Angiotensin converting enzyme (ACE) inhibitors as radiation countermeasures for long-duration space flights.

Angiotensin converting enzyme (ACE) inhibitors are effective countermeasures to chronic radiation injuries in rodent models, and there is evidence for similar effects in humans. In rodent models ACE inhibitors are effective mitigators of radiation injury to kidney, lung, central nervous system (CNS) and skin, even when started weeks after irradiation. In humans, the best data for their efficacy as radiation countermeasures comes from retrospective studies of injuries in radiotherapy patients. We propose that ACE inhibitors, at doses approved for human use for other indications, could be used to reduce the risk of chronic radiation injuries from deep-space exploration. Because of the potential interaction of ACE inhibitors and microgravity (due to effects of ACE inhibitors on fluid balance) use might be restricted to post-exposure when/if radiation exposures reached a danger level. A major unresolved issue for this approach is the sparse evidence for the efficacy of ACE inhibitors after low-dose-rate exposure and/or for high-LET radiations (as would occur on long-duration space flights). A second issue is that the lack of a clear mechanism of action of the ACE inhibitors as mitigators makes obtaining an appropriate label under the Food and Drug Administration Animal Rule difficult.

Radiation Increases Bioavailability of Lisinopril, a Mitigator of Radiation-Induced Toxicities.

There are no FDA-approved drugs to mitigate the delayed effects of radiation exposure that may occur after a radiological attack or nuclear accident. To date, angiotensin-converting enzyme inhibitors are one of the most successful candidates for mitigation of hematopoietic, lung, kidney, and brain injuries in rodent models and may mitigate delayed radiation injuries after radiotherapy. Rat models of partial body irradiation sparing part of one hind leg (leg-out PBI) have been developed to simultaneously expose multiple organs to high doses of ionizing radiation and avoid lethal hematological toxicity to study the late effects of radiation. Exposures between 9 and 14 Gy damage the gut and bone marrow (acute radiation syndrome), followed by delayed injuries to the lung, heart, and kidney. The goal of the current study is to compare the pharmacokinetics (PK) of a lead angiotensin converting enzyme (ACE) inhibitor, lisinopril, in irradiated vs. nonirradiated rats, as a step toward licensure by the FDA. Methods: Female WAG/RijCmcr rats were irradiated with 12.5-13 Gy leg-out PBI. At day 35 after irradiation, during a latent period for injury, irradiated and nonirradiated siblings received a single gavage (0.3 mg, 0.6 mg) or intravenous injection (0.06 mg) of lisinopril. Plasma, urine, lung, liver and kidney levels of lisinopril were measured at different times. PK modeling (R package) was performed to track distribution of lisinopril in different compartments. Results: A two-compartment (central plasma and periphery) PK model best fit lisinopril measurements, with two additional components, the gavage and urine. The absorption and renal clearance rates were similar between nonirradiated and irradiated animals (respectively: ratios 0.883, p = 0.527; 0.943, p = 0.605). Inter-compartmental clearance (from plasma to periphery) for the irradiated rats was lower than for the nonirradiated rats (ratio 0.615, p = 0.003), while the bioavailability of the drug was 33% higher (ratio = 1.326, p < 0.001). Interpretation: Since receptors for lisinopril are present in endothelial cells lining blood vessels, and radiation induces vascular regression, it is possible that less lisinopril remains bound in irradiated rats, increasing circulating levels of the drug. However, this study cannot rule out changes in total amount of lisinopril absorbed or excreted long-term, after irradiation in rats.

WAG/RijCmcr rat models for injuries to multiple organs by single high dose ionizing radiation: similarities to nonhuman primates (NHP).

Purpose: Defined animal models are needed to pursue the FDA Animal Rule for approval of medical countermeasure for radiation injuries. This study compares WAG/RijCmcr rat and nonhuman primate (NHP) models for acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE).Materials and methods: Irradiation models include total body irradiation, partial body irradiation with bone marrow sparing and whole thorax lung irradiations. Organ-specific sequelae of radiation injuries were compared using dose-response relationships.Results and conclusions: Rats and NHP manifest similar organ dysfunctions after radiation, starting with acute gastrointestinal (GI-ARS) and hematopoietic (H-ARS) syndromes followed by lung, heart and kidney toxicities. Humans also manifest these sequelae. Latencies for injury were earlier in rats than in NHP. After whole thorax lung irradiations (WTLI) up to 13 Gy, there was recovery of lung function from pneumonitis in rats. This has not been evaluated in NHP. The latency, incidence, severity and progression of radiation pneumonitis was not influenced by early multi-organ injury from ARS in rats or NHP. Rats developed more severe radiation nephropathy than NHP, and also progressed more rapidly. Dosimetry, anesthesia, environment, supportive care, euthanasia criteria etc., may account for the alterations in radiation sensitivity observed between species.

