Neupogen 4 , Neulasta 5 , Leukine 6 , and Nplate 7 . Neupogen (filgrastim) and Neulasta (PEGylated filgrastim) were approved in 2015 and are both recombinant forms of the cytokine, Granulocytecolony stimulating factor (G-CSF). Neupogen (filgrastim) requires daily dosing until neutrophil counts are above 1000/ml for 3 consecutive readings and Neulasta (PEGylated filgrastim) requires two doses administered a week apart. Both Neupogen and Neulasta mitigate H-ARS by facilitating bone marrow recovery (especially neutrophil recovery) which aids in maintaining immune function and capacity for fighting infections. Leukine was approved in 2018 and is a recombinant form of the Granulocyte-macrophage colony-stimulating factor

JNJ-26366821 administered 4-24 h post-TBI increases survival.
Prior to TBI studies, TPOm (single dose of 3 mg/kg) was tested for safety and tolerability in CD2F1 male mice and was found to be safe at this dose with no abnormal clinical signs of toxicity and the absence of pathological findings on gross necropsy. JNJ-26366821 (0.3 mg/kg) or saline were administered 4 h post-TBI at 9.35 Gy (LD70/30) and monitored for survival. At the conclusion of the study, 88% of animals administered JNJ-26366821 survived whereas 35% of animals administered saline survived (Fig. 1A). Comparing the survival curves using Log-rank test demonstrated a significant difference (p = 0.0002) between the saline and JNJ-26366821 curves; comparing the survival percentage in both groups at day 30 with a Fisher's exact test (88% vs 35%) was also statistically significant (p = 0.0001). Additional animals were administered either JNJ-26366821 (0.3 mg/kg or 1.0 mg/kg) or saline 24 h post-TBI; survival in animals administered 1.0 mg/kg JNJ-2636682, 0.3 mg/kg JNJ-26366821, and saline was 79%, 71%, and 29%, respectively. (Fig. 1B).
Log-rank test comparison of the survival curves demonstrated a significant increase in survival in both JNJ-26366821-treated groups compared to the saline-treated group (1.0 mg/kg JNJ-26366821 vs saline, p = 0.0008 and 0.3 mg/kg JNJ-26366821 vs saline, p = 0.0122); survival in JNJ-26366821 groups was not significantly different (p = 0.4787). Comparison of survival outcome at day 30 (Fisher's exact test), demonstrated significantly increased survival in both JNJ-26366821-treated groups over saline (1.0 mg/kg JNJ-26366821 (79%) vs saline (29%), p = 0.0012 and 0.3 mg/kg JNJ-26366821 (71%) vs saline (29%), p = 0.0087); and no significant difference in survival outcome between the two JNJ-26366821-treated groups (79% vs 71%, p = 0.7400). Additionally, survival following multiple doses of JNJ-26366821 (a 1, 2, or 3 dose regimen administered once daily) was assessed however, no additional survival benefit was observed in animals administered more than a single dose of JNJ-26366821 (Supplemental Fig. 1).   Fig. 2A). Log-rank comparison of the survival curves show statistical differences between treatment with 0.3 mg/kg and 1.0 mg/kg JNJ-26366821 (p = 0.0106 and p < 0.0001, respectively); survival in animals treated with 0.1 mg/kg and 3.0 mg/kg JNJ-26366821 was not statistically different than that in saline-treated animals (Log-rank p = 0.2515 and p = 0.0734, respectively). When comparing the survival curves (Log-rank test) for the various JNJ-26366821-treated groups, survival in the groups that received 0.1 mg/kg and 1.0 mg/kg were statistically different (p = 0.0009), whereas survival curves for animals administered 0.1 and 0.3 or 0.1 and 3.0 mg/kg were not statistically different (p = 0.1599 and p = 0.3606, respectively). Survival curves for animals that received 0.3 and 1.0 mg/kg were statistically different (p = 0.0451) whereas survival curves from animals receiving 0.3 and 3.0 mg/kg were not (p = 0.5674). Finally, survival curves for animals administered 1.0 mg/kg were statistically different from those administered 3.0 mg/kg (p = 0.0096). All log-rank statistical analyses are summarized in Table 1 (top set of values). In total, the group that was administered 1.0 mg/kg JNJ-26366821 was the only group was statistically different from all other groups.   www.nature.com/scientificreports/ Comparing the 30-day survival (Fisher's exact test) of animals administered saline or JNJ-26366821, survival was significantly increased in animals administered 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg JNJ-26366821 over the saline control (p = 0.0077, p < 0.0001, and p = 0.0355 respectively) no statistical difference was observed in the survival of animals administered 0.1 mg/kg JNJ-26366821 as compared to saline (p = 0.2124). Comparison of 30-day survival (Fisher's exact test) in the various JNJ-26366821 dose groups showed survival for animals administered 0.1 mg/kg was lower than animals administered 1.0 mg/kg JNJ-26366821 (p = 0.0020), whereas survival for animals administered 0.1 and 0.3 or 0.1 and 3.0 mg/kg JNJ-26366821 was not statistically different from one another (p = 0.2476 and p = 0.5639, respectively). Survival for animals that received 0.3 and 1.0 mg/kg or 0.3 and 3.0 mg/kg were not statistically different from one another (p = 0.0933 and p = 0.7702, respectively). Finally, survival curves for animals administered 1.0 mg/kg were statistically different from those administered 3.0 mg/kg (p = 0.0243).
