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. Author manuscript; available in PMC: 2022 Sep 5.
Published in final edited form as: Health Phys. 2015 Nov;109(5):427–439. doi: 10.1097/HP.0000000000000350

The effect of radiation dose and variation in Neupogen® initiation schedule on the mitigation of myelosuppression during the concomitant GI-ARS and H-ARS in a nonhuman primate model of high-dose exposure with marrow sparing

Thomas J MacVittie *, Alexander W Bennett *, Ann M Farese *, Cheryl-Taylor Howell *, Cassandra P Smith *, Allison M Gibbs *, Karl Prado †, William Jackson III ‡
PMCID: PMC9442798  NIHMSID: NIHMS706521  PMID: 26425903

Abstract

A nonhuman primate (NHP) model of acute high-dose, partial-body irradiation with 5% bone marrow (PBI/BM5) sparing was used to assess the effect of Neupogen® (granulocyte colony stimulating factor [G-CSF]) to mitigate the associated myelosuppression when administered at an increasing interval between exposure and initiation of treatment. A secondary objective was to assess the effect of Neupogen® on the mortality or morbidity of the hematopoietic (H)- acute radiation syndrome (ARS) and concurrent acute gastrointestinal radiation syndrome (GI-ARS). NHP were exposed to 10.0 or 11.0 Gy with 6 MV LINAC-derived photons at approximately 0.80 Gy min−1. All NHP received medical management. NHP were dosed daily with control article (5% dextrose in water) initiated on day 1 post-exposure or Neupogen® (10 μg kg−1) initiated on day 1, day 3, or day 5 until recovery (absolute neutrophil count [ANC] ≥ 1,000 cells μL−1 for 3 consecutive days).

Mortality due to GI- and H-ARS:

Mortality in both the 10.0 Gy and 11.0 Gy cohorts suggested that early administration of Neupogen® at day 1 post exposure may affect acute GI-ARS mortality, while Neupogen® appeared to mitigate mortality due to the H-ARS. However, the study was not powered to detect statistically significant differences in survival.

Neutrophil recovery:

The ability of Neupogen® to stimulate granulopoiesis was assessed by evaluating key parameters for ANC recovery: the depth of nadir, duration of neutropenia (ANC < 500 cells μL−1) and recovery time to ANC ≥ 1,000 cells μL−1.

Following 10.0 Gy PBI/BM5, the mean duration of neutropenia was 11.6 days in the control cohort, versus 3.5 days and 4.6 days in the day 1 and day 3 Neupogen® cohorts, respectively. The respective ANC nadirs were 94 cells μL−1, 220 cells μL−1, and 243 cells μL−1 for the control and day 1 and day 3 Neupogen® cohorts. Following 11.0 Gy PBI/BM5, the duration of neutropenia was 10.9 days in the control cohort, versus 2.8 days, 3.8 days, and 4.5 days in the day 1, day 3, and day 5 Neupogen® cohorts, respectively. The respective ANC nadirs for the control, and day 1, day 3, and day 5 Neupogen® cohorts were 131 cells μL−1, 292 cells μL−1, 236 cells μL−1, and 217 cells μL−1. Therefore, the acceleration of granulopoiesis by Neupogen® in this model is independent of the time interval between radiation exposure and treatment initiation up to 5 days post-exposure. The PBI/BM5 model can be used to assess medical countermeasure efficacy in the context of the concurrent GI- and H-ARS.

Keywords: health effects, laboratory animals, radiobiology, radiation effects, whole body irradiation

INTRODUCTION

The utility of medical countermeasures (MCM) must be viewed in the “context of use” as considered in the criteria set forth in the recent FDA guidance document for MCM development (Food and Drug Administration 2014). It is imperative to consider the context of use in the evolving clinical treatment scenario. In the context of a mass-casualty radiation exposure scenario, such as the detonation of an improvised nuclear device, the exposures will be ill-defined, uncontrolled, non-uniform, and heterogeneous, with the potential for physical shielding. These exposure characteristics forecast the sparing of bone marrow and GI tissue, which may enhance early recovery from the hematopoietic subsyndrome of the acute radiation syndrome (H-ARS) and possibly the gastrointestinal subsyndrome (GI-ARS) (Monroy et al. 1988; Maloney et al. 1972; Cole et al. 1967; Wang et al. 1991; Nothdurft et al. 1997; Bertho et al. 2005a; Bertho et al. 2005b; MacVittie et al. 2012a; Herodin et al. 2012; Croziat et al. 1976; Gidali et al. 1972; Farese et al. 1993; Farese et al. 1994; Herodin et al. 2007; Drouet et al. 2008). Thus, medical management, including the use of antibiotics and blood transfusions based on clinical triggers, may not be required for days or weeks post-exposure in such a scenario, due to the cellular kinetics noted after near-uniform or non-uniform irradiation (Liu et al. 2008; Baranov et al. 1990; Baranov 1996; Guskova et al. 1990; Gourmelon et al. 2010; Fliedner et al. 2002).

In this regard, medical management as the standard of care will also be combined with the administration of leukocyte growth factors (LGF) such as Neupogen®, Neulasta®, or Leukine® (Waselenko et al. 2004; Clark et al. 2005; Kuderer et al. 2007; Timmer-Bonte et al. 2005; Dainiak et al. 2011). The database in both preclinical and clinical studies is consistent and substantial in supporting the use of LGFs and prophylactic antibiotics to mitigate severe myelosuppression and mortality (Monroy et al. 1988; Neelis et al. 1997; Nothdurft et al. 1997; MacVittie et al. 2005; Gafter-Gvili et al. 2005; Clark et al. 2005; Liu et al. 2008; Timmer-Bonte et al. 2005; Bertho et al. 2005a; Farese et al. 2013; Farese et al. 2012b; Dainiak et al. 2011). The majority of preclinical studies used bilateral, uniform, total-body irradiation (TBI) at various lethal doses. However, this exposure geometry is unlikely for a nuclear scenario. Therefore, it is important to use a model that is more predictive of a clinical treatment scenario within the context of use for assessing MCM efficacy (Monroy et al. 1988; drouet et al. 2004; Nothdurft et al. 1997; Bertho et al. 2005a; Bertho et al. 2005b; MacVittie et al. 2012a; Herodin et al. 2012; Herodin et al. 2007; Farese et al. 1993; MacVittie et al. 1994; Farese et al. 1994; Drouet et al. 2008). ) This study used a high-dose partial-body irradiation (PBI) model with minimal sparing of approximately 5% of bone marrow (BM5). This model permits the study of multiple organ injuries (MOI) due to uniform exposure to key organs and dose dependent survival after exposure to doses of 10.0–12.0 Gy, which induce the acute GI-ARS with the concurrent evolution of the H-ARS and prolonged GI injury (MacVittie et al. 2012a).

