Radioprotective Efficacy of Gamma-Tocotrienol in Nonhuman Primates Author(s): Vijay K. Singh , Shilpa Kulkarni , Oluseyi O. Fatanmi , Stephen Y. Wise , Victoria L. Newman , Patricia L. P. Romaine , Howard Hendrickson , Jatinder Gulani , Sanchita P. Ghosh , K. Sree Kumar and Martin Hauer-Jensen Source: Radiation Research, 185(3):285-298. Published By: Radiation Research Society DOI: http://dx.doi.org/10.1667/RR14127.1 URL: http://www.bioone.org/doi/full/10.1667/RR14127.1

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RADIATION RESEARCH

185, 285–298 (2016)

0033-7587/16 $15.00 Ó2016 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR14127.1

Radioprotective Efficacy of Gamma-Tocotrienol in Nonhuman Primates Vijay K. Singh,a,b,1 Shilpa Kulkarni,b Oluseyi O. Fatanmi,b Stephen Y. Wise,b Victoria L. Newman,b Patricia L. P. Romaine,b Howard Hendrickson,c Jatinder Gulani,d Sanchita P. Ghosh,b K. Sree Kumarb and Martin Hauer-Jensenc a

Department of Radiation Biology, F. Edward He´bert School of Medicine, ‘‘America’s Medical School’’ Uniformed Services University of the Health Sciences, Bethesda, Maryland; b Armed Forces Radiobiology Research Institute, Bethesda, Maryland; c Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare Systems, Little Rock, Arkansas; and d Veterinary Sciences Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland

of GT3 as a promising radioprotector. Singh, V. K., Kulkarni, S., Fatanmi, O. O., Wise, S. Y., Newman, V. L., Romaine, P. L. P., Hendrickson, H., Gulani, J., Ghosh, S. P., Kumar, K. S. and Hauer-Jensen, M. Radioprotective Efficacy of Gamma-Tocotrienol in Nonhuman Primates. Radiat. Res. 185, 285–298 (2016).

Ó 2016 by Radiation

Research Society

INTRODUCTION

The search for treatments to counter potentially lethal radiation-induced injury over the past several decades has led to the development of multiple classes of radiation countermeasures. However, to date only granulocyte colonystimulating factor (G-CSF; filgrastim, Neupogen)and pegylated G-CSF (pegfilgrastim, Neulasta) have been approved by the United States Food and Drug Administration (FDA) for the treatment of hematopoietic acute radiation syndrome (ARS). Gamma-tocotrienol (GT3) has demonstrated strong radioprotective efficacy in the mouse model, indicating the need for further evaluation in a large animal model. In this study, we evaluated GT3 pharmacokinetics (PK) and efficacy at different doses of cobalt-60 gamma radiation (0.6 Gy/min) using the nonhuman primate (NHP) model. The PK results demonstrated increased area under the curve with increasing drug dose and half-life of GT3. GT3 treatment resulted in reduced group mean neutropenia by 3–5 days and thrombocytopenia by 1–5 days. At 5.8 and 6.5 Gy total-body irradiation, GT3 treatment completely prevented thrombocytopenia. The capability of GT3 to reduce severity and duration of neutropenia and thrombocytopenia was dose dependent; 75 mg/kg treatment was more effective than 37.5 mg/kg treatment after a 5.8 Gy dose. However, the higher GT3 dose (75 mg/kg) was associated with higher frequency of adverse skin effects (small abscess) at the injection site. GT3 treatment of irradiated NHPs caused no significant difference in animal survival at 60 days postirradiation, however, low mortality was observed in irradiated, vehicle-treated groups as well. The data from this pilot study further elucidate the role and pharmacokinetics of GT3 in hematopoietic recovery after irradiation in a NHP model, and demonstrate the potential

Acute radiation syndrome (ARS) occurs in humans after whole-body or significant partial-body exposure to ionizing radiation with doses greater than 1 Gy, delivered at relatively high rates. Clinical components of ARS include the hematopoietic sub-syndrome (H-ARS, 2–6 Gy), gastrointestinal sub-syndrome (GIS; 6–8 Gy) and the cerebrovascular sub-syndrome (.8 Gy) (1). In H-ARS, the number of red blood cells (RBCs) and white blood cells (WBCs), neutrophils, platelets as well as other cells decline and susceptibility to potentially fatal infection increases. In GIS, breakdown of the gastrointestinal (GI) system results in translocation of GI bacteria to other organs, which ultimately results in sepsis and eventual death. While HARS and GIS are collectively well recognized as the major sub-syndromes of ARS, this tends to oversimplify the clinical reality of ARS, which often involves complex, multi-organ dysfunctions (2–4). The cerebrovascular subsyndrome is considered incurable, whereas individuals receiving lower radiation doses that result in either the HARS alone or in combination with GIS are more likely to be responsive to countermeasures. Although efforts to produce such medical countermeasures to ARS were initiated over half a century ago, to date, only granulocyte colony-stimulating factor (G-CSF; filgrastim, Neupogen) and pegylated G-CSF (pegfilgrastim, Neulasta) have been approved for H-ARS by the U.S. Food and Drug Administration (FDA) (5–10). Most recently, natural products have been investigated for prevention and therapy of human diseases because they are ‘‘generally recognized as safe’’ and appropriate for medicinal purposes. Unlike their synthetic analogs, they are well tolerated and minimally toxic, even in the upper ranges of dietary intake (11, 12). Vitamin E represents a family of

Editor’s note. The online version of this article (DOI: 10.1667/ RR14127.1) contains supplementary information that is available to all authorized users. 1 Address for correspondence: F. Edward He ´ bert School of Medicine ‘‘America’s Medical School’’, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Ave., Bethesda, MD 208895603; email: [email protected]. 285