Chemical radiosensitizers: the Journal history.

Purpose: To review the Journal's coverage of chemical radiosensitizers. Methods: I have reviewed all the possibly-relevant papers that appeared in the Journal prior to 1970 and since 2010, plus the most highly-cited papers from the intervening years. I excluded papers that dealt only with oxygen as a sensitizer, that referred to sensitization of phototoxicity or hyperthermia, or that described interactions with antineoplastic agents unless they clearly distinguish between additive toxicity and radiosensitization. My definition of 'chemical' was very broad, so the coverage includes everything from classical hypoxic cell sensitizers to gold nanoparticles. Results: A literature search identifies ∼600 Journal articles as involving 'radiation sensitizing agents'; these articles are not common in Journals' first years but take off after 1970 with a peak in the late 1980s. Half of the highly-cited radiosensitizer papers were published between 1969 and 1974; the two most-cited radiosensitizer papers were 1969 and 1979 papers on hypoxic cell sensitizers. The third most-cited radiosensitizer paper would not come for two more decades, and it would use a physical rather than a chemical approach to radiosensitization. Conclusion: The development of an agent that would differentially sensitize tumors to irradiation remains a 'holy grail' of clinically-oriented radiobiology. Approaches to this goal have been a major feature of the Journal since its first decade, but we have yet to find such an agent. Perhaps we should be discouraged, but personally, I remain optimistic that we (or our students) will succeed.

Effects of Diet on Late Radiation Injuries in Rats.

It has been speculated that the addition of antioxidants to diet could act as either radioprotectors or as mitigators of radiation injury. In preparation for studies of the mitigation efficacy of antioxidants, rats were placed on a modified version of AIN-76A, the diet typically used in such studies. This AIN-76A diet is refined and has no synthetic antioxidants or isoflavones. Compared to the natural-ingredient Teklad 8904 diet used in previous studies, use of the AIN-76A diet from 1-18 wk after irradiation significantly reduced injury in a radiation nephropathy model. A confirmation study included an additional arm in which the AIN-76A diet was started 2 wk prior to irradiation; again, the switch to AIN-76A postirradiation mitigated radiation nephropathy (p < 0.001), but switching to the AIN-76A diet preirradiation had no effect (p > 0.2). The two diets do not differ in salt content, but the AIN-76A diet is somewhat lower in protein (18% vs. 24%). The protein source (primarily soy in Teklad 8904 vs. casein in AIN-76A) might explain the effects. However, replacing the casein in AIN-76A with soy did not change the mitigation efficacy of the diet (p > 0.2 for comparison of the different AIN-76A diets). A similar study in a rat radiation pneumonitis model also suggested mitigation by postirradiation use of AIN-76A, although the effect was not statistically significant (p = 0.07). In conclusion, base diet alone can have biologically significant effects on organ radiosensitivity, but the mechanistic basis for the effect and its dependence of timing relative to irradiation are unclear.

Radiation fractionation: the search for isoeffect relationships and mechanisms.

PURPOSE: Review the historical basis for the use of fractionated radiation in radiation oncology. CONCLUSION: The history of dose fractionation in radiation oncology is long and tortuous, and the radiobiologist's understanding of why fractionation worked came decades after radiation oncologists had adopted multi-week daily-dose fractionation as 'standard'. Central to the history is the search for 'isoeffective' formulas that would allow different radiation schedules to be compared. Initially, this meant dealing with different lengths of treatment, leading to the 1944 Strandqvist formulation that dominated thinking for decades. Concerns about the number of fractions, not just the total time, led to the 1967 Ellis NSD formulation that held sway through the 1980s. The development of experimental radiotherapy in 1970s (e.g. Fowler's work at the Gray Laboratory, and Fischer's work at Yale) led to biologically-based approaches that culminated with the Biologically Effective Dose (BED) concept. BED is the current dogma for treatment optimization, but it must be used with caution, as there are multiple formulations, and some parameters have debatable values. There is also a controversy about whether BED is biologically-based or a 'curve-fitting' exercise. These latter issues are beyond the scope of this article, but the history of fractionation models suggests that our current concepts are probably wrong, although when used with caution they are clearly useful.

Combined Hydration and Antibiotics with Lisinopril to Mitigate Acute and Delayed High-dose Radiation Injuries to Multiple Organs.

