The Toll-Like Receptor 5 Agonist Entolimod Mitigates Lethal Acute Radiation Syndrome in Non-Human Primates

Abstract

There are currently no approved medical radiation countermeasures (MRC) to reduce the lethality of high-dose total body ionizing irradiation expected in nuclear emergencies. An ideal MRC would be effective even when administered well after radiation exposure and would counteract the effects of irradiation on the hematopoietic system and gastrointestinal tract that contribute to its lethality. Entolimod is a Toll-like receptor 5 agonist with demonstrated radioprotective/mitigative activity in rodents and radioprotective activity in non-human primates. Here, we report data from several exploratory studies conducted in lethally irradiated non-human primates (rhesus macaques) treated with a single intramuscular injection of entolimod (in the absence of intensive individualized supportive care) administered in a mitigative regimen, 1-48 hours after irradiation. Following exposure to LD50-70/40 of radiation, injection of efficacious doses of entolimod administered as late as 25 hours thereafter reduced the risk of mortality 2-3-fold, providing a statistically significant (P<0.01) absolute survival advantage of 40-60% compared to vehicle treatment. Similar magnitude of survival improvement was also achieved with drug delivered 48 hours after irradiation. Improved survival was accompanied by predominantly significant (P<0.05) effects of entolimod administration on accelerated morphological recovery of hematopoietic and immune system organs, decreased severity and duration of thrombocytopenia, anemia and neutropenia, and increased clonogenic potential of the bone marrow compared to control irradiated animals. Entolimod treatment also led to reduced apoptosis and accelerated crypt regeneration in the gastrointestinal tract. Together, these data indicate that entolimod is a highly promising potential life-saving treatment for victims of radiation disasters.

Fig 1. Improved survival of non-human primates (NHPs) injected with entolimod 1–48 hours after lethal irradiation.

Kaplan-Meier plots of non-human primate (NHP) survival over the 40 days following exposure to LD_50/40 –LD_75/40 doses of total body irradiation (TBI) are shown. Time frame of entolimod efficacy (panels A, B) was evaluated in studies Rs-03 (treatment at 1 h after LD_75/40 TBI; N = 10) and Rs-06 (treatment at 16, 25, or 48 h after LD_75/40 TBI; N = 8–12). Dose-dependence of entolimod efficacy (panels C, D) was tested in studies Rs-09 (treatment at 1 h after LD_50/40 TBI; N = 18) and Rs-14 (treatment at 25 h after LD_50/40 TBI; N = 10).

Fig 2. Accelerated hematological recovery of peripheral blood in NHPs injected with entolimod 16–48 hours post-irradiation.

NHPs were treated with a single injection of entolimod at 16–48 hours after LD_50/40 or LD_75/40 TBI. A, C, E, G: Effect of 40 μg/kg entolimod administered at different time points (16, 25 or 48 hours) after 6.5 Gy TBI (LD_75/40; study Rs-06; N = 8–12). B, D, F, H: Effect of different entolimod doses (10 or 40 μg/kg) administered at 25 hours after 6.75 Gy TBI (LD_75/40; study Rs-14; N = 10). Cytopenia/anemia thresholds: dotted lines—Grade 3 (platelets <50,000/μL; neutrophils <1,000/μL; hemoglobin <80 g/L); dashed lines—Grade 4 (platelets <10,000/μL; neutrophils <500/μL; hemoglobin <65 g/L). Error bars represent standard errors.

Fig 3. Enhanced morphological recovery of hematopoietic and lymphoid organs in NHPs treated with entolimod post-irradiation.

NHPs were treated with a single injection of 40 μg/kg entolimod 16, 25 or 48 hours after LD_75/40 total body irradiation (TBI). Tissue morphology was assessed 40 days post-irradiation and compared to that in control NHPs treated with vehicle 16 hours after LD_75/40 TBI. Representative histological images (hematoxylin-eosin staining) of sternum bone marrow sections, thymuses, spleens and mesenteric lymph nodes of animals that survived to study termination on Day 40 post-TBI (study Rs-06) are shown. Scale bars: 100 μm for bone marrow, 200 μm for thymus, spleen, and lymph node.

Fig 4. Entolimod treatment ameliorates radiation damage in the gastrointestinal (GI) tract.

A, B. Small intestine sections from NHPs 8 hours after exposure to 6.5 Gy TBI and treatment with vehicle or 40 μg/kg entolimod 1 h later (study Rs-04). Blue—DAPI nuclear staining, red—smooth muscle actin immunostaining. A. TUNEL staining showing fewer apoptotic cells (green) in GI crypts of entolimod-treated NHPs (scale bar 100 μm); B. SOD2 immunostaining (green) showing more positive cells in GI villi (arrowheads) and lamina propria (arrows) of entolimod-treated NHPs (scale bar 50 μm). C, D. Small intestine sections of NHPs 7 days after exposure to 11 Gy TBI and treatment with vehicle or 40 μg/kg entolimod 4 h later (study Rs-22). C. Visualization of proliferating cells in the jejunum crypts: EdU (10 mg/kg i.v. 1 h before euthanasia) inclusion in replicating DNA (green) and phosphohistone 3 immunostaining of mitotic cells (red) showing more intensive proliferation of GI crypts in entolimod-treated NHPs (scale bar– 200 μm). D. H&E staining of ileum sections: upper panels—low magnification (scale bar– 200 μm), lower panels—high magnification (scale bar– 50 μm).

Fig 5. Effect of entolimod treatment on G-CSF and IL-6 levels in peripheral blood of irradiated NHPs.

A, B: Effect of different entolimod doses administered 1 h after LD_50/40 TBI (6.75 Gy; study Rs-09; N = 18). C, D: Effect of different entolimod doses administered 25 h after LD_50/40 TBI (6.75 Gy; study Rs-14; N = 10). E, F: Comparison of dose-dependence of background-adjusted Area Under the Curve (AUC_0-24) values for G-CSF and IL-6 after entolimod treatment given 1 h versus 25 h after LD_50/40 TBI (with dashed log-linear regression lines). Error bars represent standard errors.

Fig 6. Schematic presentation of mechanism(s) underlying anti-acute radiation syndrome (ARS) effects of entolimod.

Entolimod binding to Toll-like receptor 5 (TLR5) initiates a cascade of events, all merging at attenuation of major pathological processes—leading causes of death in ARS: damage to hematopoietic (HP) and gastrointestinal (GI) systems resulting in bleeding and sepsis. The immediate TLR5-dependent effectors include anti-oxidants (e.g., SOD2), anti-apoptotic factors (both NF-κB-dependent (i.e., IAP and Bcl family members [–70]), and NF-κB-independent (i.e., PI3K/AKT, MKP7 and STAT3 [, –73]), hematopoietic cytokines (e.g., G-CSF and IL-6 [49]), anti-infective factors [, –95] and processes (e.g., neutrophil mobilization). In addition, stimulation of TLR5 is expected to inhibit radiation-induced aseptic inflammation involved in secondary tissue damage [64] e.g. via induction of an anti-inflammatory cytokine IL-10, IL-1β antagonist (IL-1βa) [76] and stimulation of mesenchymal stem cells (MSC) known to express TLR5 [77, 78] and to have anti-inflammatory properties [79]. Together with fibroblasts that can be induced to proliferate via TLR5 stimulation [104], MSC may also contribute to wound-healing processes [79]. Dashed lines show all molecular connections downstream of TLR5 that are not directly established for entolimod, but are extrapolated from published data on TLR5-dependent effects of flagellin.