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compounds that act as antioxidants, which regulate peroxidation reactions and control free-radical production within the body (13, 14). This family of compounds has eight different isoforms that belong to two categories: four saturated (a, b, c and d) tocopherol analogs and four unsaturated tocotrienol analogs. These eight components are collectively known as tocols. Tocopherols and tocotrienols share common structural features of a chromanol ring and a 15-carbon tail at the C-2 position derived from homogentisic acid and phytyl diphosphate, respectively. Tocotrienols differ structurally from tocopherols by the presence of three trans-double bonds in the hydrocarbon tail. The isomeric forms of tocopherol and tocotrienol are distinguished by the location of methyl groups on the chromanol rings. Recent studies suggest that both the molecular and therapeutic targets of the tocotrienols are distinct from those of the tocopherols. A number of studies have shown that tocotrienols are superior antioxidants compared to tocopherols (5, 15–19). Gamma-tocotrienol (GT3), one of the tocols, is a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (20, 21). Its antioxidant activity was a compelling reason to evaluate it for radioprotective efficacy; in recent years, it has received a great deal of attention by researchers and appears to be one of the most promising radioprotective tocols tested to date. GT3 has been shown to increase survival in mice through the amelioration of the hematopoietic and GI systems. In one study, it was reported that a dose of 200 mg/kg of GT3 given subcutaneously (s.c.) 24 h before 60Co gamma irradiation significantly protected mice against radiation doses as high as 11.5 Gy, a supralethal dose, and its dose reduction factor was 1.29 (22). GT3 demonstrated dose response against different doses of radiation. GT3 treatment accelerated hematopoietic recovery as indicated by higher numbers of total white blood cells, neutrophils, monocytes, platelets and reticulocytes in peripheral blood (22), and enhanced hematopoietic progenitors (23) in the bone marrow of irradiated mice. Additionally, GT3-treated irradiated mice had higher numbers of colony-forming cells, more regenerative microfoci for myeloid and megakaryocytes, higher cellularity in bone marrow and reduced frequency of micronucleated erythrocytes compared to irradiated, vehicle-treated controls (23). Mouse survival studies with GT3 suggested the most efficacious time for drug administration was 24 h prior to radiation exposure, possibly due to the induction of key hematopoietic cytokines during that time frame. GT3 treatment resulted in significant induction of G-CSF and several other cytokines in mice; administration of G-CSF antibody completely abrogated the radioprotective efficacy of GT3 in mice (24). GT3 treatment provided protection against radiation doses close to the GIS-inducing range in mice, meriting its further development. These results prompted us to evaluate the nonhuman primate (NHP) model treated with GT3 prior to

ionizing radiation exposure. We evaluated GT3 pharmacokinetics (PK) and efficacy using different doses of drug and radiation. Our results demonstrate promising hematopoietic recovery by GT3 in irradiated NHPs. MATERIALS AND METHODS Animals Healthy naive rhesus macaques (Macaca mulatta, Chinese substrain) were obtained from Primate Products, Inc. (Miami, FL) and quarantined for 6–7 weeks prior to the start of the experiment. Clinically healthy male and female rhesus macaques 3–5 years of age, weighing 3.6–8.4 kg were used in this study. All NHPs were individually housed in stainless-steel cages in environmentally controlled rooms maintained at 228C 6 28C, 30–70% relative humidity, 10–15 air change cycles per hour and a 12:12 h light-dark cycle. Animals were fed primate diet (Teklad T.2050 diet; Harlant Laboratories Inc., Madison, WI) twice daily and received drinking water ad libitum. All the animals received enrichment food (fresh fruits and vegetables, prima treats, peanuts, marshmallows, etc.) once daily Monday–Friday. Melon was restricted to animals expected to be evaluated for citrulline. They received mirrors, toys and challenge balls for enrichment. Televisions were used for sensory enrichment for 4–5 h at least 3 times a week. All the animals were able to see, hear and/or touch the conspecifics through the cages. All animals were serologically negative for Macacine herpesvirus 1 (herpes B virus), simian retrovirus (SRV), simian T-cell leukemia virus (STLV) and simian immunodeficiency virus (SIV). They were vaccinated with positive antibody titers for measles and tested negative for tuberculin. Animals were stratified by gender and body weight increases during the quarantine period and then assigned to different treatment groups. This animal study was conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Experimental Design Twelve NHPs were used for the pharmacokinetics study (four NHP each injected s.c. with 9.375, 18.75 and 37.5 mg/kg GT3). To assess the radioprotective efficacy of GT3 as a radiation countermeasure, 48 NHPs were divided into three groups of 16 animals and each group was exposed to 5.8 (LD30), 6.5 (LD50) or 7.2 Gy (LD70) (0.6 Gy/min) dose of radiation. The NHPs were administered GT3 s.c. 24 h prior to exposure. The NHPs exposed to 5.8 Gy were further divided into two groups (eight NHPs each) with one group receiving 37.5 mg/kg GT3 and the other receiving 75 mg/kg. All animals exposed to 6.5 and 7.2 Gy received 37.5 mg/kg GT3. In all experiments, 50% of the animals were injected with GT3 and the remaining 50% with vehicle control. Drug Preparation and Administration GT3 and olive oil formulations (50 and 100 mg/ml) in 5% Tweent 80 in saline was purchased from Yasoo Health Inc. (Johnson City, TN). Olive oil formulation was used as vehicle control. The quantity of GT3 and vehicle for each NHP was based on individual NHP weight (GT3 dosing details are found in the Experimental Design section). Drug and vehicle were administered at the dorsal scapular area (between the shoulder blades). The area surrounding the injection site was shaved at least 48 h before injection, so the site could be easily observed for any adverse skin reactions such as rash/eruption, inflammation, irritation and abscess formation after GT3 and vehicle administration. Immediately prior to drug injection, the site was wiped with 70% isopropyl rubbing alcohol and allowed to air dry; the drug was administered s.c. using a 3 ml disposable luer-lock syringe with 25 G 5/8 inch needle.

