Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes

Abstract

There is a need for countermeasures to mitigate lethal acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE). In WAG/RijCmcr rats, ARS occurs by 30-days following total body irradiation (TBI), and manifests as potentially lethal gastrointestinal (GI) and hematopoietic (H-ARS) toxicities after >12.5 and >7 Gy, respectively. DEARE, which includes potentially lethal lung and kidney injuries, is observed after partial body irradiation >12.5 Gy, with one hind limb shielded (leg-out PBI). The goal of this study is to enhance survival from ARS and DEARE by polypharmacy, since no monotherapy has demonstrated efficacy to mitigate both sets of injuries. For mitigation of ARS following 7.5 Gy TBI, a combination of three hematopoietic growth factors (polyethylene glycol (PEG) human granulocyte colony-stimulating factor (hG-CSF), PEG murine granulocyte-macrophage-CSF (mGM-CSF), and PEG human Interleukin (hIL)-11), which have shown survival efficacy in murine models of H-ARS were tested. This triple combination (TC) enhanced survival by 30-days from ∼25% to >60%. The TC was then combined with proven medical countermeasures for GI-ARS and DEARE, namely enrofloxacin, saline and the angiotensin converting enzyme inhibitor, lisinopril. This combination of ARS and DEARE mitigators improved survival from GI-ARS, H-ARS, and DEARE after 7.5 Gy TBI or 13 Gy PBI. Circulating blood cell recovery as well as lung and kidney function were also improved by TC + lisinopril. Taken together these results demonstrate an efficacious polypharmacy to mitigate radiation-induced ARS and DEARE in rats.

Keywords: acute radiation syndrome; delayed effects of acute radiation exposure; hematopoietic growth factor; lisinopril; mitigation; polypharmacy; radiation pneumonitis; supportive care.

FIGURE 1

Experimental design. To study the Acute Radiation Syndrome (ARS) (A), female WAG/RijCmcr rats (11–12 weeks of age) were irradiated with 7.5 Gy total body irradiation (TBI). At 24-h post-irradiation, all rats received a subcutaneous (SQ) injection of either the triple combination (TC, consisting of PEG-hG-CSF, PEG mGM-CSF and PEG hIL-11) or vehicle and the ACE inhibitor lisinopril was started in the drinking water (24 mg m⁻² d⁻¹) in two groups at 7 days post-irradiation. A subset of each treatment group was bled at days 10, 18, 25, and 30-days and complete blood counts were analyzed. The ARS experiment was terminated at 30-days.To study the Delayed Effects of Acute Radiation Syndrome (DEARE) (B), female WAG/RijCmcr rats (11–12 weeks of age) were irradiated with 13 Gy leg-out partial body irradiation (leg-out PBI). At 24-h post-irradiation, all rats received a subcutaneous (SQ) injection of either the triple combination (TC, consisting of PEG-hG-CSF, PEG mGM-CSF and PEG hIL-11), PEG-hG-CSF or vehicle and the ACE inhibitor lisinopril was started in the drinking water (24 mg m⁻² d⁻¹) in two groups at 7 days post-irradiation. Supportive care consisting of antibiotics (enrofloxacin, 10 mg kg⁻¹ d⁻¹, days 2–14) in the drinking water, SQ fluid hydration (saline, 40 ml kg⁻¹ d⁻¹, days 3–7) and supplemental powdered diet (days 35–70) were provided to all rats in the DEARE study. Radiation pneumonitis was monitored weekly by recording breathing rates (weeks 4–16) and while radiation nephropathy was monitored by measuring the blood urea nitrogen (BUN, days 90, 120, and 150). Lungs and kidneys were harvested at 56 and 120 days respectively to observe lung and renal radiation injury. The DEARE studies were terminated at 120 or 160-days.