The NIAID Radiation and Nuclear Countermeasures Program is developing medical agents to mitigate the acute and delayed effects of radiation that may occur from a radionuclear attack or accident. To date, most such medical countermeasures have been developed for single organ injuries. Angiotensin converting enzyme (ACE) inhibitors have been used to mitigate radiation-induced lung, skin, brain, and renal injuries in rats. ACE inhibitors have also been reported to decrease normal tissue complication in radiation oncology patients. In the current study, the authors have developed a rat partial-body irradiation (leg-out PBI) model with minimal bone marrow sparing (one leg shielded) that results in acute and late injuries to multiple organs. In this model, the ACE inhibitor lisinopril (at ~24 mg m d started orally in the drinking water at 7 d after irradiation and continued to ≥150 d) mitigated late effects in the lungs and kidneys after 12.5-Gy leg-out PBI. Also in this model, a short course of saline hydration and antibiotics mitigated acute radiation syndrome following doses as high as 13 Gy. Combining this supportive care with the lisinopril regimen mitigated overall morbidity for up to 150 d after 13-Gy leg-out PBI. Furthermore, lisinopril was an effective mitigator in the presence of the growth factor G-CSF (100 µg kg d from days 1-14), which is FDA-approved for use in a radionuclear event. In summary, by combining lisinopril (FDA-approved for other indications) with hydration and antibiotics, acute and delayed radiation injuries in multiple organs were mitigated.

Clinically Relevant Doses of Enalapril Mitigate Multiple Organ Radiation Injury.

Angiotensin-converting enzyme inhibitors (ACEi) are effective mitigators of radiation nephropathy. To date, their experimental use has been in fixed-dose regimens. In clinical use, doses of ACEi and other medication may be escalated to achieve greater benefit. We therefore used a rodent model to test the ACEi enalapril as a mitigator of radiation injury in an escalating-dose regimen. Single-fraction partial-body irradiation (PBI) with one hind limb out of the radiation field was used to model accidental or belligerent radiation exposures. PBI doses of 12.5, 12.75 and 13 Gy were used to establish multi-organ injury. One third of the rats underwent PBI alone, and two thirds of the rats had enalapril started five days after PBI at a dose of 30 mg/l in the drinking water. When there was established azotemic renal injury enalapril was escalated to a 60 mg/l dose in half of the animals and then later to a 120 mg/l dose. Irradiated rats on enalapril had significant mitigation of combined pulmonary and renal morbidity and had significantly less azotemia. Dose escalation of enalapril did not significantly improve outcomes compared to fixed-dose enalapril. The current data support use of the ACEi enalapril at a fixed and clinically usable dose to mitigate radiation injury after partial-body radiation exposure.

Epoxyeicosatrienoic acid analogue mitigates kidney injury in a rat model of radiation nephropathy.

Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by CYP epoxygenases, and EETs are kidney protective in multiple pathologies. We determined the ability of an EET analogue, EET-A, to mitigate experimental radiation nephropathy. The kidney expression of the EET producing enzyme CYP2C11 was lower in rats that received total body irradiation (TBI rat) compared with non-irradiated control. At 12 weeks after TBI, the rats had higher systolic blood pressure and impaired renal afferent arteriolar function compared with control, and EET-A or captopril mitigated these abnormalities. The TBI rats had 3-fold higher blood urea nitrogen (BUN) compared with control, and EET-A or captopril decreased BUN by 40-60%. The urine albumin/creatinine ratio was increased 94-fold in TBI rats, and EET-A or captopril attenuated that increase by 60-90%. In TBI rats, nephrinuria was elevated 30-fold and EET-A or captopril decreased it by 50-90%. Renal interstitial fibrosis, tubular and glomerular injury were present in the TBI rats, and each was decreased by EET-A or captopril. We further demonstrated elevated renal parenchymal apoptosis in TBI rats, which was mitigated by EET-A or captopril. Additional studies revealed that captopril or EET-A mitigated renal apoptosis by acting on the p53/Fas/FasL (Fas ligand) apoptotic pathway. The present study demonstrates a novel EET analogue-based strategy for mitigation of experimental radiation nephropathy by improving renal afferent arteriolar function and by decreasing renal apoptosis.

Simvastatin mitigates increases in risk factors for and the occurrence of cardiac disease following 10 Gy total body irradiation.