Use of a PBI/BM5 model could address several knowledge gaps relative to the use of LGFs in the nuclear radiation exposure scenario. These are: a) assessing the efficacy of LGFs with an increasing interval between irradiation and treatment initiation to mitigate myelosuppression and/or mortality of the H-ARS; b) determining the effect of Neupogen® administration on other organ system sequelae, e.g., the incidence and severity of the acute GI-ARS; and c) assessing the effect of early Neupogen® administration during the concurrent evolution of the GI- and H-ARS on the latency, onset, and progression of MOI (co-morbidities and mortality) induced by the threshold exposure for overt manifestation of the delayed effects of acute radiation exposure on lung, heart, and kidney injury (DEARE). A strategic view of organ-specific MCM development must approach the question of efficacy from a different perspective. Does administration of a MCM such as Neupogen® affect the MOI which characterizes the link between ARS and DEARE?

The primary effort focused on the efficacy of Neupogen® when administered at an increasing interval between exposure and treatment, to mitigate the myelosuppression of the H-ARS. A secondary objective included early analysis of the effects of Neupogen®, on mortality associated with the concomitant progression of the acute GI-ARS and H-ARS after high-dose exposure with minimal BM sparing.

MATERIAL AND METHODS

The animal husbandry, irradiation, medical management, and hematology procedures summarized below for the PBI/BM5 model have been described in detail previously (Farese et al. 2013; MacVittie et al. 2012a).

Animals:

Male Chinese rhesus macaques (Macaca mulatta, 5.3 – 10.4 kg, n = 48) were used. NHP housing and care was performed in accordance with the Animal Welfare Act at the University of Maryland’s Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility. Data from historical controls (PBI/BM5: n = 25; TBI: n = 102) are included in the analyses presented herein.

Irradiation:

NHP were exposed to 10.0 Gy or 11.0 Gy PBI/BM5 utilizing 6 MV LINAC-derived photons (2 MV average) at a dose rate of approximately 0.80 Gy min−1 (TrueBeam™, Varian Medical Systems, Palo Alto, CA). Radiation dose calculations were performed by a board-certified medical physicist, based on a sagittal measurement taken at the xiphoid process. Dose was calculated for delivery to midline tissue, including correction factors to account for the inverse-square distance, tissue depth, and sources of photon scatter. Prior to irradiation, NHP were sedated with ketamine, secured in a supine restraint device, and then administered xylazine to ensure proper radiation field placement would be maintained. The NHP were positioned with their tibiae, ankles, and feet outside of the beam field. Half of the prescribed dose was delivered with an anteroposterior (AP) beam, and half with a posteroanterior (PA) beam. All irradiations were performed between the hours of 7:00 AM and 1:00 PM. In vivo dosimetry was performed using a microStar® system with nanoDots™ (Landauer, Inc., Glenwood, IL).

Animal cage-side observations:

Cage-side observations of the animals were performed twice daily by the research staff. Activity, posture, stool consistency, emesis, hemorrhage, alopecia, respiratory status, and seizure activity were observed, scored, and recorded. The animals were observed at least one additional time each day to assess their general health.

Medical Management:

All NHP were administered medical management according to an Institutional Animal Care and Use Committee (IACUC)-approved protocol of defined clinical triggers to start and stop treatments. This included hydration fluids, antibiotics, analgesics, anti-diarrheals, antipyretics, anti-emetics, anti-ulceratives, nutritional support, and blood transfusions. Specific details as to initiation, dosages, and duration of treatments were described previously (Farese et al. 2013). During the conduct of the study presented herein, Invanz® (ertapenem sodium) (Merck & Co. Inc., Whitehouse Station, NJ) was administered in place of Primaxin® when microbial resistance was demonstrated to enrofloxacin, gentamicin, and ceftriaxone.

Survival:

Acute GI-ARS survival was defined at 15 days post-irradiation, and acute H-ARS survival was defined at 60 days post-irradiation. Animals were euthanized according to a set of IACUC-designated clinical criteria.

Neupogen® Administration:

Neupogen® (Amgen, Inc., Thousand Oaks, CA) was dosed according to the previous day’s body weight (10.2 ± 0.3 μg kg−1), and injected subcutaneously once a day starting at 24 (day 1), 72 (day 3), or 120 (day 5) hours post-irradiation. Control animals were dosed with an identical volume of 5% dextrose in water starting at 24 hours post-irradiation. Dosing was stopped when an absolute neutrophil count (ANC) ≥ 1,000 cells μL−1 was observed for 3 consecutive days.

Hematology and bacteriology:

Peripheral blood was obtained from the saphenous vein for complete blood (CBC) (AcT diff2™, Beckman Coulter, Inc., Miami, FL) and manual white blood cell (WBC) differential counts (Wright-Giemsa Stain). The ANC was calculated using the WBC count obtained by CBC and the neutrophil count obtained by differential. Peripheral blood samples for bacteriology cultures were collected on days when febrile neutropenia (FN), defined as an ANC < 500 cells μL−1 coincident with a rectal body temperature ≥ 103.0 °F (39.4 °C), was observed. Blood was cultured in aerobic and anaerobic bottles and analyzed with a BACTEC 9050 Microbial Detection System (Becton Dickinson, Franklin Lakes, NJ).

Statistics:

Probit fits were made to mortality data, and confidence intervals were calculated for lethal dose (LD) and slope values according to the methods of Finney using the R statistical software (version 2.13.1.) (Finney 1947). A comparison of slopes and LD50s was made using Wald statistics. Means values and associated standard errors for continuous and count data were calculated and plotted using Microsoft Excel 2010.

RESULTS

The Dose Response Relationships (DRRs) for GI-ARS and H-ARS using TBI or PBI/BM5 plus Medical Management.

The GI-ARS DRR.

