Introduction
Acute radiation syndrome (ARS) is a life-threatening illness caused by whole body or significant partial-body exposure to radiation doses > 1 Gy over a short period of time, as would occur in the event of a nuclear accident or attack[
1,
2]. The pathophysiology of ARS is well understood, and is similar across all mammals, involving detrimental effects on the hematopoietic, gastrointestinal, central nervous and cutaneous systems[
3]. In the hematopoietic subsyndrome of ARS (HSARS), toxicity is due to rapid bone marrow ablation[
1,
2], leading to pancytopenia. HSARS ultimately results in death due to infection and/or hemorrhage over the range of 2 weeks to 2 months, depending on the radiation exposure level[
1,
2]. While the availability of a radiation medical countermeasure (R-MCM) in the event of a large scale radiation emergency is critical for saving lives, currently no treatments are approved as R-MCMs by the US Food and Drug Administration (FDA). Since early 1990s, preclinical studies have demonstrated radioprotective and/or radiomitigating properties of various cytokines and cytokine cocktails, but their further clinical development was hindered by adverse reactions[
3‐
5]. A cocktail containing stem cell factor, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and interleukin-3 with or without a long-acting pegylated form of granulocyte-colony stimulating factor (G-CSF) has been shown to improve survival in murine and primate models of acute radiation[
6]. However, compared to the single agent approach, multicytokine combinations impose significant difficulties from the drug development perspective as well as logistical challenges for administration in the mass casualty scenario.
Typical treatment guidelines for HSARS include short- or long-term cytokine administration, depending on the radiation exposure level[
3]. While available cytokine products support the growth of some individual cell types (such as G-CSF for neutrophils)[
1], they do not consistently reduce overall mortality after TBI[
7]. An optimal R-MCM against HSARS would be able to regenerate all blood cell lineages, and, given the expected logistic impediments of a mass casualty scenario, should be effective when administered hours to days after exposure, preferably as a single dose, and in the absence of intensive supportive care. These requirements are not fulfilled by G-CSF, which affects only the granulopoietic lineage, requires multiple daily administrations, and improves survival only in combination with intensive, trigger-based medical management[
8]. Additionally, use of G-CSF in the context of radiation exposure was associated with delayed adverse effects, such as long-standing isolated thrombocytopenia[
9] and lung toxicity[
10].
We previously reported that a single administration of recombinant human IL-12 (rHuIL-12) given 24–25 hours after irradiation, in the absence of antibiotics, fluids or blood products, improved survival in both a murine HSARS model and in a proof-of-concept, open-label, male-only study in non-human primates (NHP)[
11]. These findings supported the further development of rHuIL-12 as an R-MCM for HSARS under the FDA Animal Rule, where efficacy is proven in an appropriate animal model (eg, non-human primates [NHP]) and safety is demonstrated in humans. Herein, we describe results of a randomized, blinded, efficacy study of rHuIL-12 as an R-MCM in a larger group of male and female rhesus monkeys performed under Good Laboratory Practice (GLP). This study advances rHuIL-12 towards approval under the Animal Rule.
Discussion
The data from this randomized, blinded, placebo-controlled study demonstrate a positive and significant effect of a single, subcutaneous injection of rHuIL-12, over a 10-fold dose range, on survival following lethal TBI (700 cGy; LD
90/60) in the rhesus monkey model of HSARS. The animal model used in this study has been validated at CiToxLAB North America as an established model of human HSARS, based on the occurrence of similar hematologic effects, infection and hemorrhage following TBI as reported for humans[
8,
11]. Compared with other studies using a similar NHP model[
8], our study did not include supportive care in the form of antibiotics, blood products and intravenous fluids, and therefore allows for a clearer demonstration of the effects of mitigating treatments and affords greater relevance for the mass casualty scenarios following a nuclear attack or accident, where intensive, hospital-based care would be significantly delayed and/or limited.
The current study, conducted under the FDA Animal Rule, provides a more robust demonstration of efficacy than our previously published proof-of-concept study[
11], as it was blinded, randomized, and GLP compliant, and involved more than twice the number of animals.
