HemaMax™, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8–9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit–expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD50/30) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12–14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin

m-HemaMax administration increased plasma m-HemaMax and IFN-γ levels in irradiated and non-irradiated mice.

<p>Animals received m-HemaMax subcutaneously at a dose of (a) 10 ng/mouse, (b) 20 ng/mouse, (c) 40 ng/mouse, or (d) 200 ng/mouse in the absence of irradiation or at 24 hours after an LD_90/30 of TBI. The plasma concentrations of m-HemaMax and IFN-γ were determined by ELISA in blood samples withdrawn at the indicated times. The y-axis scale in (d) is 8 times greater than those in (a) and (b) and 5 times greater than that in (c). n = 3 per timepoint in each group.</p

Plasma PK Characteristics of m-HemaMax in Irradiated and Non-Irradiated Mice.

<p>Animals received m-HemaMax subcutaneously at a dose of 10 ng/mouse, 20 ng/mouse, 40 ng/mouse, or 200 ng/mouse in the absence of irradiation or at 24 hours after an LD_90/30 of TBI. The plasma concentrations of m-HemaMax were determined by ELISA.</p><p>AUC = area under the curve; C_max = maximum plasma concentrations; NR = no irradiation; R = irradiation; TBI = total body irradiation; T_max = time to achieve the maximum plasma concentration; t_1/2 = half life.</p

HemaMax initiated at least 24 hours after irradiation increased percentage of survival of unsupported monkeys.

<p>Individual dosing groups (a) and the pooled HemaMax dosing group (b) are shown. Animals were subjected to an LD_50/30 of TBI at day 0 and subsequently received either vehicle (P5.6TT) or HemaMax subcutaneously at the indicated dosing regimens. Supportive care was prohibited during the study. Animals were monitored for survival up to 30 days. ^a One animal was excluded from the study due to a broken tooth.</p

m-HemaMax at low dose suppresses radiation-induced intestinal injury in mice.

<p>The IL-12Rβ2 expression in jejunal crypts (a) and the suppression of jejunal expression of LGR5 (b), a GI stem cell injury marker, are shown. Mice received vehicle (P5.6TT) or m-HemaMax subcutaneously at the indicated doses either in the absence of irradiation or 24 hours after TBI (8.6 Gy). Three days after irradiation, jejunum tissues were removed and immunohistochemically stained for IL-12Rβ2 (a) or LGR5 (b). Representative images show LGR5 in brown as indicated with arrows. Magnification = 400.</p

Irradiated rhesus monkeys receiving HemaMax had less body weights loss than animals receiving vehicle.

<p>Body weights in Kg (a and b) and in percentage (c and d) are shown for the 100 ng/Kg and 250 ng/Kg dose groups. Monkeys were subjected to an LD_50/30 of TBI at day 0 and subsequently received either vehicle (P5.6TT) or HemaMax subcutaneously at the indicated dosing regimens. Supportive care was prohibited during the study. Body weights were recorded every other day for up to day 30.</p

m-HemaMax promotes hematopoietic recovery in irradiated mice.

<p>Representative sections of femoral bone marrow from non-irradiated, untreated mice that were stained for IL-12Rβ2 (orange color) are shown in (a). Animals were subjected to TBI (8.0 Gy) and subsequently received vehicle (P5.6TT) or m-HemaMax (20 ng/mouse) subcutaneously at the indicated times post irradiation (b–f). An additional group of mice received HemaMax at 24 hours after TBI (g). Femoral bone marrow was immunohistochemically stained for IL-12Rβ2 (orange color) 12 days after irradiation. While bone marrow from mice treated with vehicle lacked IL-12Rβ2–expressing cells and showed no signs of hematopoietic regeneration (b), mice treated with m-HemaMax showed hematopoietic reconstitution and the presence of IL-12Rβ2–expressing megakaryocytes, myeloid progenitors, and osteoblasts (c–f). Mice treated with HemaMax showed IL-12Rβ2–expressing osteoblasts but lacked megakaryocytes (g). Magnification = 100×.</p

Similar exposures to m-HemaMax and HemaMax at species-specific equivalent doses in mice and rhesus monkeys.

<p>The plot of plasma AUC_last of m-HemaMax versus the dose administered to mice in the absence of irradiation was linear at doses from 10 ng/mouse to 40 ng/mouse. The plasma AUC_last of HemaMax at monkey equivalent doses of 20 ng/Kg and 80 ng/Kg was in good agreement with the extend of dose-dependent increases in m-HemaMax exposure in mice.</p