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TABLE 1 Pharmacokinetic Parameters of GT3 in Rhesus Macaques Dose (mg/kg) 9.375 18.75 37.5

Cmax (ng/ml)

Tmax (h)

T1/2 (h)

AUC (lg*h/ml)

Cl (ml/h/kg)

MRT (h)

MRT (days)

734.9 6 375.8 997.4 6 745.1 1142.0 6 883.5

265 866 12 6 5

30.4 6 13.3 34.6 6 9.1 40.9 6 12.6

26.2 6 13.5 43.7 6 25.9 82.2 6 57.6

0.358 6 0.094 0.429 6 0.152 0.456 6 0.236

48.6 6 28.7 48.0 6 18.1 273.9 6 132.1

2.0 6 1.2 2.0 6 0.8 11.4 6 5.5

Notes. AUC values were calculated using the trapezoid rule. Cmax was determined using the maximum average value corresponding to each dose. Tmax was the time point affiliated with Cmax. The half-life (T1/2) of GT3 was determined by utilizing the line of best fit for the portion of the curve following the second peak concentration and by picking a concentration with a known associated time point along that portion of the curve. We calculated the value of half chosen concentration (C1/2) and used the equation of the line of best fit to calculate that concentration’s associated time point. The difference in these two time points is T1/2. Drug systemic clearance (Cl) was calculated by dividing amount of drug delivered by AUC then by average NHP weight. MRT was calculated by dividing the area under the first moment curve (AUMC) by AUC. AUMC was determined using PK Solutions 2.0. Radiation Exposures A maximum of four NHPs were irradiated per day. Food was withheld from each animal approximately 12–18 h prior to exposure to minimize the occurrence of radiation-induced vomiting. Approximately 30–45 min prior to irradiation, NHPs were given 10–15 mg/kg of ketamine hydrochloride intramuscularly for sedation, then placed in custom-made Plexiglas irradiation boxes and secured in a seated position. Two NHPs were placed on the irradiation platform facing away from each other and exposed with a midline dose of 5.8, 6.5 or 7.2 Gy at a dose rate of 0.6 Gy/min. To deliver the precise dose, the abdominal width of each animal was measured with digital calipers. Animals were observed throughout the irradiation procedure via inroom cameras. After irradiation, animals were returned to the transport cart and to their cages in the housing area and monitored for recovery from the procedure. Dose rate measurements were based primarily on the alanine/EPR (electron paramagnetic resonance) system (25, 26), currently accepted as one of the most accurate methods for relatively high radiation doses and used for comparisons among national metrology institutes. The calibration curves (EMXmicro spectrometer, Bruker Inc., Billerica, MA) used in dose measurements at the Armed Forces Radiobiology Research Institute are based on standard alanine calibration sets purchased from the U.S. National Institute of Standards and Technology (NIST; Gaithersburg, MD). The alanine dosimeters obtained from NIST had been calibrated in terms of absorbed dose to water using the U.S. National Standard Radiation Sources. At AFRRI, identical alanine dosimeters were placed midline within NHP phantoms (Plexiglas cylinders 6.9, 10 and 12.5 cm in diameter and 34.5 cm length) and irradiated to approximately 100 Gy. Measurement of their EPR signals was performed using the calibration curve constructed with alanine dosimeters from NIST-provided dose rates to water in the core bodies of NHP. A small correction was subsequently applied for the difference in mass energy absorption coefficients between water and soft tissue. Pharmacokinetic Analysis For pharmacokinetic analysis, three doses of GT3 (9.375, 18.75 and 37.5 mg/kg) were selected based on efficacy/toxicity studies previously completed using the mouse model. Each dose was administered to four NHPs and blood samples were collected at various time points (0.25, 0.5, 1, 2, 4, 8 and 12 h and 1, 2, 3, 4, 5, 6, 7, 10, 14 and 20 days) after s.c. GT3 injection. The PK study included determination of plasma concentration of GT3 at various time points by liquid chromatography-mass spectrometry (LC-MS). The area under the curve (AUC) values were calculated using the trapezoid rule (GraphPad Prism 5; GraphPad Software Inc., La Jolla, CA). The maximum systemic drug concentration (Cmax) was determined using the maximum average value corresponding to each dose. The maximum time (Tmax) was the time point affiliated with Cmax. The half-life (T1/2) of GT3 was determined by utilizing the line of best fit

for the portion of the curve following the second peak concentration and by selecting a concentration with a known associated time point along that portion of the curve. We calculated the value of half of the chosen concentration (C1/2) and used the equation of the line of best fit to calculate that concentration’s associated time point. The difference in these two time points is T1/2. Drug systemic clearance rate (Cl) was calculated by dividing the amount of drug delivered by the AUC then by the average NHP weight. Mean residence time (MRT) was calculated by dividing the area under the first moment curve (AUMC) by AUC. AUMC was determined using PK Solutions 2.0 (Summit Research Services, Montrose, CO). Additional details of assays and experiments that yielded insignificant results are provided in the Supplementary material (http://dx.doi.org/10.1667/RR14127.1.S1). Statistical Analysis Fold change (FC) is the ratio of GT3 group mean to vehicle group mean. Average fold change (AFC) is the average of all fold changes for a given time period. A Kaplan-Meier plot and log-rank test were used to visualize and to compare survival data, respectively. Fisher’s exact test was used to compare survival rates at the end of day 60, with a Bonferroni correction used to control for type I error if multiple comparisons were made. For PK and efficacy data, mean values with standard errors (SE, when applicable) are reported. Independent sample t tests and analysis of variance (ANOVA) was used to detect if there were significant differences among experimental groups. When significance was indicated, a Tukey’s post hoc test was used to determine where the significant differences were located among groups. All statistical tests were two-sided, with a 5% significance level. Statistical software SPSSt v.22 (IBM, Armonk, NY) and GraphPad Prism 5 software were used for analyses.