The mechanism by which IL-12 rescues animals following TBI involves the multiple effects of IL-12 on hematopoieses and immune function. Radiation-induced bone marrow suppression was mitigated by rHuIL-12: 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. Further, the increase, relative to controls, in mean platelet volume among animals treated with rHuIL-12 suggests that rHuIL-12 promoted release of newly formed platelets from the bone marrow. Quantitative analysis of the number and size of bone marrow regenerative pockets supports the conclusion that rHuIL-12 alone stimulates hematopoiesis, allowing for recovery of all major blood cell components.
Our in vivo observation that rHuIL-12 induced recovery of multiple hematopoietic lineages is consistent with previous reports in which IL-12 stimulated growth of hematopoietic stem cells and progenitors in vitro[
12‐
14], and prevented radiation-induced death of hematopoietic stem cells in vivo in a murine model of HSARS[
15]. This multilineage hematopoietic effect is also consistent with our previous findings in studies of tumor-bearing mice[
16] and with our observation that IL-12 receptors are present on hematopoietic stem cells[
11]. The observations that rHuIL-12 increased proportion of animals with higher blood counts, and higher blood counts were associated with survival, support the conclusion that rHuIL-12 improved survival by mediating early regeneration of multilineage bone marrow hematopoiesis, as compared to control.
Decrease of lymphocyte counts below 0.25 × 10
9/L has been established as a marker of irreparable lethal bone marrow damage in a large database of human victims of acute radiation[
17]. As such, it is important to note that in our study, the average lymphocyte nadir was 0.09 × 10
9/L among decedents in the control group, 0.14 × 10
9/L among decedents in the rHu-IL12-treated groups, and 0.27 × 10
9/L among survivors in all groups. These findings further support 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.
Consistent with the reduction in severe neutropenia and lymphopenia, the incidence of blood culture positivity for infection was significantly lower in rHuIL-12-treated groups 4 and 5 (47% and 44%, respectively) compared with than in the control group (86%). These data demonstrate that rHuIL-12, administered 24 hours after TBI, in the absence of antibiotics, decreased infectivity of broad-spectrum bacteria. These effects are consistent with and are likely due to the well-known multiple stimulatory effects of IL-12 on innate and adaptive immunity[
18]. On critical days 14–18 associated with deadly infections in this HSARS NHP model, the average lymphocyte counts in rHuIL-12 treated groups were higher than in control animals. The improvement of lymphocyte counts in this critical period may have provided enhancement of the T-cell-mediated immunity and B-cell antibody-mediated immunity (B cells) defense system, thereby contributing to higher survival in the treated groups as compared to the placebo group. Previous studies in mice have shown that during the early stages following exposure to lethal radiation, type 1 T-helper cell (Th1) function is reduced due to the suppression of endogenous IL-12 secretion from antigen presenting cells[
19,
20]. IL-12 administration may alleviate the radiation-induced impairment of Th1 function by promoting proliferation and activation of the NK cells, macrophages, and dendritic cells[
21], which can be damaged by radiation[
22]. Here we demonstrated that in irradiated monkeys rHuIL-12 increases plasma levels of IFN-γ, the hallmark of NK cell activation, as well as IL-18, and IP-10, similarly as we observed previously in non-irradiated monkeys[
11]. The tri-directional cross-talk between NK, macrophages and dendritic cells further promotes their maturation[
23], leading to the restoration of Th1 function and the establishment of early immune competence following TBI[
11]. Further, continuous production of endogenous IL-12 from pathogen-activated dendritic cells serves as a positive feedback loop and plays a key role in sustaining the initial response to exogenous IL-12[
24]. Taken together, these IL-12 generated immune-mediated effects can account in large part for the positive survival benefit observed in this study.