JNJ-26366821 has a favorable dose reduction factor and enhances long term survival of C57BL/6 male mice. Male C57BL/6 mice were administered saline or JNJ-26366821 (1.0 mg/kg) 24 h after TBI at various radiation doses to determine the dose reduction factor (Fig. 5A). By plotting survival for the various radiation dose groups treated with either saline or JNJ-26366821, the dose of radiation correlating to 50% lethality over 30 days (LD50/30) was determined by probit analysis. For animals administered saline, the LD50/30 dose was 7.78 Gy (95% confidence interval: 7.65-7.90 Gy) and for animals administered JNJ-26366821 the LD50/30 dose was 8.65 Gy (95% confidence interval: 8.52-8.79 Gy). In conjunction, these values indicated a dose reduction factor of 1.113 (95% confidence interval: 1.063-1.197). From this study, the survival of animals irradiated at 8.0 and 8.5 Gy was monitored for 6 months ( Fig. 5B and C, respectively). Analysis of the survival curves for animals irradiated at 8.0 Gy indicated the curves were significantly different between saline and JNJ-2636682-treated groups as determined by log-rank test (p < 0.0001) and that survival was significantly higher at 6 months in animals administered JNJ-26366821 compared to those administered saline (94.7% vs 14.3%, p < 0.0001). Analysis of the survival curves for animals irradiated at 8.5 Gy indicated the curves were significantly different between saline and JNJ-2636682-treated groups as determined by log-rank test (p = 0.0003) and that survival was significantly higher at 6 months in animals administered JNJ-26366821 compared to those administered saline (52.6% vs 0%, p < 0.0001).

Discussion
Based on its favorable safety and tolerability profile in animals 15 and humans 16  www.nature.com/scientificreports/ with JNJ-26366821 in our irradiated mouse model did not show additional benefit over a single administration 24 h post-TBI. It is known that radiation-induced neutropenia and thrombocytopenia contribute significantly to lethality as a result of damage to the progenitor cells in the bone marrow [18][19][20] . To test the hypothesis that JNJ-26366821 increases thrombopoiesis in irradiated animals, which may partly contribute to survival benefit, we analyzed circulation peripheral blood cells in non-lethally irradiated (7 Gy) mice at 8 time points throughout the 30-day period following irradiation. This dose of radiation in the CD2F1 strain has been shown to induce severe bone marrow myelosuppression without associated mortality 21 . Since blood was collected through submandibular vein (only 20 µl) without sacrificing the animals, this model allows for multiple sampling times with surviving irradiated controls. The increase in circulating platelets by JNJ-26366821 supports our hypothesis that improvement in survival after JNJ-26366821 administration may be partly due to the thrombopoietic nature of the drug. The increased neutrophil counts observed after JNJ-26366821 administration may be due to stem cell mobilization into peripheral blood. Since JNJ-26366821 is shown to function by interaction through cMpl receptor, the drug's ability to rescue mice from radiation-induced lethality could be attributed to stimulation of bone marrow to promote faster recovery of progenitor cells [22][23][24] . In addition, the effect of JNJ-26366821 in protection of vasculature integrity may have also contributed to the survival benefit 25 .
There are data showing that patients who switched between romiplostim or the FDA approved TPO mimetic eltrombopag, either due to lack of efficacy or for other reasons such as adverse side effects, had a significant clinical benefit from the second drug compared to the one that was taken initially 9,26-30 . These results support the potential value of developing additional novel TPO agonists, even though Romiplostim and Eltromboag have already been approved for use in humans.

Conclusion.