The DRR determined for PBI/BM5-induced GI-ARS (n = 111) resulted in an estimated LD50 at 15 day (LD50/15) of 11.97 Gy [95% confidence interval: 11.64, 12.62], which was significantly different (p = 0.02) than the estimated value of 11.33 Gy for TBI-induced GI-ARS (n = 61). The corresponding slope for the PBI/BM5 DRR was 1.03 [0.59, 1.47], compared to 0.92 [0.70, 1.34] for the TBI DRR (Table 1, Fig. 1). The slopes were not significantly different (p = 0.46) from each other (MacVittie et al. 2012b; MacVittie et al. 2012a). However, the sparing of approximately 5% BM significantly increased the LD50/15 to 11.97 Gy relative to 11.33 Gy for the TBI-induced GI-ARS (MacVittie et al. 2012a).

Table 1.

Comparison of mortality values for GI-ARS and H-ARS post-TBI or PBI/BM5.

Model LD10 (Gy) LD50 (Gy) LD90 (Gy) Slope n
H-ARS TBI 6.41[4.57,6.89] 7.54[7.24,7.75] 8.66[8.24,10.27] 1.14[0.45,1.82] 133
H-ARS PBI/BM5 9.21[7.87,9.79] 10.88[10.48,11.3] 12.56[11.97,13.91] 0.76[0.44,1.09] 92
GI-ARS TBI 9.93[8.47,10.54] 11.33[10.81,11.75] 12.73[12.21,13.9] 0.92[0.70,1.34] 61
GI-ARS PBI/BM5 10.73[10.12,11.06] 11.97[11.64,12.62] 13.22[12.6,14.7] 1.03[0.59,1.47] 111
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Rhesus macaques were exposed to TBI or PBI/BM5 utilizing 6 MV LINAC-derived photons. Animals were euthanized according to a set of IACUC-approved clinical criteria. Mortality due to GI-ARS and H-ARS was defined at 15 days and 60 days post-irradiation, respectively. The values displayed are derived from linear (no dose transform) normal probit fits. Values in brackets are 95% confidence intervals.

Figure 1. TBI vs. PBI/BM5 GI- and H-ARS survival curves.

Figure 1.

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Rhesus macaques were exposed to TBI or PBI/BM5 utilizing 6 MV LINAC-derived photons. Animals were euthanized according to a set of IACUC-approved clinical criteria. Probit curves of 15- and 60-day survival are plotted.

The H-ARS DRR.

Sparing approximately 5% BM had significant effects on the DRR for the H-ARS subsyndrome (Fig. 1). The database used to determine the H-ARS DRR for TBI and PBI/BM5 includes additional NHP from recent efficacy studies (Farese et al. 2013; Farese et al. 2014; Hankey et al. 2015). These additional data supported the original DRR.

The DRR for TBI H-ARS (n = 133) established the LD50/60 at 7.54 Gy [7.24, 7.75] with a corresponding slope of 1.14 [0.45, 1.82] (Table 1, Fig. 1). The DRR determined for the PBI/BM5 H-ARS plus GI damage (n = 92) resulted in respective LD50/60 and slope of 10.88Gy [10.48, 11.30] and 0.76 [0.44, 1.09] (Table 1, Fig. 1). This represented a significant increase over the TBI LD50/60 (p < 0.001). Animals exposed to PBI/BM5 at the TBI LD50/60 of 7.54 Gy would all survive through 60 days if administered only medical management. It would require approximately 9.0 Gy PBI/BM5 to induce 5–10% mortality using the medical management alone (Fig. 1). The slope for the H-ARS after TBI was not significantly different from the PBI/BM5 value (p = 0.33). Hematopoietic activity in the spared BM permitted study of animals that survived the acute GI-ARS. However, it is important to note that the acute GI-ARS resulted in prolonged GI damage that occurred concomitantly with the development of H-ARS in the PBI/BM5 model. Survivors of the acute GI-ARS in the PBI/BM5 protocol would succumb to the H-ARS if exposed to uniform TBI at the same dose levels (MacVittie et al. 2012a).

GI-ARS mortality and survival time: Neupogen® administration at increasing intervals post-exposure.

GI-ARS mortality at day 15 in the 10.0 Gy control cohort was 7% (n = 1/15) relative to the Neupogen® cohorts of 29% (2/7), and 0% (0/8), when starting administration on day 1 or day 3, respectively. The mortality at day 15 in the 11.0 Gy control cohort was 5% (1/22) relative to the Neupogen® cohorts of 20% (1/5), 0% (0/8), and 0% (0/8) when starting administration on day 1, day 3, or day 5 respectively (Table 2). This is approximate to the respective mortalities of 10% (10.0 Gy) and 15% (11.0 Gy) previously observed in the PBI/BM5 model (MacVittie et al. 2012a). The study was not powered to detect the effect on mortality; however, the data suggested that early administration of Neupogen® may exacerbate the acute GI-ARS mortality.

Table 2.

Mortality rates and MST of decedents post-PBI/BM5.

PBI/BM5 Dose Treatment 0–15 Day Mortality MST of 0–15 Day Decedents 0–60 Day Mortality MST of 0–60 Day Decedents
10.0 Gy Control (n=15) 7% 13.0 (n=1) 28% 26.3 ± 6.3
Neupogen® on day 1 (n=7) 29% 9.0 ± 2.0 29% 9.0 ± 2.0
Neupogen® on day 3 (n=8) 0% NA 13% 38.0 (n=1)
11.0 Gy Control (n=22) 5% 9.0 (n=1) 41% 25.3 ± 4.5
Neupogen® on day 1 (n=5) 20% 9.0 (n=1) 60% 24.0 ± 10.4
Neupogen® on day 3 (n=8) 0% NA 25% 29.5 ± 8.5
Neupogen® on day 5 (n=8) 0% NA 38% 28.7 ± 4.5
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Rhesus macaques were exposed to 10.0 or 11.0 Gy PBI/BM5 utilizing 6 MV LINAC-derived photons. Animals were administered control article (5% dextrose in water) or Neupogen® beginning at the indicated times post-PBI/BM5, through the third consecutive day an ANC ≥ 1,000 cells μL−1 was observed post-nadir. Animals were euthanized according to a defined set of clinical criteria per IACUC protocol. Mortality rates were collated at 15 days post-PBI/BM5 and 60 days, inclusive. The MST of the decedents in each interval is displayed ± sem.

H-ARS Mortality: PBI/BM5 at 10.0 Gy plus Neupogen®.