Consistent with the reduction in severe thrombocytopenia, rHuIL-12 treatment in this study was associated with lower severity of hemorrhage for animals that died or were euthanized prior to the scheduled termination on Day 60. In support of our finding that treatment with rHuIL-12 was associated with reduced severe thrombocytopenia and hemorrhage, we recently reported that hematopoietic stem cells, megakaryocytes and osteoblasts in the bone marrow express the IL-12 receptor β2 subunit (IL-12Rβ2)[
11], which is primary subunit for IL-12 signaling[
25]. The presence of IL-12Rβ2 receptors on these key bone marrow cells suggest that through its receptors, rHuIL-12 may promote proliferation and differentiation of the surviving stem cells and megakaryocytes following exposure to lethal radiation, thereby enhancing platelets regeneration and reducing severe thrombocytopenia. Indeed, quantitative analysis of the bone marrow in our current companion study showed that relative to controls, rHuIL-12 treated groups had higher numbers of megakaryocytes. The ability of rHuIL-12 to facilitate regeneration of platelets may be of clinical importance in indications other than HSARS mitigation, such as cancer, as there is currently no available drug that can facilitate platelet recovery following myelosuppressive therapies.
While leucocyte growth factors are recommended for use in victims of radiation, they are not approved by FDA for this indication. One published study of radiation mitigation in NHP demonstrated improved survival following exposure to lethal radiation for animals treated with rHuG-CSF in combination with intensive, trigger-based medical management (antibiotics, intravenous blood product transfusions, intravenous fluid replacement) compared with that of control animals that received only the medical management[
8]. We recently completed a randomized, blinded study comparing a single injection of rHuIL-12 or vehicle with 18 injections of rHuG-CSF in the NHP model without supportive care. Survival analysis confirmed superior survival in the rHuIL-12-treated group vs. both the control group and a G-CSF-treated group. Notably, G-CSF did not provide any survival benefit compared to control (Gluzman-Poltorak et al., submitted for publication).
In parallel to the animal efficacy studies, the safety and tolerability of rHuIL-12 has been examined in normal healthy subjects per the Animal Rule. A first in human (FIH) study was conducted to determine the safe and well-tolerated doses of rHuIL-12 via dose escalation (at doses ranging from 2 to 20 μg). The FIH study was followed by a phase 1b expansion study at the highest safe and well-tolerated dose from the FIH study of 12 μg (Gokhale et al., accepted for publication). The 12 μg unit human dose for a 70 kg adult can be converted to 171 ng/kg rhesus monkey dose using a weight based conversion and this dose is within the efficacious dose range as determined in our rhesus monkeys studies.
Methods
Ethics statement
All procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of CiToxLAB Research, Inc.
Animals
Studies were conducted in compliance with the Good Laboratory Practice (21 CFR Part 58) at CitoxLAB North America (a Contract Research Organization, Montreal, Quebec, Canada). Rhesus monkeys (Macaca mulatta) were obtained from the Yongfu County Xingui Wild Animals Raising Ltd., China. Monkeys (3 to 5 years old, and 3.0 to 5.7 kg at the start of treatment) were housed individually and acclimated for ≥ 5 weeks prior to irradiation. Harlan Teklad Certified Hi-Fiber Primate Diet #7195C (Harlan Laboratories, Indianapolis, Indiana) was provided twice daily.
Experiment design
The dose of 700 cGy (60 cGy/minute from a Theratron 1000 Co
60 source [Best Theratronics; Ottawa, Ontario, Canada]) was based on available historical data from CiToxLAB North America. TBI was conducted with animals in a vertical position, as described previously[
11]. For homogenous dose distribution, the first half-dose was delivered anteroposterior and the second half-dose was delivered posteroanterior. Dosimetry was verified to be within 10% of prescribed dose using nanodot chips (Landauer, Inc., Glenwood, Illinois, USA) positioned on the front and back of each animal.