RESULTS

Pharmacokinetic Assessment of GT3 in NHPs

Three drug doses (9.375, 18.75 and 37.5 mg/kg) were selected for PK studies based on earlier efficacy and toxicity studies conducted in the mouse model. Each dose was administered s.c. to four NHPs and blood samples were collected at various time points after GT3 injection. Studies were terminated at the end of day 20. PK study data are shown in Table 1 and Fig. 1. Table 1 displays maximum blood plasma concentrations (Cmax), the time at which Cmax was obtained (Tmax), half-life (T1/2), AUC, Cl and MRT for the various doses. Figure 1 tracks the blood plasma levels of GT3 throughout the period of the study. PK results demonstrated increased AUC and half-life with increasing drug dose.

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FIG. 1. Pharmacokinetics and pharmacodynamics of GT3 in blood plasma of NHP. Three different doses of GT3 (9.375, 18.75 and 37.5 mg/ml) were administered to NHPs. Blood samples were collected at various time points after GT3 administration. Averaged data are displayed with individual NHP data. Plasma samples were analyzed for PK. AUC ¼ area under the curve.

Effects of GT3 on Complete Blood Count (CBC) in Irradiated NHPs

The data in Figs. 2–5, Table 2 and Supplementary Figs. S1–4 (http://dx.doi.org/10.1667/RR14127.1.S1) demonstrate the accelerated hematopoietic recovery in GT3-treated animals compared to their respective vehicle-treated controls after three different radiation doses. Efficacy of GT3 on CBC after 5.8 Gy (LD30/60) Radiation Exposure

The NHPs treated with 37.5 mg/kg GT3 had a higher level of WBCs beginning at day 16 postirradiation compared to vehicle-treated controls (FC 1.12); this difference became significant at day 21 postirradiation (FC 2.55) and continued inconsistently until day 38 (FC 2.36, AFC 2.34), at which time the average WBC levels of both groups returned to pre-exposure levels (Fig. 2). GT3treated NHPs had a higher number of neutrophils beginning at day 16 (FC 2.67) postirradiation. The GT3-treated group was considered neutropenic from day 6 to 16; the vehicletreated group was neutropenic from day 6 to 20, indicating that GT3 treatment shortens the duration of neutropenia (Table 2). The reticulocyte response curve for NHPs treated with 37.5 mg/kg GT3 closely resembles the response curve for those treated with the vehicle. The platelet counts for the GT3-treated NHPs were also higher from day 14 (FC 1.82) to day 60 (FC 1.24, AFC 1.80), but the difference between treatments was not significant at any time. RBCs of animals administered 37.5 mg/kg GT3 were significantly higher than the vehicle-treated NHPs at day 1 (FC 1.08) and day 14 (FC 1.07) postirradiation (Fig. 2). GT3-treated NHPs had significantly higher hemoglobin (Hgb) at day 10 (FC 1.06) postirradiation compared to those treated with vehicle.

There was no significant difference of hematocrit (Hct) levels between GT3- and vehicle-treated NHPs. The GT3treated NHPs had significantly higher levels of monocytes at two time points: day 17 (FC 10.62) and day 18 (FC 6.61) (Supplementary Fig. S1; http://dx.doi.org/10.1667/ RR14127.1.S1). Non-human primates treated with 75 mg/kg GT3 had higher levels of WBCs than the vehicle-treated NHPs at day 14 (FC 1.28); this improvement was significant at day 15 (FC 2.29) and continued until day 24 (FC 2.38, AFC 5.09) when counts for both groups returned to normal levels (Fig. 3). Additionally, GT3-treated NHPs were neutropenic for less time compared to those that were vehicle-treated (10 vs. 14 days) (Table 2). Recovery from neutropenia occurred on day 14 for the GT3-treated group and day 19 for the vehicle-treated group. NHPs treated with 75 mg/kg clearly had improved reticulocyte recovery compared to the vehicle-treated group by day 16 postirradiation (FC 1.73); the significant difference by GT3 treatment ranged from day 19 (FC 13.38) to day 22 (FC 4.5, AFC 7.70) postirradiation. The GT3-treated NHPs also had significantly higher platelet counts than the vehicle-treated NHPs from day 15 (FC 7.77) to day 30 (FC 2.67, AFC 4.52). The GT3-treated group did not become thrombocytopenic whereas the vehicle-treated group was in a state of thrombocytopenia from day 12 to 16 postirradiation. RBC levels of GT3-treated groups were comparable to those of vehicle-treated NHPs for the first 26 days of the observation period, after which GT3-treated NHP RBC levels were on average 1.22-fold higher. This difference was significant only on day 42 (FC 1.24) and day 50 (FC 1.14). Hct levels followed a pattern similar to the RBCs, with GT3-treated NHPs having higher levels only after day 24 and until the end of the study period, with an average fold change of 1.16. These levels were significant

RADIOPROTECTIVE EFFICACY OF GAMMA-TOCOTRIENOL

289

FIG. 2. GT3 induced changes in complete blood counts as well as differential counts in NHPs. NHPs received GT3 (37.5 mg/kg) or vehicle 24 h before irradiation (5.8 Gy). Blood was collected at various time points. Cells were counted using a Bayer Advia-120 cell counter. The data for each time point are shown as the mean 6 standard error. *The difference between GT3-treated and vehicle-treated groups was significant when equal variance between groups was assumed (P , 0.05).

only on day 28 and 38. There was no significant difference of Hgb among treatment groups at this radiation dose. After day 16, the NHPs receiving 37.5 mg/kg had only two time points with significantly higher levels of monocytes compared to the vehicle-treated controls (FC 10.65 and 6.61, respectively), whereas the group that received 75 mg/kg produced 7 time points with significantly higher levels compared to the control group (FC 6.15, 3.62, 2.87, 2.89, 2.16, 1.76 and 1.70) (Supplementary Figs. S1 and S2; http://dx.doi.org/10.1667/RR14127.1.S1). Monocytes showed a much more dramatic dose response than the other parameters in NHPs exposed to 5.8 Gy (Supplementary Fig. S2). When Figs. 2 and 3 and Supplementary Figs. S1 and S2 are compared, a dose response of GT3 can be

clearly seen; 75 mg/kg was more efficacious than 37.5 mg/ kg. Efficacy of GT3 on CBC after 6.5 Gy (LD50/60) Radiation Exposure