It was estimated from the DRR that 10.0 Gy PBI/BM5 with medical management alone would result in approximately 25% mortality within 60 days post-exposure. The 10.0 Gy control cohort in the current study (n = 15) had 28% mortality (4/15) within 60 days. The mean survival time (MST) for decedents was 26.3 days. The cohort administered Neupogen® from day 1 had 29% (2/7) mortality at day 60. These NHP succumbed at day 7 and day 11 (MST = 9 days) from the acute GI-ARS. There were no mortalities noted throughout the ensuing 60 day period during the H-ARS. The cohort administered Neupogen® from day 3 had 13% (1/8) mortality at day 60, due to a single mortality at day 38 post-exposure (Table 2). The study was not powered to detect a significant difference in mortality; however, the trend suggested that Neupogen® improved survival during the duration of the H-ARS, and did not exacerbate mortality when administered in concert with an active acute GI-ARS subsyndrome.

H-ARS Mortality: PBI/BM5 at 11.0 Gy plus Neupogen®.

It was estimated from the DRR that 11.0 Gy PBI/BM5 with medical management alone would result in approximately 50% mortality within 60 days post-exposure. The 11.0 Gy control cohort in the current study had 41% mortality (9/22) within 60 days. The mean survival time was 25.3 days, with a single (1/22) NHP succumbing at day 9 within the GI-ARS. The cohorts administered Neupogen® initiated at day 1, day 3, or day 5 had respective mortality rates of 60% (3/5), 25% (2/8), and 38% (3/8) at day 60, with MSTs of 24.0, 29.5, and 28.7 days. As noted above, the study was not powered to detect a significant difference in mortality; however, the trend suggested that Neupogen® improved survival relative to the control cohort, and did not exacerbate mortality when administered in a delayed schedule and in concert with an active acute GI-ARS subsyndrome.

H-ARS Mortality: TBI at 7.5 Gy, 10.0 Gy, and 11.0 Gy.

TBI at 7.5 Gy plus supportive care is the approximate LD50/60 for the H-ARS (Farese et al. 2013; Farese et al. 2012a; Farese et al. 2014). Neupogen® administration significantly increased survival when initiated on day 1 after 7.5 Gy TBI, from 41% compared to 79% for TBI with medical management alone (Farese et al. 2013). TBI at 10.0 Gy or 11.0 Gy would be 100% lethal for the H-ARS and approximately 10% and 50% lethal for the GI-ARS, respectively (MacVittie et al. 2012b; Farese et al. 2012a).

Hematologic parameters and neutrophil kinetics: TBI at 7.5 Gy, 10.0 Gy, and 11.0 Gy vs. PBI/BM5 at 10.0 Gy and 11.0 Gy.

TBI:

The current database of hematologic parameters at 7.5 Gy (n = 85) was compiled during three previous GLP-compliant studies (Farese et al. 2013; Farese et al. 2014; Hankey et al. 2015). There is no difference in the initial slope of ANC decrease after exposure relative to all doses referenced herein (Fig. 2a). The mean first day of ANC to < 500 cells μL−1 was day 4.9. However, the ANC continued to decrease and reached the mean nadir, 2 cells μL−1 on day 12.8 post-exposure. The mean duration of ANC < 500 cells μL−1 was 17.4 days, and the mean first day post-exposure to reach a sustained ANC ≥ 1,000 cells μL−1 was day 24.6 (Fig. 2a, Table 3).

Figure 2. ANC kinetics post-PBI/BM5 or TBI with Neupogen® treatment.

Figure 2.

Figure 2.

Figure 2.

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Rhesus macaques were exposed to 10.0 Gy or 11.0 Gy PBI/BM5 or TBI, or 7.5 Gy TBI utilizing 6 MV LINAC-derived photons. Animals were administered control article (5% dextrose in water) or Neupogen® beginning at the indicated times post-irradiation, through the third consecutive day an ANC ≥ 1,000 μL−1 was observed post-nadir. Peripheral blood was drawn daily, and the ANC was calculated using values obtained from an automated CBC and manual WBC differential. The plotted values are means. A comparison between irradiation models and doses is shown in (a), while comparisons between control and Neupogen®-treated cohorts exposed to 10.0 Gy or 11.0 Gy are shown in (b) and (c), respectively.

Table 3.

Latency, duration, and severity of neutropenia post-PBI/BM5 or TBI.

Latency
 Duration
 Severity
Exposure Treatment 1st Day of ANC < 500 μL−1 # of Days of ANC < 500 μL−1 1st Day of Recovery Incidence of Recovery Nadir (cells μL−1) Day of Nadir
7.5 Gy TBI Control (n=85) 4.9 ± 0.1 17.4 ± 0.4 24.6 ± 0.7 55% 2 ± 1 12.8 ± 0.2
10.0 Gy PBI/BM5 Control (n=15) 4.5 ± 0.2 11.6 ± 1.1 24.2 ± 0.9 87% 94 ± 14 7.9 ± 0.5
Neupogen® on day 1 (n=7) 3.9 ± 0.1 3.5 ± 0.7 9.5 ± 1.1 86% 220 ± 29 5.0 ± 0.2
Neupogen® on day 3 (n=8) 4.8 ± 0.2 4.6 ± 1.4 12.4 ± 1.8 100% 243 ± 65 6.0 ± 0.2
11.0 Gy PBI/BM5 Control (n=22) 5.0 ± 0.1 10.9 ± 1.2 23.2 ± 2.5 86% 131 ± 17 8.5 ± 0.3
Neupogen® on day 1 (n=5) 4.2 ± 0.2 2.8 ± 0.3 10.0 ± 1.4 80% 292 ± 42 5.0 ± 0.4
Neupogen® on day 3 (n=8) 5.0 ± 0.3 3.8 ± 0.8 12.1 ± 1.2 100% 236 ± 32 6.0 ± 0.5
Neupogen® on day 5 (n=8) 4.9 ± 0.4 4.5 ± 0.6 13.0 ± 1.4 100% 217 ± 32 6.1 ± 0.4
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Rhesus macaques were exposed to 10.0 or 11.0 Gy PBI/BM5 or 7.5 Gy TBI utilizing 6 MV LINAC-derived photons. Animals were administered control article (5% dextrose in water) or Neupogen® beginning at the indicated times post-irradiation, through the third consecutive day an ANC ≥ 1,000 cells μL−1 was observed post-nadir. The ANC was calculated daily using values obtained from an automated CBC and manual WBC differential. Recovery was defined as the observation of an ANC ≥ 1,000 cells μL−1 for 3 consecutive days of post-nadir. The ANCs of 2 NHP in the day 3 Neupogen® cohort were never observed to be < 500 cells μL−1. NHP that did not recover were excluded from the duration calculations, and NHP that did not reach a nadir were excluded from the severity calculations. The values displayed are means ± sem.