In the main study, male and female animals (45 each; 9 animals per sex per dose group) were randomized, stratified by body weight to the following doses of clinical grade rHuIL-12 administered by SC injection between the scapulas approximately 24–25 hours following TBI: vehicle control (Group 1), 50 ng/kg (Group 2), 100 ng/kg (Group 3), 250 ng/kg (Group 4), or 500 ng/kg (Group 5). The concentration of the test item in each dosing sample was verified by Intertek Pharmaceutical Services (San Diego, CA) using the Quantikine® Human IL-12 ELISA Kit (R&D Systems Inc., Minneapolis, USA). The pathologist and study staff other than the study team leader and those involved with irradiation were blinded. Specifically, animal care and euthanasia decisions were made by the blinded personnel.
Symptomatic care
The following products were permitted as symptomatic care: buprenorphine (0.01-0.015 mg/kg/dose BID or TID, SC) for pain; bupivacaine (0.25%) topically for management of mouth ulcers; Pepto-Bismol for management of diarrhea; snacks or supplements (Rhesus Liquid Diet [BIO-SERV; Frenchtown, New Jersey], Ensure® [Abbott Laboratories, Abbott Park, Illinois, USA], vegetables, juices, or crushed cookies with banana) for anorexia; topical hydrotherapy and/or iodine 1% for wounds.
Assessments
Decreases in appetite (based on food intake) and physical activity were recorded daily and scored as follows: 1 = slight; 2 = moderate; and 3 = severe. A detailed physical examination was performed prior to rHuIL-12 dosing and twice weekly thereafter. Body temperature (auricular) was taken prior to irradiation and on Days 3–10, 12, 14, 16, 18, 30, 45, and 60, or when clinically justified. Blood sampling (0.5 mL) for peripheral blood counts was performed prior to irradiation and at Days 5, 10, 12, 14, 16, 18, 30, 45, and 60. Blood was collected for hemoculture in cases of febrile neutropenia (absolute neutrophil count <0.05 109/L together with rectal body temperature ≥104° F/40.0°C) and at necropsy.
Terminal procedure
Animals were euthanized prior to Day 60 if any of the following criteria were observed: respiratory distress; complete anorexia for 3 day; loss of > 20% of initial body weight over a 3 day period; severely decreased activity level (recumbent during an entire observation period or unresponsiveness to touch); acute loss of > 20% estimated blood volume; generalized seizure activity; abnormal appearance (posture, rough coat, head down, exudates around eyes and nose, pallor, tucked abdomen and clinical appearance) associated with abnormal vital signs: severe dehydration with hypothermia (decreasing rectal temperature reaching <34.6°C and severely decreased activity level) or hyperthermia (temperature >40.1°C and severely decreased activity level). Euthanasia decisions were made by a team of technicians and veterinarians blinded to the animal group assignment. Surviving animals were euthanized at Day 60 following TBI.
Necropsy comprised an external macroscopic examination, a detailed internal examination, evaluation of organ weights and gross pathology, and collection of tissues for histopathology. Presence of hemorrhage was scored for major organs as follows: 0 = absence; 1 = minimal; 2 = slight; 3 = moderate; 4 = marked; 5 = severe. For histological examination, tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin-phloxin (H & E).
Microbiological analysis was conducted on brain, heart, kidney, liver, both lungs, and spleen. Bacterial growth was scored (0 to 4) for each organ. The total score was summed for each animal; the mean score was calculated for each treatment group.