In conclusion, the current study reports the first evaluations of JNJ-26366821 as a radiation mitigator in mice. The results demonstrate a single dose of JNJ-26366821 provides a significant survival benefit in lethally irradiated mice, with a window of protection from 4 to 24 h post-TBI and potentially beyond. Administration of JNJ-26366821 also mitigates platelet loss and enhances the rate of platelet and neutrophil recovery in CD2F1 mice. Confirmation of efficacy was conducted in male C57BL/6 male mice as seen by the observation of trends towards increased recovery of peripheral blood cell counts at the conclusion of the 30-day study, significant increases in colony forming units and megakaryocytes in animals administered JNJ-26366821 compared to those administered saline, and recovery of FLT3-L levels in animals administered JNJ-26366821. Probit analysis of survival in animals administered JNJ-26366821 or saline revealed a dose reduction factor of 1.113 and animals exposed to lethal radiation doses showed significant increases in survival up to 6 months following irradiation and subsequent administration of JNJ-26366821 compared to animals administered saline after irradiation. Taken together, these results demonstrate increased survival and recovery with JNJ-26366821-treatment following exposure to ionizing radiation highlighting the promise of JNJ-26366821 as a radiation countermeasure.

Materials and methods
Animals. Male CD2F1 mice (10-11 weeks old) were purchased from Envigo (Indianapolis, Indiana) and male C57BL/6 mice (10-11 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were housed in the Armed Forces Radiobiology Research Institute's (AFRRI) vivarium (accredited by the Association for Assessment and Accreditation of Laboratory Animal Care-International). Experimental animals were identified by tail tattoo and housed 4 per box in sterile polycarbonate boxes with filter covers (Microisolator, Lab Products Inc., Seaford, DE) with autoclaved hardwood chip bedding. The animals received Harlan Teklad Rodent Diet 8604 and acidified water (pH 2.5-3.0) ad libitum and were acclimatized for 1-2 weeks prior to the start of each study. The animal rooms were maintained at 21 ± 2 °C and 50 ± 10% relative humidity with 10-15 cycles of fresh air hourly and a 12:12 h light:dark cycle. All procedures pertaining to animals were reviewed and approved by the AFRRI Institutional Animal Care and Use Committee (IACUC) using the principles outlined in the National Research Council's Guide for the Care and Use of Laboratory Animals and performed in accordance with relevant guidelines and regulations. Animal studies were conducted in compliance with ARRIVE guidelines. Total body irradiation (TBI) studies. Since clinical trials in which humans are exposed to lethal doses of TBI cannot ethically be conducted, the approval process for radiation countermeasures is governed by guidance issued by the FDA known as the Animal Rule 31 . Under this guidance, pre-clinical data in one or more species along with safety and dosing data in humans is used to support the approval of drugs for use in this indication. The mouse is an accepted model for development of radiation countermeasures and it was therefore used to evaluate JNJ-26366821.
Mice were irradiated bilaterally at an estimated dose rate of ~ 0.6 Gy/min in the Cobalt-60 γ-irradiation facility at AFRRI (Bethesda, MD). Animals were placed in well-ventilated plexiglass chambers made specifically for irradiating mice. An alanine/Electron Spin Resonance (ESR) dosimetry system (American Society for Testing and Material Standard E 1607) was used to measure the dose rates in the cores of acrylic phantoms ( www.nature.com/scientificreports/ and 1 inch in diameter) located in all empty slots of the exposure rack in the plexiglass chamber. ESR signals were measured with a calibration curve based on standard calibration dosimeters provided by the National Institute of Standard and Technology (Gaithersburg, MD). The calibration curve was verified by inter-comparison with the National Physical Laboratory in the United Kingdom. The corrections applied to the measured dose rates in phantoms were for decay of the Co-60 source and for a small difference in mass-energy absorption coefficients for water and soft tissue at the Co-60 energy level. The radiation field was previously reported to be uniform within ± 2% 19,32 . Housing and care of animals after irradiation. After irradiation, animals were returned to their cages and monitored three to four times daily (early morning, late morning, late afternoon and evening). Sick animals were monitored closely and a health score was given at each time of monitoring in accordance with pre-defined criteria described and approved in the IACUC protocol. A predetermined threshold health score that necessitated euthanasia was also approved in the IACUC protocol; mice that reached this threshold health score were humanely euthanized. Pain and distress were monitored using several criteria including unresponsiveness, abnormal posture, unkempt