Table 1.
Efficacy of a single injection of entolimod in increasing 40-day survival of lethally irradiated NHPs when administered at different dose levels within 1–48 hours after TBI.
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 LD50/40 –LD75/40 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 LD75/40 TBI; N = 10) and Rs-06 (treatment at 16, 25, or 48 h after LD75/40 TBI; N = 8–12). Dose-dependence of entolimod efficacy (panels C, D) was tested in studies Rs-09 (treatment at 1 h after LD50/40 TBI; N = 18) and Rs-14 (treatment at 25 h after LD50/40 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 LD50/40 or LD75/40 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 (LD75/40; 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 (LD75/40; 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.
Table 2.
Mean nadir values of neutrophils, platelets and hemoglobin in peripheral blood following total body irradiation and vehicle or entolimod treatment.
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 LD75/40 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 LD75/40 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.
Table 3.
Histological evaluation of hematopoietic/lymphoid organs from NHPs that survived to day 40 after 6.5 Gy TBI and vehicle or entolimod treatment (study Rs-06)
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).
Table 4.
Histological evaluation of GI tract segments on day 7 after 11 Gy TBI and vehicle or 40 μg/kg entolimod treatment at +4 hours (study Rs-22, N = 4/group).
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 LD50/40 TBI (6.75 Gy; study Rs-09; N = 18). C, D: Effect of different entolimod doses administered 25 h after LD50/40 TBI (6.75 Gy; study Rs-14; N = 10). E, F: Comparison of dose-dependence of background-adjusted Area Under the Curve (AUC0-24) values for G-CSF and IL-6 after entolimod treatment given 1 h versus 25 h after LD50/40 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 [67–70]), and NF-κB-independent (i.e., PI3K/AKT, MKP7 and STAT3 [54, 71–73]), hematopoietic cytokines (e.g., G-CSF and IL-6 [49]), anti-infective factors [54, 90–95] 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.