Pharmacokinetics and pharmacodynamics of rHuIL-12 and bone marrow histopathology in irradiated rhesus monkeys
An evaluation of pharmacokinetics and pharmacodynamics of rHuIL-12 in irradiated rhesus monkeys was conducted using separate animals randomized to the same doses of rHuIL-12 as in the survival cohort (2 per sex per group). Blood samples from animals treated with SC rHuIL-12 or placebo were collected at the following time points: pretreatment (approximately 2 weeks prior to irradiation), 24 hours after irradiation immediately before rHuIL-12 dosing, and at 1, 3, 5, 8, 12, 24, 48, 72, 96, 120, 144, 240, and 264 hours after rHuIL-12 dosing. The concentrations of rHuIL-12 and IFN- γ in monkey plasma were determined by validated GLP ELISA methods at Intertek ALTA Analytical Laboratory (San Diego, CA). rHuIL-12 was measured using the Human IL-12 HS ELISA kit (catalog # PHS120, HS120, or SS120 or equivalent; R&D Systems). The lower limit of quantitation was 3.5 pg/mL in 100% monkey plasma. IFN-γ was measured using the Monkey IFN-γ ELISA kit (catalog # 3420 M-1H-20, or equivalent; Mabtech, Inc., Mariemont, OH ). The lower limit of quantitation was 7.5 pg/mL in 100% monkey plasma. Interleukin-18 (IL-18) and interferon γ-induced protein (IP-10) levels were determined using non-GLP qualified ELISA methods at Neumedicines, Inc. IL-18 was assayed using the MBL International Corporation Human IL-18 ELISA (R&D Systems, Minneapolis, MN). The lower limit of quantitation was 120 pg/mL in 100% monkey plasma. IP-10 concentrations were determined in plasma using a Quantikine Human CXCL10/IP-10 ELISA (R&D Systems, Minneapolis, MN). The lower limit of quantitation was 15 pg/mL in 100% monkey plasma. Standard non-compartmental analyses were performed using Phoenix™ WinNonlin® Version 6.3 (WinNonlin; Pharsight Corporation, Mountain View, CA).
The separate cohort animals were euthanized on Day 12 after TBI, except one animal that underwent unscheduled euthanasia on Day 11. All animals were included in the bone marrow analysis. Two H & E femur sections for each animal were scanned on an Olympus BX41 compound microscope. Images of approximately 40 fields of view encompassing each femur section in its entirety were acquired on Infinity Analyze software v5.0 at a magnification of 10×. The number of bone marrow regeneration islands was determined by visual quantification in each field of view in each section. The total area of bone marrow regeneration was determined using ImageJ software, version 1.46. The mean number of regeneration islands and mean area of regeneration from two sections per animal were used in the statistical analyses. The number of megakaryocytes was determined visually in each femur section.
Statistical analysis
All statistical comparisons were conducted for sex and for the entire study population. Survival functions were estimated using the Kaplan-Meier product-limit method applied on daily intervals. The control group was compared to each of the other treated groups using the Mantel log-rank test. Predefined GLP survival analysis was performed at CiToxLAB.
Group comparisons for incidences of severe neutropenia (defined as neutrophil count < 0.05 × 109/L), severe thrombocytopenia (defined as platelet count < 10 × 109/L), and hemoculture positivity (sepsis) were performed using Fisher Exact test. If the overall comparison was significant (p ≤ 0.05), pair-wise comparisons between control group and each dosing group was done using the Fisher Exact test.
Group means for bone marrow regeneration data (number and area of regeneration islands) were compared by a one-tailed t test using the statistical software program Prism version 6 (GraphPad, San Diego, CA). Differences with p <0.05 were considered significant.
Acknowledgements
These studies were supported by the Biomedical Advanced Research and Development Authority (BARDA), US Dept of Health and Human Services Contract Nos. HHSO100200800060C and HHSO100201100037C. The content is solely the responsibility of the authors and does not necessarily represent the official views of BARDA.
The authors thank Simon Authier , DVM, MBA, PhD, and Alexis Ascah, PhD, (CiToxLAB North America; Laval, Quebec, Canada) for support in designing and conductance of the animal studies and Martha Sensel, PhD, a medical writer, for assistance in writing the manuscript.
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Competing interests
All authors are employees of and own equity of $10,000 or more in Neumedicines, Inc. The authors declare that they have no competing interests.
Authors’ contributions
ZGP designed research, performed research, collected data, analyzed and interpreted data, performed statistical analysis, and wrote the manuscript. SRM performed research, collected, analyzed and interpreted data, and wrote the manuscript. VV analyzed and interpreted data, performed statistical analysis, and wrote the manuscript. HK collected and analyzed data. LAB designed research, analyzed and interpreted data, and wrote the manuscript. All authors read and approved the final manuscript.