Association of Hematological Nadirs and Survival in a Non-Human Primate Model of Hematopoietic Syndrome of Acute Radiation Syndrome

Recombinant human interleukin-12 (rHuIL-12, HemaMax^TM) is being developed for mitigation of HSARS under the FDA Animal Rule using a NHP model of HSARS for proof of efficacy and clinical studies in healthy human subjects to demonstrate safety. We have shown previously that a single injection of rHuIL-12 administered 24-25 hours after lethal total body irradiaton (TBI), in the absence of antibiotics, fluids or blood products, resulted in improved survival while filgrastim (G-CSF) did not provide any survival benefit in our NHP HSARS model (Basile et al 2012, Gluzman-Poltorak et al 2014, Gluzman-Poltorak et al 2014).

The mechanism by which IL-12 mitigates HSARS following TBI appears to involve multiple effects of IL-12 on hematopoieses. In our previous studies, animals treated with rHuIL-12 showed statistically significant reductions in the occurrence of severe neutropenia and severe thrombocytopenia, as well as attenuated nadirs for lymphocytes, neutrophils, platelets, and reticulocytes.

To characterize further relationship between survival and hematological nadirs (lymphocytes, neutrophils, platelets, RBCs and reticulocytes) in the rhesus model of HSARS and to better understand the effects of rHuIL-12 versus G-CSF on blood cell nadirs, we undertook a meta-analysis analysis across three studies in irradiated rhesus monkeys.

Animals used in this analysis were irradiated (700 cGy) and treated with a single subcutaneous injection of vehicle (n=64) or rHuIL-12 50-500ng/kg (n=108) 24-25 hours after irradiation, or daily subcutaneous injections of G-CSF at 10μg/kg/d for 18 days starting 24-25 hours after irradiation (n=26). Males and females were equal in each group.

Lymphocytes, neutrophils and platelets were significantly lower in decedents versus monkeys that survived to day 60 overall and this was true in each treatment group (p<0.001, Wilcoxon rank-sum test). The mean RBC nadir was significantly higher in survivors compared to non-survivors in the rHuIL-12 group (p = 0.008), but not in the control group or the G-CSF group.

Lymphocytes nadir appears to be the strongest and most consistent predictor of death followed by neutrophils and platelets (Spearman’s rank correlation). RBCs and reticulocytes are less informative in terms of predicting survival status.

In addition, the receiver operating characteristic (ROC) curve area under the curve (AUC) by nadir value was assessed. The operating characteristics for lymphocytes, neutrophils and platelets allow for relatively large positive predictive values (PPV) of death with relatively high sensitivity. A cutoff value for lymphocytes nadir of 0.08x10⁹/L (values less than or equal predicting death and higher values predicting life) allows for largest PPVs (97.2% and 92.5%) with 76.1% and 62.7% sensitivities for control and rHuIL-12 treatments, respectively. A cutoff value for neutrophils nadir of 0.03x10⁹/L permits for 84% and 71.1% PPVs with sensitivities of 91.3% and 91.5 % for control and rHuIL-12 treatments, respectively. A cutoff value for platelets nadir of 9x10⁹/L permits for 84.1% and 76.8% PPVs with sensitivities of 80.4% and 72.9 % for control and rHuIL-12 treatments, respectively. RBCs and reticulocytes were found less informative.

To conclude, in the rhesus model of HSARS we have observed that an augmented hematological nadirs generally predicts an increased potential for survival as this effect reflects early bone marrow regeneration. The nadir for lymphocytes appears to be the strongest and most consistent predictor of death. Decrease of lymphocyte counts has been established as a best marker of bone marrow damage in a large database of human victims of acute radiation (METREPOL, Fliedner et al 2001). Thus, the correlation of our results with the human data supports the validity of our animal model as an accurate representation of human HSARS and its ability to predict effectiveness in humans exposed to lethal radiation. These data also suggest that the significant increase in early bone marrow regeneration seen in our studies, resulting in increases in nadir values for all major blood cell types, may be the main mechanism of action by which rHuIL-12 mitigates the lethality of HSARS.

This project has been entirely funded with Federal funds from BARDA/ASPR/DHHS under Contract No. HHSO100201100037C.