The trend of accelerated recovery in GT3-treated NHPs exposed to 5.8 Gy radiation was also noted at the higher dose of 6.5 Gy. Figure 4 shows that WBC levels were significantly higher in GT3-treated animals from day 14 (FC 2.04) to day 28 (FC 2.08, AFC 4.56) postirradiation compared to the vehicle-treated group. GT3-treated NHPs had significantly higher neutrophil counts than the vehicletreated NHPs continuously from day 14 (FC 6.63) to day 30 (FC 2.14, AFC 9.58). The GT3-treated group was

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FIG. 3. GT3 induced changes in complete blood counts as well as differential counts in NHPs. NHPs received GT3 (75 mg/kg) or vehicle 24 h before irradiation (5.8 Gy). Blood was collected at various time points. Cells were counted using a Bayer Advia-120 cell counter. The data for each time point are shown as the mean 6 standard error. *The difference between GT3-treated and vehicle-treated groups was significant when equal variance between groups was assumed (P , 0.05).

considered neutropenic from day 3 to 14, whereas the vehicle-treated group was in a state of neutropenia from day 5 to 19 and was considered severely neutropenic from day 10 to 17. GT3-treated animals never became severely neutropenic. Reticulocytes in the GT3-treated NHPs were consistently higher than the control over the course of the first 28 days postirradiation (AFC 4.37). GT3-treated NHP reticulocytes returned to pre-exposure levels more quickly than the vehicle-treated NHPs (day 19 vs. day 24). Reticulocytes had significantly higher levels in GT3-treated NHPs than in vehicle-treated NHPs from day 17 (FC 6.55)

to day 24 (FC 3.94, AFC 7.80). The platelet recovery also reflected the significant advantage provided by GT3 administration in irradiated NHPs from day 16 (FC 8.05) to day 24 (FC 2.25, AFC 4.93), GT3-treated NHPs never became thrombocytopenic, whereas the vehicle-treated group was in a state of thrombocytopenia from day 12 to 17 and was in a state of severe thrombocytopenia on day 14 for one day (Fig. 4 and Table 2). RBC levels of GT3-treated NHPs were higher from day 19 (FC 1.10) to day 24 (FC 1.17), with an AFC of 1.12 for this period. This difference was significant on day 26, 28 and 30. Hgb and Hct showed

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291

FIG. 4. GT3 induced changes in complete blood counts as well as differential counts. NHPs received GT3 (37.5 mg/kg) or vehicle 24 h before irradiation (6.5 Gy). Blood was collected at various time points. Cells were counted using a Bayer Advia-120 cell counter. The data for each time point are shown as the mean 6 standard error. *The difference between GT3-treated and vehicle-treated groups was significant when equal variance between groups was assumed (P , 0.05).

very similar patterns, with higher levels from GT3-treated NHPs starting at day 19 until day 42 with average fold improvement of 1.15 and 1.16, respectively. Both parameters were significantly improved by GT3 treatment from day 24 to 30, with AFC for this time period for each parameter at 1.25 and 1.26, respectively. Animals treated with 37.5 mg/kg GT3 prior to a 6.5 Gy dose of radiation had monocyte levels significantly higher than the vehicle-treated controls at day 17 (FC 9.21) to day 28 (FC 2.43, AFC 5.90) (Supplementary Fig. S3; http://dx.

doi.org/10.1667/RR14127.1.S1). GT3 did not show such efficacy for other parameters (eosinophils, lymphocytes and basophils) at this radiation dose; significantly higher levels of these parameters in GT3-treated NHPs did not last longer than three consecutive time points. Efficacy of GT3 on CBC after 7.2 Gy (LD70/60) Radiation Exposure

The effect of GT3 in NHPs exposed to 7.2 Gy was minimal in improving WBC and reticulocyte levels or

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FIG. 5. GT3 induced changes in complete blood counts as well as differential counts. NHPs received GT3 (37.5 mg/kg) or vehicle 24 h before irradiation (7.2 Gy). Blood was collected at various time points. Cells were counted using a Bayer Advia-120 cell counter. The data for each time point are shown as the mean 6 standard error. *The difference between GT3-treated and vehicle-treated groups was significant when equal variance between groups was assumed (P , 0.05).

neutropenia and thrombocytopenia (Fig. 5). WBC levels in GT3-treated NHPs were significantly higher at day 14 (FC 1.87) and day 16 (FC 3.89) postirradiation. Neutrophil levels in GT3-treated NHPs were significantly higher than vehicle-treated NHPs on day 14 postirradiation (FC 3.10). Reticulocyte levels were significantly higher on day 12 (FC 5.20) and day 16 (FC 4.08) postirradiation. Platelet levels in GT3-treated NHPs were not significantly improved compared to vehicle-treated NHPs (Fig. 5). RBC levels were higher in GT3-treated animals from the beginning of the study to day 50 (AFC 1.18) but the difference was

significant only on day 42 (FC 1.24). Again at this radiation dose, Hgb and Hct had very similar patterns; levels from GT3-treated NHPs were consistently higher than vehicletreated NHPs starting at day 16 until day 60 with average fold difference of 1.22 and 1.27, respectively. Both Hgb and Hct in the GT3-treated NHPs were significantly higher than in the vehicle-treated NHPs at day 42; AFCs for this time point for each parameter are 1.30 and 1.33, respectively. Overall, the response curves of these parameters were similar in both of the treatment groups at this radiation dose (Fig. 5).