The ability of simvastatin to mitigate the increases in risk factors for and the occurrence of cardiac disease after 10 Gy total body irradiation (TBI) was determined. This radiation dose is relevant to conditioning for stem cell transplantation and threats from radiological terrorism. Male rats received single dose TBI of 10 Gy. Age-matched, sham-irradiated rats served as controls. Lipid profile, heart and liver morphology and cardiac mechanical function were determined for up to 120 days after irradiation. TBI resulted in a sustained increase in total- and LDL-cholesterol (low-density lipoprotein-cholesterol), and triglycerides. Simvastatin (10 mg/kg body weight/day) administered continuously from 9 days after irradiation mitigated TBI-induced increases in total- and LDL-cholesterol and triglycerides, as well as liver injury. TBI resulted in cellular peri-arterial fibrosis, whereas control hearts had less collagen and fibrosis. Simvastatin mitigated these morphological injuries. TBI resulted in cardiac mechanical dysfunction. Simvastatin mitigated cardiac mechanical dysfunction 20-120 days following TBI. To determine whether simvastatin affects the ability of the heart to withstand stress after TBI, injury from myocardial ischemia/reperfusion was determined in vitro. TBI increased the severity of an induced myocardial infarction at 20 and 80 days after irradiation. Simvastatin mitigated the severity of this myocardial infarction at 20 and 80 days following TBI. It is concluded simvastatin mitigated the increases in risk factors for cardiac disease and the extent of cardiac disease following TBI. This statin may be developed as a medical countermeasure for the mitigation of radiation-induced cardiac disease.

Whole-thorax irradiation induces hypoxic respiratory failure, pleural effusions and cardiac remodeling.

To study the mechanisms of death following a single lethal dose of thoracic radiation, WAG/RijCmcr (Wistar) rats were treated with 15 Gy to the whole thorax and followed until they were morbid or sacrificed for invasive assays at 6 weeks. Lung function was assessed by breathing rate and arterial oxygen saturation. Lung structure was evaluated histologically. Cardiac structure and function were examined by echocardiography. The frequency and characteristics of pleural effusions were determined. Morbidity from 15 Gy radiation occurred in all rats 5 to 8 weeks after exposure, coincident with histological pneumonitis. Increases in breathing frequencies peaked at 6 weeks, when profound arterial hypoxia was also recorded. Echocardiography analysis at 6 weeks showed pulmonary hypertension and severe right ventricular enlargement with impaired left ventricular function and cardiac output. Histologic sections of the heart revealed only rare foci of lymphocytic infiltration. Total lung weight more than doubled. Pleural effusions were present in the majority of the irradiated rats and contained elevated protein, but low lactate dehydrogenase, when compared with serum from the same animal. Pleural effusions had a higher percentage of macrophages and large monocytes than neutrophils and contained mast cells that are rarely present in other pathological states. Lethal irradiation to rat lungs leads to hypoxia with infiltration of immune cells, edema and pleural effusion. These changes may contribute to pulmonary vascular and parenchymal injury that result in secondary changes in heart structure and function. We report that conditions resembling congestive heart failure contribute to death during radiation pneumonitis, which indicates new targets for therapy.

Model development and use of ACE inhibitors for preclinical mitigation of radiation-induced injury to multiple organs.

The NIH/NIAID initiated a countermeasure program to develop mitigators for radiation-induced injuries from a radiological attack or nuclear accident. We have previously characterized and demonstrated mitigation of single organ injuries, such as radiation pneumonitis, pulmonary fibrosis or nephropathy by angiotensin converting enzyme (ACE) inhibitors. Our current work extends this research to examine the potential for mitigating multiple organ dysfunctions occurring in the same irradiated rats. Using total body irradiation (TBI) followed by bone marrow transplant, we tested four doses of X radiation (11, 11.25, 11.5 and 12 Gy) to develop lethal late effects. We identified three of these doses (11, 11.25 and 11.5 Gy TBI) that were lethal to all irradiated rats by 160 days to test mitigation by ACE inhibitors of injury to the lungs and kidneys. In this study we tested three ACE inhibitors at doses: captopril (88 and 176 mg/m(2)/day), enalapril (18, 24 and 36 mg/m(2)/day) and fosinopril (60 mg/m(2)/day) for mitigation. Our primary end point was survival or criteria for euthanization of morbid animals. Secondary end points included breathing intervals, other assays for lung structure and function and blood urea nitrogen (BUN) to assess renal damage. We found that captopril at 176 mg/m(2)/day increased survival after 11 or 11.5 Gy TBI. Enalapril at 18-36 mg/m(2)/day improved survival at all three doses (TBI). Fosinopril at 60 mg/m(2)/day enhanced survival at a dose of 11 Gy, although no improvement was observed for pneumonitis. These results demonstrate the use of a single countermeasure to mitigate the lethal late effects in the same animal after TBI.