TBI with 10.0 Gy or 11.0 Gy caused severe myelosuppression, with the ANC decreasing to absolute values within 10 days to 14 days post exposure (Fig. 2a).

PBI/BM5:

The sparing of approximately 5% of active BM significantly reduced the myelosuppressive effect of 10.0 Gy and 11.0 Gy exposures (Fig. 2a). All neutrophil parameters indicative of recovery were enhanced relative to TBI at 7.5 Gy, 10.0 Gy, or 11.0 Gy, with the exception of the recovery time to an ANC ≥ cells 1,000 μL−1 (Fig. 2a, Table 3). The mean ANC recovery kinetics slowed somewhat after returning to the 500 cells μL−1 level at day 14 and day 17 post-PBI/BM5, respectively. ANC recovery to a level ≥ cells 1,000 μL−1 required an additional week. There is an interesting difference in neutrophil recovery kinetics between the PBI/BM5 cohorts exposed to 10.0 Gy or 11.0 Gy relative to the TBI cohort exposed to 7.5 Gy (Fig. 2a).

Hematologic parameters and neutrophil kinetics: PBI/BM5 at 10.0 Gy plus Neupogen®.

The administration of Neupogen® initiated on day 1 or day 3 in the PBI/BM5 protocol resulted in a substantial mitigation of neutropenia relative to the control cohort (Fig. 2a, Table 3).

The 10.0 Gy cohorts: First day to ANC < 500 cells μL−1.

PBI/BM5 at 10.0 Gy reduced the ANC in the control cohort to < 500 cells μL−1 on day 4.9 on average. Mean ANC values did not decrease to < 100 cells μL−1 post-exposure. In the cohorts administered Neupogen® from day 1 or day 3, the ANC was reduced to < 500 cells μL−1 on day 3.9 and day 4.8 on average, respectively. There was no apparent difference from the control cohort (Table 3).

The 10.0 Gy cohorts: Mean duration of neutropenia and nadir.

The mean duration of ANC < cells 500 μL−1 was 11.6 days in the control cohort. Neupogen® administration initiated at either day 1 or day 3 reduced the duration of mean ANC < cells 500 μL−1 to 3.5 days and 4.6 days, respectively. The mean nadir and the mean day the nadir was reached was 94 cells μL−1 at day 7.9 for the control cohort, and 220 cells μL−1 at day 5.0 and 243 cells μL−1 at day 6.0 for the cohorts where Neupogen® was initiated on day 1 or day 3, respectively (Table 3, Fig. 2b).

The 10.0 Gy cohorts: The mean time to recovery to ANC ≥ cells 1,000 μL−1.

The mean time to recovery to an ANC ≥ 1,000 μL−1 was 24.2 days for the control cohort. Neupogen administration initiated at day 1 or day 3 substantially reduced the respective mean recovery times to an ANC ≥ cells 1,000 μL−1 to 9.5 days and 12.4 days (Table 3, Fig. 2b).

The 10.0 Gy cohorts: incidence of FN.

The incidence (the number of animals who met the criteria on at least one study day) of FN in the 10.0 Gy control cohort was 47% (7/15). The incidences of FN in the day 1 and day 3 Neupogen® cohorts were 14% (1/7) and 13% (1/8), respectively. The mean first day of FN was day 15.9 in the control cohort and FN was observed on day 4 and day 12 in the day 1 and day 3 Neupogen® cohorts, respectively (Table 4).

Table 4.

Incidence, latency, and duration of FN with bacteriology post-PBI/BM5.

Exposure Treatment Incidence of FN (% of NHP) First Day of FN # of Days Incidence of Infection (% of Cultured NHP)
Gram+ Gram−
10.0 Gy Control (n=15) 47% 15.9 ± 2.8 1.1 ± 0.1 29% 0%
Neupogen® on
day 1 (n=7)		 14% (n=1) 4 1 0% 0%
Neupogen® on day 3 (n=8) 13% (n=1) 12 1 0% 0%
11.0 Gy Control (n=22) 32% 16.1 ± 2.8 2.1 ± 0.5 57% 0%
Neupogen® on day 1 (n=5) 0% NA NA NA NA
Neupogen® on day 3 (n=8) 25% 8.0 ± 1.0 1.5 ± 0.5 50% 0%
Neupogen® on day 5 (n=8) 0% NA NA NA NA
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Rhesus macaques were exposed to 10.0 or 11.0 Gy PBI/BM5 utilizing 6 MV LINAC-derived photons. Animals were administered control article (5% dextrose in water) or Neupogen® beginning at the indicated times post-irradiation, through the third consecutive day an ANC ≥ 1,000 cells μL−1 was observed post-nadir. FN was defined as ANC < 500 cells μL−1 with rectal body temperature ≥ 103.0 °F (39.4 °C), and blood cultures were collected on days when FN was observed. The time values displayed are means ± sem.

Hematologic parameters and neutrophil kinetics: PBI/BM5 at 11.0 Gy plus Neupogen®:

The administration of Neupogen® starting at day 1, day 3, or day 5 in the PBI/BM5 protocol resulted in a mitigation of neutropenia relative to the control cohort (Fig. 2c, Table 3).

The 11.0 Gy cohorts: Mean first day to ANC < 500 cells μL−1.

PBI/BM5 at 11.0 Gy reduced the ANC in the control cohort to < 500 cells μL−1 by day 5.0 post-exposure. The mean first days of ANC < 500 cells μL−1 in the day 1, day 3, and day 5 Neupogen®-treated cohorts were day 4.2, day 5.0, and day 4.9, respectively. There was no apparent difference in the ANC decrease to < 500 cells μL−1 from the control cohort (Fig. 2c, Table 3).

The 11.0 Gy cohorts: Mean duration of neutropenia and nadir.