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TABLE 2 Nadir Values and Durations of Neutropenia (,0.5 3 103/ll) and Thrombocytopenia (,20 3 103/ll) of each Radiation Dose and Treatment Group Radiation dose 5.8 Gy

6.5 Gy

7.2 Gy

Parameter

Treatment

Durationa (days)

Mean value and rangea (3103/ll)

Neutrophils Platelets Neutrophils Platelets Neutrophils Platelets Neutrophils Platelets Neutrophils Platelets Neutrophils Platelets Neutrophils Platelets Neutrophils Platelets

Vehicle (n ¼ 4) GT3 (37.5 mg/kg) (n ¼ 4) Vehicle (n ¼ 4) GT3 (37.5 mg/kg) (n ¼ 4) Vehicle (n ¼ 4) GT3 (75 mg/kg) (n ¼ 4) Vehicle (n ¼ 4) GT3 (75 mg/kg) (n ¼ 4) Vehicle (n ¼ 8) GT3 (37.5 mg/kg) (n ¼ 8) Vehicle (n ¼ 8) GT3 (37.5 mg/kg) (n ¼ 8) Vehicle (n ¼ 8) GT3 (37.5 mg/kg) (n ¼ 8) Vehicle (n ¼ 8) GT3 (37.5 mg/kg) (n ¼ 8)

6–20 6–16 NA NA 5–19 4–14 12–16 NA 5–19 3–14 12–17 NA 5–19 4–15 12–15 10–14

0.23 (0.12–0.42) 0.28 (0.11–0.49) NA NA 0.18 (0.04–0.38) 0.30 (0.10–0.50) 14.88 (10.50–18.50) NA 0.16 (0.04–0.43) 0.29 (0.15–0.44) 13.50 (9.63–17.75) NA 0.17 (0.02–0.47) 0.17 (0.10–0.26) 7.12 (6.17–8.20) 15.20 (13.60–18.00)

Nadira (mean 6 SE) (3103/ll) 0.12 0.11 20.75 29.25 0.04 0.07 10.50 25.80 0.04 0.15 9.63 59.88 0.02 0.10 6.17 13.60

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.04 0.05 10.46 12.74 0.012 0.025 5.69 6.80 0.01 0.10 2.76 32.75 0.00 0.02 1.54 5.59

Note. The group average did not fall below the given threshold for neutropenia and thrombocytopenia. NA ¼ not applicable. a Durations, mean values, ranges and nadirs are calculated using the group mean of each treatment group.

Additional parameters showed little difference between the GT3- and vehicle-treated groups (Supplementary Fig. S4; http://dx.doi.org/10.1667/RR14127.1.S1). Lymphocytes showed significant improvement during day 17 (FC 2.24) to day 24 (FC 1.83, AFC 1.97) and again from day 34 (FC 1.86) to day 42 (FC 1.81, AFC 1.89), although by day 60 postirradiation, the vehicle-treated NHPs had higher levels of lymphocytes. None of the additional parameters discussed, except lymphocytes, had significantly higher levels for more than three consecutive time points. Effect of GT3 on Survival of Irradiated NHPs

The primary end point used in the efficacy study was mortality; however, moribundity (early end point) was used as a surrogate for mortality. Moribund animals were euthanized to minimize pain and distress. Moribundity, where animals had reached ‘‘the point of no return’’, was defined by a combination of criteria, including abnormal clinical signs (inappetence, minimal or absence of response to stimuli, etc.), vital parameters [significant weight loss (20% over 3 days)], core body temperature below 96.68F after a period of febrile neutropenia, etc.) and laboratory parameters (neutropenia, thrombocytopenia, anemia, etc.). No single parameter listed above led to euthanasia, which was instead decided by a team comprised of the institutional veterinarian, principal investigator, research staff, veterinary technicians and husbandry staff and based on the combination of criteria described above. In brief, 16 out of 24 vehicle-treated animals survived while 18 out of 24 GT3-treated animals survived, and differences between survivors of GT3- and vehicle-treated animals were not significant (Fig. 6). Of the four NHPs that received 37.5 mg/kg GT3 and were exposed to 5.8 Gy

radiation, three survived; the fourth was euthanized due to moribundity on day 20. Of the four vehicle-treated control NHPs, three survived while the fourth NHP was euthanized on day 18. All four GT3-treated NHPs that received 75 mg/ kg drug and irradiated with 5.8 Gy survived; three of the four control NHPs survived. The fourth animal was euthanized on day 20. In the experiment with 6.5 Gy, six out of eight NHPs treated with 37.5 mg GT3 survived and the remaining two NHPs were euthanized on day 12 and 30. Of the control animals, six NHPs survived and the remaining two were euthanized on day 17 due to moribundity. In the experiment with 7.2 Gy, five out of eight GT3-treated NHPs survived and the remaining three NHPs were euthanized, two on day 13 and one on day 15. Of the eight 7.2 Gy control animals, four survived and the other four were euthanized, one each on day 14, 16, 17 and 26 due to moribundity. Additional details of experiments and results with figures are available in the Supplementary material (http://dx.doi. org/10.1667/RR14127.1.S1). DISCUSSION

Injury by whole-body irradiation implicates immune, hematopoietic and GI tissues, which are the most radiosensitive tissues in the body. Lymphocytes are first to diminish from circulation after exposure to radiation (.2 Gy). Lymphocyte loss is followed by neutrophil, then platelet loss over the course of days. Immune and hematopoietic injury may cause death as a result of impaired immunity and subsequent infection or thrombocytopenia and later, hemorrhages. The averages of all individual AUCs in the PK study are directly related to the dose of GT3 that was given. The

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FIG. 6. Survival curves of GT3- or vehicle-treated irradiated NHPs. NHPs received GT3 (37.5 and 75 mg/kg) or vehicle 24 h prior to irradiation (5.8, 6.5 and 7.2 Gy). Survival was monitored for 60 days postirradiation.