FIGURE 2

Mitigation of hematopoietic-acute radiation syndrome (H-ARS) by triple combination with and without lisinopril. Kaplan-Meier plots show morbidity through 30-days after 7.5 Gy total body irradiation (TBI). The triple combination (TC, consisting of PEG-hG-CSF, PEG mGM-CSF and PEG hIL-11) or vehicle were given subcutaneously 24-h after TBI (designated by PEG-HGF) and the ACE inhibitor, lisinopril, was started in the drinking water 7 days after irradiation. The number of rats in each group is designated by the “n.” Non-irradiated controls are represented with the blue line. Morbidity was not different in the three irradiated groups given 7.5 Gy only, with vehicle or lisinopril, but survival was enhanced in the group which received the TC (p = 0.05, denoted by * compared to 7.5 Gy + vehicle group). Survival was increased in the irradiated group receiving TC and lisinopril compared to the irradiated rats receiving the vehicle for TC and lisinopril (p < 0.05, denoted by #).

FIGURE 3

Neutrophil recovery through 30-days post 7.5 Gy total body irradiation (TBI) is shown on a log-scale for the neutrophil count (×10³). The horizontal blue bar represents the neutrophil counts for non-irradiated controls. Asterisks (*) represent p < 0.02 as compared to non-irradiated controls. At 18 days, the two irradiated groups given the triple combination (TC, consisting of PEG-hG-CSF, PEG mGM-CSF and PEG hIL-11) with (red) and without lisinopril (black), had higher neutrophil counts (p ≤ 0.005) than the irradiated groups given the vehicle and lisinopril (yellow). Neutrophil counts returned to levels resembling non-irradiated controls by 25-days post-irradiation. The “n” values represent the number of rats alive in each group between 10 and 30 days after irradiation which decreased over time due to rats becoming moribund. Error bars represent 95% CIs for the mean.

FIGURE 4

Kaplan-Meier plot representing morbidity from DEARE up to 120 days post 13 Gy partial body irradiation with one hind limb shielded (leg-out PBI). The triple combination (TC, consisting of PEG-hG-CSF, PEG mGM-CSF and PEG hIL-11), vehicle or PEG-hG-CSF (BBT-015) were given subcutaneously 24 h post leg-out PBI (designated by PEG-HGF) and the ACE inhibitor, lisinopril, was started in the drinking water 7 days post irradiation (24 mg m⁻² d⁻¹). All irradiated rats were given supportive care with subcutaneous hydration (40 ml kg⁻¹ d⁻¹) and enrofloxacin (10 mg kg⁻¹ d⁻¹) from days 3–7 to 2–14, respectively,. There was trend in lower morbidity in irradiated rats that received TC + lisinopril compared to irradiated rats that received PEG-hG-CSF, but this was not significant (p = 0.07). Shaded (gray) areas represent the timing for the lung and renal injuries.

FIGURE 5

Radiation pneumonitis is mitigated by lisinopril after 13 Gy leg-out PBI. Graphical representation of breathing intervals as a secondary and functional endpoint for lung injury were obtained from all 13 Gy leg-out PBI irradiated treatment groups and non-irradiated controls. Breathing rates were recorded biweekly from weeks 4 to 16 post-irradiation and converted to breathing intervals (see Methods). The bars represent means with 95% CIs. Rats moribund with lung injury confirmed at necropsy (13 Gy + vehicle n = 2, 13 Gy + PEG-hG-CSF n = 6, 13 Gy + TC n = 2) were given a breathing interval of 0 at 12 weeks to account for attrition. Numbers in the bars represent N in each group. Asterisks represent p < 0.05 between treatment groups at 12 weeks, in brackets: 0 Gy + vehicle vs. 13 Gy + PEG-hG-CSF, 0 Gy vehicle vs. 13 Gy + vehicle, 13 Gy + TC + lisinopril vs. 13 Gy + PEG-hG-CSF.