The mean duration of ANC < 500 cells μL−1 was 10.9 days in the control cohort. Neupogen® administration initiated on day 1, day 3, or day 5 reduced the mean duration of ANC < 500 cells μL−1 to 2.8 days, 3.8 days, and 4.5 days, respectively. The respective mean nadirs and days the nadirs were reached were 131 cells μL−1 at day 8.5 for the control cohort, and 292 cells μL−1 at day 5.0, 236 cells μL−1 at day 6.0, and 217 cells μL−1 at day 6.1 for the day 1, day 3, and day 5 Neupogen® cohorts, respectively (Fig. 2c, Table 3). Neupogen® administration substantially reduced the mean duration of neutropenia as well as the nadirs irrespective of the delay in initiation of treatment.

The 11.0 Gy cohorts: The mean time to recovery to ANC ≥ 1,000 cells μL−1.

The mean time to recovery to ANC ≥ 1,000 cells μL−1 was 23.2 days for the control cohort. Neupogen® administered from either day 1, day 3, or day 5 reduced the recovery time to 10.0 days, 12.1 days, and 13.0 days, respectively (Fig. 2c, Table 3).

The 11.0 Gy cohorts: Incidence of Febrile Neutropenia (FN).

The incidence of FN in the 11.0 Gy control cohort was 32% (7/22). Incidences of FN in the day 1, day 3, and day 5 Neupogen® cohorts were 0% (0/5), 25% (2/8), and 0% (0/8), respectively. The mean first day of FN was day 16.1 in the control cohort and day 8.0 in the day 3 Neupogen® cohort (Table 4).

Platelet recovery kinetics: PBI/BM5 and TBI post-10.0 Gy or 11.0 Gy.

TBI at either 10.0 Gy or 11.0 Gy markedly reduced platelet levels to absolute values within 10 days to 12 days post-exposure (Fig. 3a). Recovery did not occur due to ensuing mortality.

Figure 3. Platelet kinetics post-PBI/BM5 with Neupogen® treatment.

Figure 3.

Figure 3.

Figure 3.

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Rhesus macaques were exposed to 10.0 Gy or 11.0 Gy PBI/BM5 or TBI utilizing 6 MV LINAC-derived photons. Animals were administered control article (5% dextrose in water) or Neupogen® beginning at the indicated times post-irradiation, through the third consecutive day an ANC ≥ 1,000 μL−1 was observed post-nadir. Peripheral blood was drawn daily, and the platelet counts were obtained from an automated CBC. The plotted values are means. A comparison between irradiation models and doses is shown in (a), while comparisons between control and Neupogen®-treated cohorts exposed to 10.0 Gy or 11.0 Gy are shown in (b) and (c), respectively.

The sparing of approximately 5% of active BM significantly reduced the severely thrombocytopenic effect of TBI (Fig. 3a). All platelet-related parameters indicative of recovery were substantially enhanced post-PBI/BM5 relative to TBI at 10.0 Gy or 11.0 Gy (Fig. 3a, Table 5). The respective platelet nadirs and subsequent recovery kinetics were equivalent in both 10.0 Gy and 11.0 Gy control cohorts.

Table 5.

Incidence, latency, duration, and severity of thrombocytopenia post-PBI/BM5.

Latency
 Duration
 Severity
Exposure Treatment Incidence (% of NHP) 1st Day of Plt < 20 × 103 μL−1 # of Days of Plt < 20 × 103 μL−1 1st Day of Recovery Nadir (103 μL−1) Day of Nadir
10.0 Gy Control (n=15) 80% 9.5 ± 0.4 7.3 ± 1.2 17.6 ± 1.2 10.9 ± 2.8 11.3 ± 0.4
Neupogen® on day 1 (n=7) 71% 9.4 ± 0.5 5.8 ± 0.9 15.8 ± 0.9 9.8 ± 3.8 10.8 ± 0.5
Neupogen® on day 3 (n=8) 75% 9.2 ± 0.3 6.5 ± 1.5 16.5 ± 2.0 13.4 ± 4.0 10.8 ± 0.3
11.0 Gy Control (n=22) 77% 9.9 ± 0.2 7.5 ± 1.5 19.8 ± 2.6 10.4 ± 2.7 11.1 ± 0.3
Neupogen® on day 1 (n=5) 80% 10.0 ± 0.9 2.8 ± 0.9 12.8 ± 0.6 13.3 ± 3.3 10.3 ± 0.8
Neupogen® on day 3 (n=8) 75% 9.8 ± 0.5 4.8 ± 0.7 14.7 ± 0.6 13.9 ± 2.5 10.5 ± 0.3
Neupogen® on day 5 (n=8) 75% 9.5 ± 0.3 5.3 ± 0.6 14.8 ± 0.8 12.8 ± 2.2 11.1 ± 0.5
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Rhesus macaques were exposed to 10.0 or 11.0 Gy PBI/BM5 utilizing 6 MV LINAC-derived photons. Animals were administered control article (5% dextrose in water) or Neupogen® beginning at the indicated times post-irradiation, through the third consecutive day an ANC ≥ 1,000 cells μL−1 was observed post-nadir. Platelet counts were obtained from an automated CBC. Recovery was defined as the observation of a platelet count ≥ 20 × 103 μL−1 for 3 consecutive days post-nadir. The “# of Days of Plt < 20 × 103 μL−1” parameter represents a raw count, while the “1st Day of Recovery” parameter accounts for the effect of whole blood transfusions according to the following rule: if the NHP’s platelet count was increased to ≥ 20 × 103 μL−1 as a result of a transfusion, and it did not decrease to < 20 × 103 μL−1 again thereafter (i.e., the stated criterion for recovery was met), the 3rd day following the transfusion was counted as the 1st day of recovery. NHP that did not recover were excluded from the duration calculations, and NHP that did not reach a nadir were excluded from the severity calculations. The values displayed are means ± sem.

Platelet recovery kinetics PBI/BM5 post 10.0 Gy or 11.0 Gy plus Neupogen®.

The administration of Neupogen® beginning on day 1, day 3, or day 5 in the 10.0 Gy or 11.0 Gy PBI/BM5 protocols stimulated a modest increase in platelet recovery relative to the marked thrombopoietic effect of the spared BM in the control cohorts (Figs. 3b, 3c, Table 5). The respective nadirs relative to Neupogen® treatment were not different within either the 10.0 Gy or 11.0 Gy cohorts.