appearance, immobility, labored breathing, respiratory distress, and lack of coordination. A mouse exhibiting any of the following symptoms was determined to be moribund and was euthanized: inability of the mouse to right itself, limb paralysis, abdominal breathing, a constant twitching, trembling, or tremor that lasted for more than 10 s, or greater than 35% weight loss as per IACUC policy. Animals were eutha- The efficacy of JNJ-26366821 as a radiation mitigator against morbidity and mortality was then further confirmed in C57BL/6 male mice. The C57BL/6 mice were administered 1 mg/kg JNJ-26366821 24 h post-TBI (8.0 Gy, ~ LD70/30) in the same manner as the CD2F1 mice. Mice in all studies were weighed prior to start of the study, animals outside ± 10% of the mean weight were excluded, and selected mice were randomized into groups of four animals per box. Survival was monitored up to four times a day when warranted for 30 days and surviving animals were euthanized at the completion of the observational period. Survival data was plotted as Kaplan-Meier plots and statistical significance of the survival differences was determined by log-rank and Fisher's exact tests using GraphPad Prism 8 software. Hematology studies with JNJ-26366821. To study the effects of JNJ-26366821 on recovery from hematopoietic injury following TBI, CD2F1 male mice (n = 10/group) were treated with either JNJ-26366821 (1 mg/kg) or its vehicle (saline) 24 h after TBI at a non-lethal dose of 7.0 Gy. A non-lethal dose was used to allow for animal survival while following the recovery from the hematopoietic injury that takes place following TBI. In addition, a group of sham-irradiated CD2F1 male mice were given either the drug or saline. Additionally, blood was collected from C57BL/6 male mice irradiated at an LD70/30 dose (8.0 Gy). Mice were anesthetized with isoflurane (Hospira Inc., Lake Forest, IL) and blood was collected from the submandibular vein using a 23 G needle at 2 h and 1, 3, 7, 10, 14, and 21 days post-TBI. Mice were allowed to recover fully from anesthesia and monitored closely for any signs of a post-anesthesia reaction or bleeding at the collection site before being returned to group-housed cages. Approximately 20 µL of blood was collected into tubes containing EDTA and blood was continually rotated until CBC/differential analysis of white blood cell (WBC), absolute neutrophil (NEU), monocytes (MON), lymphocyte (LYM), and platelet (PLT) counts with the HESKA Element HT™ 5 Analyzer system (HESKA Corporation, Loveland, CO).
FLT3-L determination in serum by ELISA. Blood was collected from male C57BL/6 mice via cardiocentesis into Microtainer serum collection tubes (BD item #36596, Franklin Lakes, New Jersey) from animals anesthetized with isoflurane (5% induction, 2% maintenance); following blood collection animals were euthanized. Serum was collected following centrifugation for 10 min at 2400 × g. Circulating FLT3-L levels were determined by ELISA analysis (R&D Systems item#MFK00, Minneapolis, MN) performed according to the manufacturer's instructions; serum was diluted 1:3. www.nature.com/scientificreports/ Colony forming unit assay from femoral bone marrow. Following euthanasia, femurs were collected and the bone marrow was extracted by flushing with IMDM with 2% FBS then plated (2 × 10 5 cells / plate) following protocols from the manufacturer (Mouse Colony-Forming Cell Assays Using MethoCult, Stem Cell Technologies, Cambridge, MA). Cultures were incubated for 14 days after plating and Granulocyte-macrophage colony forming units (CFU-GM), granulocyte-erythrocyte-monocyte-macrophage CFU (CFU-GEMM), colony-forming unit-erythroid (CFU-E) and erythroid burst-forming units (BFU-E) were identified and quantified using a Nikon TS100F microscope. Fifty or more cells were considered one colony. Data are show as mean ± standard error of mean (SEM) and statistical significance was determined between irradiated vehicle-treated and drugtreated groups 33 .
Megakaryocytes from Sternal bone marrow. Also following euthanasia, sterna were collected on 30 days post-TBI. The sterna were fixed in a 20:1 volume of fixative (10% buffered formalin) to tissue then embedded in paraffin and longitudinal 5 μm sections were stained with regular hematoxylin and eosin (H&E) stain following standard protocol 33 . Megakaryocyte numbers were counted from each slide.
Determination of dose reduction factor (DRF)/long term survival. Male C57BL/6 mice were distributed into one of twelve groups with 6 groups administered saline and 6 groups administered 1.0 mg/kg JNJ-26366821 24 h after TBI. The groups that ultimately received saline were irradiated at the following doses: 7. Statistical analysis. Survival data was plotted as Kaplan-Meier plots; GraphPad Prism 9 software was utilized to perform Fisher's exact test to compare survival at 30 days and a log-rank test to compare survival curves. Probit analysis was conducted with IBM SPSS Statistics 25.0 and a student's t-test was used to assess differences between groups. Averages were reported + /− standard error of the mean (SEM).