FIGURE 6

Lisinopril mitigates histological lung injury after 13 Gy leg-out PBI. Representative histological sections of lung tissue harvested at 56-days after 13 Gy leg-out PBI were stained with H&E for each treatment group (A). Irradiated rats given the vehicle or PEG-hG-CSF showed increased alveolar wall thickness cellularity (green arrow), increased vessel wall thickness (black arrow) and foamy macrophages (red arrow). Black bars represent 100 μm. Graphical representations of the H&E-stained lung sections are shown for vessel wall thickness (B), alveolar wall thickness (C) and foamy macrophages (D). Vessel wall thickness (B) were increased in irradiated rats given the vehicle or PEG-hG-CSF, whereas TC + lisinopril mitigated lung injury (p < 0.05, denoted by * compared to controls and # compared to TC + lisinopril). Irradiation increased alveolar wall thickness (C) in all irradiated groups compared to control (p < 0.05, denoted by *) except for the TC + lisinopril group. Alveolar wall thickness was also increased compared to the TC + lisinopril group in the irradiated vehicle and PEG-hG-CSF groups (p < 0.05, denoted by #). Foamy macrophages (D) were significantly increased in irradiated rats given the vehicle, TC or PEG-hG-CSF (p < 0.05, denoted by * compared to controls and # compared to TC + lisinopril). Numbers in the bars represent N in each group and bars are means with standard deviations.

FIGURE 7

Mitigation of radiation nephropathy after 13 Gy leg-out PBI by lisinopril. The graph shows medians and 20–80% ranges for blood urea nitrogen (BUN in mg dL⁻¹) in rats at 90- and 120-days post irradiation. Asterisks (*) represent p < 0.05 as compared to irradiation only, irradiation with triple combination (TC) and irradiation with PEG-hG-CSF groups at the corresponding times show increased BUN levels. The normal BUN for non-irradiated rats ranges between 18 and 21 mg dl⁻¹ and is represented by the shaded, horizontal blue bar. Numbers in the bars represent N in each group. The decrease in N’s between 90- and 120-days post-irradiation were not due to radiation nephropathy; therefore, they were not given a value of 120 mg dl⁻¹.

FIGURE 8

Kidney injury mitigated by lisinopril after 13 Gy leg-out PBI. Representative histological sections of kidneys for each treatment group (A) were harvested at the termination of the study (120-days) after 13 Gy leg-out PBI, fixed and stained with H&E. Irradiated rats given the vehicle or PEG-hG-CSF showed increased protein casts (green arrow), glomerular sclerosis (black arrow) and glomerular mesangiolysis (red arrow) compared to non-irradiated rats. The irradiated and triple combination (TC) group showed increased protein casts and glomerular sclerosis while the addition of lisinopril mitigated these histological changes. Graphical representations of the H&E-stained kidney sections are shown in (B) as a composite histological score. Renal injury was increased with irradiation, the asterisks (*) represents p < 0.05 as compared to non-irradiated controls while the pound symbol (#) represents p < 0.05 as compared to the irradiated + TC + lisinopril. Numbers in the bars represent N in each group and bars are means with standard deviation.

FIGURE 9

Kaplan-Meier plot representing morbidity from DEARE up to 160 days post 13 Gy partial body irradiation with one hind limb shielded (leg-out PBI). The triple combination (TC, consisting of PEG-hG-CSF, PEG mGM-CSF and PEG hIL-11), or vehicle were given subcutaneously 24 h post PBI and the ACE inhibitor, lisinopril, was started in the drinking water 7-days post-irradiation. All irradiated rats were given supportive care. All irradiated rats that received TC + lisinopril survived to 160 days as compared to 100% morbidity for irradiated rats that received TC alone (p < 0.0001). Survival of irradiated rats given vehicle + lisinopril was over 90%, while irradiated rats given only the vehicle were moribund before 160 days. Shaded (gray) areas represent the timing for the lung and renal injuries.

FIGURE 10

Mitigation of radiation nephropathy after 13 Gy leg-out PBI by lisinopril (∼24 mg m⁻² d⁻¹). The graph shows medians and 20–80% ranges for blood urea nitrogen (BUN in mg dL⁻¹) in rats at 90-, 120- and 150-days post-irradiation. Asterisks (*) represent p < 0.05 as compared to 13 Gy + TC (triple combination, black bar) at the corresponding times. The normal BUN for non-irradiated rats ranges between 18–21 mg dl⁻¹ and is represented by the shaded, horizontal blue bar. All rats with 13 Gy + TC and 13 Gy + vehicle were moribund after 120 days with a BUN ≥ 120 mg dl⁻¹ and were given a BUN ≥ 120 mg dl⁻¹ at 150 days. Numbers in the bars represent N in each group.