Mitigation of experimental radiation nephropathy by renin-equivalent doses of angiotensin converting enzyme inhibitors.

PURPOSE: We tested five different angiotensin converting enzyme inhibitors (ACEI) as mitigators of experimental radiation nephropathy at drug doses calibrated to the plasma renin activity (PRA). This was done to determine whether all ACEI had the same efficacy as mitigators of radiation nephropathy when used at drug doses that gave equivalent suppression of the renin angiotensin system. METHOD: 10 Gy total body irradiation with bone marrow transplantation was used to cause radiation nephropathy in barrier-maintained rats. Equivalent ACEI doses were determined based on their effect to inhibit angiotensin converting enzyme (ACE) and raise the PRA in unirradiated animals. RESULTS: PRA-equivalent doses were found for captopril, lisinopril, enalapril, ramipril and fosinopril. These doses overlap the human doses of these drugs on a body surface area basis. All ACE inhibitors, except fosinopril, mitigated radiation nephropathy; captopril was a somewhat better mitigator than lisinopril, enalapril or ramipril. CONCLUSIONS: Most, but not all, ACEI mitigate radiation nephropathy at doses that overlap their clinically-used doses (on a body surface area basis). Fosinopril is known to be an ineffective mitigator of radiation pneumonitis, and it also does not mitigate radiation nephropathy. These pre-clinical data are critical in planning human studies of the mitigation of normal tissue radiation injury.

2013 Dade W. Moeller lecture: medical countermeasures against radiological terrorism.

Soon after the 9-11 attacks, politicians and scientists began to question our ability to cope with a large-scale radiological terrorism incident. The outline of what was needed was fairly obvious: the ability to prevent such an attack, methods to cope with the medical consequences, the ability to clean up afterward, and the tools to figure out who perpetrated the attack and bring them to justice. The medical response needed three components: the technology to determine rapidly the radiation doses received by a large number of people, methods for alleviating acute hematological radiation injuries, and therapies for mitigation and treatment of chronic radiation injuries. Research done to date has shown that a realistic medical response plan is scientifically possible, but the regulatory and financial barriers to achieving this may currently be insurmountable.

Enhanced survival from radiation pneumonitis by combined irradiation to the skin.

PURPOSE: To develop mitigators for combined irradiation to the lung and skin. METHODS: Rats were treated with X-rays as follows: (1) 12.5 or 13 Gy whole thorax irradiation (WTI); (2) 30 Gy soft X-rays to 10% area of the skin only; (3) 12.5 or 13 Gy WTI + 30 Gy skin irradiation after 3 hours; (4) 12.5 Gy WTI + skin irradiation and treated with captopril (160 mg/m(2)/day) started after 7 days. Our end points were survival (primary) based on IACUC euthanization criteria and secondary measurements of breathing intervals and skin injury. Lung collagen at 210 days was measured in rats surviving 13 Gy WTI. RESULTS: After 12.5 Gy WTI with or without skin irradiation, one rat (12.5 Gy WTI) was euthanized. Survival was less than 10% in rats receiving 13 Gy WTI, but was enhanced when combined with skin irradiation (p < 0.0001). Collagen content was increased at 210 days after 13 Gy WTI vs. 13 Gy WTI + 30 Gy skin irradiation (p < 0.05). Captopril improved radiation-dermatitis after 12.5 Gy WTI + 30 Gy skin irradiation (p = 0.008). CONCLUSIONS: Radiation to the skin given 3 h after WTI mitigated morbidity during pneumonitis in rats. Captopril enhanced the rate of healing of radiation-dermatitis after combined irradiations to the thorax and skin.

Safety and blood sample volume and quality of a refined retro-orbital bleeding technique in rats using a lateral approach.

The collection of blood samples from laboratory rats requires the use of bleeding techniques that provide quality samples of sufficient volume for analysis without injury to the animal. Retro-orbital bleeding (ROB) is a phlebotomy technique that can yield high-quality samples of adequate volume, but it has been criticized for its potential to cause injury. To evaluate the injury-causing potential of their refined ROB method using a lateral approach, the authors retrospectively reviewed ROB procedures carried out in their colony during an 18-month period and found that 0.6% of these procedures were associated with ocular injury. The authors also compared the quality of blood samples collected by ROB and by saphenous phlebotomy and found that ROB yielded samples of better quality. The authors conclude that, when done using a lateral approach and by an experienced technician, ROB is humane and safe and provides blood samples of adequate volume and quality for analysis.