absence of linear and predictable GT3 absorption rate (Tmax) and extent of absorption (Cmax) can be attributed to the inherent variability of s.c. administration which results in variations of pharmacokinetic measures, such as AUC, within a single individual and between individuals of the same species. The variability is made apparent by the small sample size used in this study. Others have also noted variability of s.c. administration; administration of cefovecin to NHPs showed intraindividual coefficient of variations in pharmacokinetic parameters, which were significantly higher than those obtained after intravenous (i.v.) injection (27, 28). While the purpose of the work cited here by Raabe et al. (27) and Papp et al. (28) was to show interspecies differences in cefovecin pharmacokinetics, it is clear from these data that there was higher variability after s.c. versus i.v. administration. The technique of s.c. administration is highly dependent on the angle and speed of injection, as well as the localized blood flow at the site of injection. These technical factors cannot be controlled completely. Intraindividual variation in the absorption of insulin after s.c. injection is also well recognized, but complicated by formulation (i.e., suspension vs. solution) and absorption mechanism differences that are related to the high molecular weight of insulin relative to GT3 (29). Therefore, the cefovecin comparison discussed above is more descriptive of the intraindividual differences observed for GT3. Our study demonstrates significantly decreased severity and duration of neutropenia and thrombocytopenia in GT3treated irradiated NHPs compared to the vehicle-treated irradiated controls. As shown in Figs. 2–5 and Supplementary Figs. S1–S4 (http://dx.doi.org/10.1667/RR14127.1.S1), at some time points, CBC parameter values (i.e., WBC,

neutrophils, platelets, reticulocytes) of the GT3-treated group are lower than those of the vehicle-treated group. To minimize the potential for confusion, we have not discussed these situations under the Results section. Our studies used a NHP model similar to that reported in earlier studies (30–36); however, our study excluded supportive care (antibiotics, blood products and i.v. fluids) to allow for a better demonstration of the radioprotective effects of GT3 and to more closely simulate a mass casualty scenario where these products would be limited, if available at all. In this study, GT3 treatment did not significantly improve survival of irradiated animals; this may be due to small sample size of four or eight animals per group. We plan to conduct a study utilizing a larger sample size in the future. There are three published studies using the NHP model to test efficacy of recombinant human interleukin-12 (IL-12); one using 6.7 Gy and others with 7.0 Gy (Theratron 1000 60 Co source) (33, 34, 37). All these studies have been conducted without supportive care. It was also reported that granulocyte-macrophage colony-stimulating factor (GMCSF) and pegylated G-CSF demonstrated comparable results with respect to improving neutropenia and thrombocytopenia in irradiated NHPs (9, 31, 32). Pegylated GCSF has been tested with exposure to doses of 6 and 7.5 Gy. In the case of the GM-CSF study, the radiation dose was 8 Gy partial-body exposure (tibiae shielded with lead walls) (32). In a recently published study, G-CSF failed to demonstrate radiomitigative efficacy in the NHP model (37). Further, administration of G-CSF failed to improve upon the survival benefit already provided by recombinant human IL-12 in NHPs (37). The cause for the disparity in results

RADIOPROTECTIVE EFFICACY OF GAMMA-TOCOTRIENOL

remains to be assessed but may have been due to the lack of supportive care. Although G-CSF has been approved by FDA for hematopoietic ARS and has already been procured, along with GM-CSF, for the strategic national stockpile for use in a radiological emergency (38), the adverse consequences of G-CSF need to be taken into consideration. G-CSF administration after cytotoxic agents that damage bone marrow stem cells exacerbates long-term stem cell damage through excessive differentiation stimulation (39). Additional concerns stem from its role in exacerbating delayed lung damage in an animal model of ARS (40). The effect of G-CSF has been evaluated on bleomycin-induced lung injury that developed diffuse alveolar damage and subsequent pulmonary fibrosis of varying severity in a rat model. The results indicated that G-CSF treatment in rats with minor lung injury bearing no pulmonary fibrosis does not worsen the lung injury. The exacerbating effects of G-CSF seem to be associated not only with the marked infiltration of activated neutrophils but also with the severity of the underlying lung injury (41). The International Atomic Energy Agency recommends that platelet counts be monitored during G-CSF administration (42). Low lymphocyte counts were found to be the best early phase (,6 days) predictor of radiation-induced human lethality in medical treatment protocols for radiation accident victims (METREPOL) (43). Consistent with the improvement in neutropenia and cytopenia, lymphocyte counts in GT3-treated groups (5.8 Gy–75 mg/kg, 6.5 and 7.2 Gy–37.5 mg/kg) were significantly higher than in controls for one (19 days), three (17, 18, and 19 days) and nine (18, 19, 20, 21, 22, 24, 34, 38 and 42 days) time points, respectively (Supplementary Figs. S2–S4; http://dx.doi.org/ 10.1667/RR14127.1.S1). We did not observe improvement in lymphopenia by GT3 during early time points (,4 days) except at one time point (day 3) with 5.8 Gy irradiation and 37.5 mg/kg GT3. WBC counts were also significantly improved by GT3 treatment. We are currently investigating the mechanism by which a single injection of GT3 elicits radioprotective efficacy. Among the eight naturally occurring vitamin E analogs, GT3 is a particularly potent radioprotective agent in vivo (12). GT3 protects endothelial cells from radiation-induced injury not only by virtue of its antioxidant properties, but also by inhibition of HMG-CoA reductase and improving the availability of the nitric oxide synthase cofactor tetrahydrobiopterin (44). GT3 induces multiple changes in functional genetic pathways known to be of critical importance in the cellular responses to radiation exposure, such as oxidative stress, DNA damage, cell cycle phase, regulation of cell death, cell proliferation, hematopoiesis and blood vessel development (45). Recently, it has been reported that GT3-mediated protection of intestinal cells occurs via up-regulation of anti-apoptotic and downregulation of pro-apoptotic factors (46). GT3 treatment combined with pentoxifylline increased postirradiation