DISCUSSION

MCM efficacy:

Studies were initiated to assess the efficacy of Neupogen® against the H-ARS in accordance with recommendations provided in recent FDA guidance documents (Food and Drug Administration 2009; Food and Drug Administration 2014). Additionally, an FDA Advisory committee meeting focused on the efficacy of Neupogen® and other LGFs provided a number of questions that suggested viewing the efficacy of LGFs in the larger context of MCM development (Food and Drug Administration 2013). MCM efficacy and administration must be considered in the context of the clinical scenario consequent to a nuclear event. Will the MCM mitigate severe myelosuppression in the following conditions: a) following exposure to radiation doses associated with lethality greater than an LD50, b) with a delay in the initiation of treatment, c) in the presence of other organ co-morbidities or subsyndromes and d) in the combined injury situation?

The post-nuclear exposure environment:

Accounts of nuclear weapon detonations and radiation accident scenarios have characterized the post-radiation environment as ill-defined relative to exposure geometry (Liu et al. 2008; Guskova et al. 1990; Gourmelon et al. 2010; Hirama et al. 2003). The exposures will be non-uniform and heterogeneous, with a variable dose distribution to the key organ systems responsible for the ARS and DEARE. The advantageous aspect afforded by this type of exposure relative to TBI is the coincident tissue-sparing and enhanced potential for mitigating myelosuppression, morbidity, and mortality. The effect of marrow sparing via shielding and heterogeneous or unilateral exposure on the severity of myelosuppression and mortality has been described adequately in the mouse, canine, and NHP (Monroy et al. 1988; Cole et al. 1967; Maillie et al. 1966; Nothdurft et al. 1997; Bertho et al. 2005b; Rauchwerger 1972; MacVittie et al. 2012b; Herodin et al. 2007; Croziat et al. 1976; Gidali et al. 1972; Farese et al. 1993; MacVittie et al. 1994; Drouet et al. 2008).

An animal model research platform: Marrow sparing, high-dose exposure, concurrent ARS subsyndromes, and delayed effects.

A partial-body model with minimal bone marrow sparing permits high-dose exposure within the dose range that induces the GI-ARS and DEARE, characterized by MOI to the lungs, heart, and kidneys, and prolonged effects to the immune, GI, and hematopoietic systems (MacVittie et al. 2012a; Booth et al. 2012; Chua et al. 2012; MacVittie et al. 2014). Furthermore, it allows development of the H-ARS subsequent to survival from the acute GI-ARS (MacVittie et al. 2012b; MacVittie et al. 2012a). Therefore, the PBI/BM5 geometry established a model in which the key sequelae of the ARS and DEARE develop concurrently in a dose- and time-dependent fashion. The ARS is evolving during the coincident organ-dependent latent period for the DEARE against the backdrop of severe and prolonged immunosuppression. This post-exposure environment provides a platform to assess the organ-specific efficacy and multi-organ effects of single and combined MCM. In this context of high-dose exposure, the H-ARS evolves in the company of a coincident GI-ARS resulting in a condition of prolonged, dose-dependent GI damage maintained through at least 180 days post-exposure (MacVittie et al. 2012a). Conversely, the acute GI-ARS evolves in an environment of early regenerating hematopoiesis focused within the spared tibial marrow. The PBI/BM5 exposure protocol is therefore amenable to addressing three of the four questions posed relative to using LGFs in a more realistic context of use and clinical support scenario. Herein, we addressed two questions: 1) will Neupogen® mitigate severe myelosuppression when administered with an increasing interval between exposure and treatment and 2) will Neupogen® influence the morbidity and mortality associated with the concurrent GI-ARS?

The H-ARS with concomitant GI damage: Effect of Neupogen® relative to early and delayed administration schedule.

Neupogen®, administered early, within 24 h following an LD50/60 dose of TBI, significantly increased survival and diminished all secondary parameters associated with severe myelosuppression (Farese et al. 2013). However, when administration of Neupogen® was delayed until 48 h post an LD50/60 dose of TBI, its mitigation of mortality and hematologic parameters was marginalized (Farese et al. 2014). Uniform TBI is an unlikely consequence of a nuclear device detonation or accidental radiation exposure event. A partial-body model with marrow sparing minimizes exposure variables and permits analysis of MCM efficacy in a more radiologically- and clinically-relevant scenario. This objective – marrow sparing – may also be achieved using unilateral, non-uniform TBI. However, a full DRR has not been performed in these models (Farese et al. 1993; MacVittie et al. 1994; Drouet et al. 2004; Herodin et al. 2007; Drouet et al. 2008). The PBI/BM5 model allowed determination of MCM efficacy against the H-ARS relative to the concurrent GI-ARS in the context of an early and markedly delayed initiation of Neupogen® administration post-exposure.

H-ARS and GI-ARS mortality.

Furthermore, the PBI/BM5 model demonstrated the significant effect of BM sparing alone on H-ARS and GI-ARS mortality (MacVittie et al. 2012a). The BM sparing alone shifted the DRR for H-ARS and increased the LD50/60 from 7.45 Gy after TBI to 11.01 Gy; the DRR and LD50/15 for GI-ARS was also significantly shifted from 11.33 Gy after TBI to 11.95 Gy (MacVittie et al. 2012b; MacVittie et al. 2012a; Farese et al. 2012a). The approximate 5% marrow sparing was intended to eliminate the noted marrow-sparing effect on the GI-ARS (Vriesendorp et al. 1992; Terry et al. 1989; Mason et al. 1989; Handford et al. 1961). However, it is apparent that a minimal amount of BM sparing can mitigate GI-ARS mortality. Interestingly, the analysis of GI crypt damage and recovery appeared comparable in the TBI and PBI/BM5 models (MacVittie et al. 2012a). The BM-sparing effect on GI-ARS mortality forecast that the administration of effective MCM against the H-ARS could also mitigate the severity of the GI-ARS. However, the data herein provided evidence that the administration of Neupogen® in an early or delayed schedule did not mitigate the GI-ARS despite its significant enhancement on hematopoietic recovery and mitigation of myelosuppression.

H-ARS myelosuppression.

The hematopoietic effect of the spared BM was significant. However, the spared BM was exposed to a mean dose of approximately 0.50 Gy. This is close to the expected d37 or d0 of 0.60 Gy for hematopoietic stem and progenitor cells and will result in a dose-dependent loss of these cells (van Bekkum 1991). The surviving progenitor cells provided for the early production of neutrophils that reduced the level but not the timing of the ANC nadir relative to comparable 10.0 Gy and 11.0 Gy doses of TBI. The 10.0 Gy and 11.0 Gy TBI cohorts continued to absolute neutropenia and lethality, whereas the 10.0 Gy and 11.0 Gy PBI/BM5 cohorts reduced the loss of ANC within 6 days post exposure, and stabilized production through to 10 days, followed by increased production to reach ANC levels ≥ 500 cells μL−1 within approximately 15 days post-exposure. Production then continued over the next week to reach ANC levels ≥ 1,000 cells μL−1. The response suggested that the tibial marrow-derived progenitor cells responded to demand from a myeloablated system with a multi-phasic response to secure recovery within 30 days post-exposure. It is notable that the dose differential had no apparent effect on hematopoietic recovery since an equivalent amount to BM was spared. The total radiation dose would only influence the dose-dependent sequelae of acute GI-ARS and delayed MOI (MacVittie et al. 2012b; MacVittie et al. 2012a).

Administration of Neupogen® stimulated an earlier recovery of all granulopoietic parameters relative to the 10.0 Gy and 11.0 Gy control cohorts. At both dose levels, administration of Neupogen® improved the nadir and diminished the time to recovery from neutropenia to ANCs ≥ 500 cells μL−1 and ≥ 1,000 cells μL−1, regardless of when treatment was initiated from 1 to 5 days post-exposure. The ability to evoke an equivalent granulopoietic response is likely due to the fact that the spared BM had regenerated with each day post-exposure to form a larger LGF-responsive pool of progenitors. The responsive progenitor cells respond in situ with increased regeneration and differentiation, as well as mobilization and seeding of myeloablated marrow sites.

The marrow-sparing effect has been demonstrated in mice, canines, and NHP with and without the use of LGFs (Monroy et al. 1988; Maloney et al. 1972; Nothdurft et al. 1997; Bertho et al. 2005a; Croziat et al. 1976; Gidali et al. 1972; Farese et al. 1993; MacVittie et al. 1994; Farese et al. 1994; Drouet et al. 2008; Drouet et al. 2004; Drouet et al. 2014; Zhuge et al. 2012). Monroy et al. and Bertho et al. provided evidence of similar responses to GM-CSF or G-CSF in non-uniform and heterogeneous PBI models in the NHP (Monroy et al. 1988; Bertho et al. 2005a). Monroy et al. exposed control non-shielded and partially shielded NHP at a high dose rate (Co-60 gamma radiation, 5.0–7.35 Gy min−1) to 8.0 Gy to the torso with femoral and tibial sparing. The control 8.0 Gy uniform TBI exposure without BM sparing plus medical management was 100% lethal. The non-uniform exposure with BM sparing plus medical management resulted in 20% mortality. The tibiae were estimated to have received 4.35 Gy relative to the 8.0 Gy delivered to the whole torso. Monroy et al. actually delayed initiation of GM-CSF until day 3 post-exposure, but used continuous administration of GM-CSF via osmotic mini-pumps to stimulate increased production of neutrophils and platelets relative to the control with BM sparing. Bertho et al. utilized a model of Co-60 gamma radiation with uniform 8.5 Gy (0.24 Gy min−1) exposure to the torso and approximately 4.5 Gy to the right arm. The exposed arm was estimated to contain approximately 5% of active BM, similar to Monroy et al. and the study herein (Monroy et al. 1988; Bertho et al. 2005a; MacVittie et al. 2012a). Lenograstim was started at 6 h post-exposure, and the dose was divided between two subcutaneous injections per day. The LGF shifted the nadir and recovery of leukocytes to earlier time points. Bertho et al. also noted a coincident development of GI-ARS in the 10.0 Gy cohort in which only partial hematopoietic recovery was stimulated by lenograstim (Bertho et al. 2005b).

The marrow sparing effect is also achieved in models of unilateral, total-body, non-uniform exposure by virtue of differential dose distribution through the body (Bond et al. 1967; Bond et al. 1991). These models have been effectively used to assess the survival and hematological effects of a full range of growth factor treatment (Drouet et al. 2004; Drouet et al. 2008; Drouet et al. 2014; Herodin et al. 2007; Farese et al. 1993; MacVittie et al. 1994; Farese et al. 1994). Significant differences in the models are noted. Farese et al. used a reactor-derived, mixed gamma and neutron exposure in the PA exposure aspect delivered at a “pulse” (50 msec) dose rate. Herodin and Drouet et al. exposed animals in the AP aspect at a moderate dose rate (0.20 Gy min−1).

The differences in models can be dramatic, including the following factors: a) the radiation dose to the region of spared BM, relative to the total dose; b) the dose rate employed; c) the region of BM spared; d) the variability in uniform, non-uniform and heterogeneous exposure; e) the choice of LGF; f) the LGF schedule and route of administration; and g) the number of animals utilized. However, the similarities rest in the approximate amount of BM spared based on the earlier study by Taketa et al. and the basic assumptions asserted by Bond and Robinson that survival from the H-ARS is dependent on the dose- and time-dependent survival of a critical fraction of stem and progenitor cells that are equally distributed throughout the active BM sites, and that surviving progenitor cells will contribute to regeneration of the hematopoietic system at a rate independent of their anatomic location (Bond et al. 1967; Taketa et al. 1970; Bond et al. 1991).

The effort herein used the established DRR for a PBI/BM5 model to further assess: a) the efficacy of BM sparing on the severity and time course of the H-ARS; b) the efficacy of the LGF, Neupogen®, in a more realistic model of high-dose exposure with minimal BM sparing and extended delays between exposure and the initiation of treatment; and c) to determine if administration of Neupogen® influenced the severity and time course of the GI-ARS. These questions are critical to provide realistic data on the efficacy of LGFs as current MCM to be employed in the context of a clinical scenario imposed by a nuclear radiation event. Spared BM will provide a ready source of multi-lineage and lineage-specific progenitor cells as targets for autologous recovery, as well as exogenous use of LGFs to accelerate hematopoietic recovery. The timely recovery of hematopoiesis and utility of LGFs and other MCM will likely depend of the amount of BM spared.

Acknowledgements:

We acknowledge the continued discussion, insight and constructive critique of our NIAID colleagues, Drs. David Cassatt and Bert Maidment. The study could not be performed without the tremendous support and expertise of the research staff of the UMB Preclinical Radiobiology lab and the MCART radiation physics core.

Source of Funding

This work was supported by NIAID contract HHSN272201000046C.

Footnotes

Conflicts of Interest

The authors declare no conflicts of interest.

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