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survival over that of GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli (47). It has been shown that s.c. GT3 treatment induced G-CSF in a dose-dependent manner in mice (48). G-CSF antibody administration completely abrogates the radioprotective efficacy of GT3 in mice, suggesting that G-CSF may play an important role in the radioprotective efficacy of GT3 (24). Similar radioprotective efficacy abrogation has been reported with other G-CSF-inducing radiation countermeasures; CBLB502, 5-androstenediol, d-tocotrienol and tocopherol succinate data indicate they may work through a similar mechanism (12, 49–51). GT3 induced G-CSF in irradiated and nonirradiated NHPs, however, these levels were less dramatic than those observed in mice (for more information on cytokines, see Supplementary Fig. S5 for nonirradiated animals and Figs. S6–S9 for irradiated animals; http://dx.doi.org/10.1667/RR14127.1.S1). It remains unknown whether GT3-induced G-CSF plays any role in radioprotective efficacy of GT3 in NHPs. Lethal, whole-body ionizing radiation exposure induces polymicrobial sepsis as a result of intestinal epithelium injury and subsequent bacterial translocation. An optimal radiation countermeasure would protect from GI injury, enhance resistance to bacterial infections, reduce translocation of bacteria and eliminate infections, leading to increased survival and a longer window of opportunity for additional intervention to victims. When we studied gut bacterial translocation to peripheral circulation, we found several bacterial species in the peripheral circulation of irradiated NHPs, but there was no significant difference between GT3-treated and vehicle-treated control groups irradiated with different doses (Supplementary Fig. S10 and Table S1; http://dx.doi.org/10.1667/RR14127.1.S1). There are several published studies of countermeasures inhibiting translocation of coliform bacteria to peripheral circulation in animals exposed to high doses of radiation (52–56). There was a noticeable dose response to GT3 in NHPs irradiated with 5.8 Gy where neutropenia and thrombocytopenia were improved, with a dose of 75 mg/kg being more beneficial than 37.5 mg/kg. However, skin irritation (abscess formation) was observed at the injection site when the concentration was increased to 75 mg/kg. Currently, we are working with various formulations so that higher doses of GT3 can be administered without any local adverse effects. We observed lower than expected mortality rates of animals irradiated with 5.8, 6.5 and 7.2 Gy 60Co (Fig. 6). As stated above, based on the available literature, 6.5 Gy (without supportive care) is expected to be the LD50 for rhesus macaques (30). We have come across similar observations in the past. There have been reports that indicate varying NHP mortality rates when NHPs are exposed to the same radiation dose under similar experimental conditions. In one published study with 6.7 Gy (dose rate of 0.55 Gy/min) without any supportive care, 50% of the control animals died (33). In two more studies by the

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same investigators using similar experimental conditions, untreated control groups of subjects exposed to 7 Gy had mortality rates of 64 and 90% (34, 37). In our study, mortality was significantly lower compared to these studies. Such variation in mortality may be due to GI flora of NHPs procured from different geographic locations or to other unknown reasons. Although this study was focused on the evaluation of GT3 in an ARS model, it would be interesting to extend the animal observation period beyond 60 days in a future study, to evaluate the effects of GT3 against the delayed effects of acute radiation exposure. In the era of increased risk for radiological terrorism or accident, medical contingency plans and preparedness are critical to saving human lives, and the availability of effective radiation countermeasures is key to the preparedness success. To our knowledge, this is the first published study on the radioprotective efficacy of GT3 in NHPs, an animal model that is closely related to humans. We have accumulated a wealth of data demonstrating hematopoietic recovery after irradiation and on the PK of GT3 in NHPs. In a mass casualty scenario, one would expect that the availability of intensive clinical care will be limited, owing to logistic constraints of patient evacuation from contaminated areas to hospitals and potential shortages of blood products and other medical supplies. Therefore, an optimal frontline radiation medical countermeasure would be efficacious in the absence of intensive care. Since GT3 is a prophylactic agent, it is more suitable for military personnel, first responders and individuals anticipating radiation exposure. The radioprotective efficacy of GT3 in NHPs assumes greater significance since radiation doses used in this study are equivalent to lethal human doses. Our results with GT3 demonstrate its potential as another potent, naturally occurring agent that could be further developed as a radioprotector. Currently, GT3 is in a clinical trial to investigate its efficacy in combination with pentoxifylline for delayed radiation enteropathy. This agent needs to be developed in accordance with the FDA Animal Efficacy Rule. Phase I safety studies are required to assess the safety and PK profiles of GT3, as well as additional animal efficacy and mechanistic studies. The culmination of human and animal studies will help determine the predictive efficacious dose of GT3 in humans under the Animal Efficacy Rule (57). ACKNOWLEDGMENTS We are grateful to the Veterinary Sciences Department (VSD) and the Cobalt Radiation Facility for their experimental support, Kevin Hieber, Jessica Scott and LTC Christine Christensen for their assistance in the animal study and Dr. Thomas B. Elliott for help in bacterial translocation experiments. This study was supported by a grant from the Defense Threat Reduction Agency (CBM.RAD.01.10.AR.005) administered by The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. SK, SYW, VLN, PLPR and JG are affiliated with The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. The opinions or assertions contained herein are the private views of the authors and are not necessarily those of the U.S. Armed Forces

Radiobiology Research Institute, the Uniformed Services University of the Health Sciences, or the Department of Defense. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. Received: April 27, 2015; accepted: December 30, 2015; published online: March 1, 2016

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improve intestinal integrity after whole body irradiation. Int J Radiat Biol 2013; 89:334–45. 56. Singh PK, Wise SY, Ducey EJ, Fatanmi OO, Elliott TB, Singh VK. Alpha-tocopherol succinate protects mice against radiationinduced gastrointestinal injury. Radiat Res 2012; 177:133–45. 57. Singh VK, Newman VL, Patricia LP, Hauer-Jensen M, Pollard HB. Use of biomarkers for assessing radiation injury and efficacy of countermeasures. Expert Rev Mol Diagn 2015; 16:65–81.