Abstract
This workshop examined the effects of ionizing radiation on certain understudied populations, including pregnant/lactating, in utero, pediatric, and geriatric populations. Research using animal models has revealed significant age and condition-related differences in radiation-induced injuries, highlighting the need for tailored triage and treatment strategies. Historical data from Hiroshima, Nagasaki, and Chernobyl further support these findings, demonstrating that radiation effects lead to wide-ranging issues with unique profiles during pregnancy, childhood and elderly age. While some research has been conducted on these groups, ethical and logistical challenges make it difficult to study these populations extensively. Therefore, developing alternative approaches that offer promising avenues for further research is critical. Radiation-induced biomarkers and biodosimetry also show age-related differences, including distinctive metabolic disruptions, necessitating further validation of biodosimetry tools. These findings emphasize the importance of considering age, sex, and demographic factors in preclinical and clinical radiation research to develop treatments that improve outcomes of understudied populations after a radiological or nuclear public health emergency.
MEETING PROGRAM OVERVIEW
The global response to the COronaVIrus Disease of 2019 (COVID-19) pandemic highlighted many deficiencies regarding emergency preparedness for understudied populations. While crucial lessons were learned in this case, radiation incidents will likely present an even more critical window for intervention, with the additional complexity of the immediacy of the event, physical destruction, and persistent radiation danger. These incidents will require a prompt response, intensifying the need for adequate preparedness for all populations.
On April 10–11, 2024, the Radiation and Nuclear Countermeasures Program (RNCP) within the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH) hosted a workshop “Understudied Populations in Radiation Exposure Research: Needs, Challenges, and Mitigation Strategies.” The workshop was held in partnership with three other NIH Institutes: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the National Institute on Aging (NIA), and the National Cancer Institute (NCI). The goal of this workshop was to identify specific challenges and needs of understudied populations (pregnant/lactating women, in utero, pediatric, and geriatric) after exposure to radiation during a public health emergency. The level of radiation injury is dependent on age and sex, as well as the underlying level of stress and damage, which normally increases with age (Fig. 1). Additionally, as people age, the effectiveness of resolution and repair processes diminishes, leading to increased senescence, inflammation, tissue damage, immune deficiency, and stem cell exhaustion. The workshop also covered the state of the science for radiation medical countermeasures (MCMs) and biomarkers/biodosimetry in these understudied populations to better understand these processes. Biomarkers of radiation injury are crucial in product development and serve various functions, including: 1. Diagnostic biomarkers, which can confirm the presence of radiation injury; 2. Pharmacodynamic/response biomarkers that can determine an MCM’s biological effect; 3. Predictive biomarkers that can be used to predict radiation exposure (e.g., triage biodosimetry assay); 4. Prognostic biomarkers, which may predict acute and delayed biological effects of radiation (e.g., lung or kidney injury) (1). The workshop also discussed the biological differences and policies that influence the inclusion of these populations in the development of therapeutics and diagnostics.
FIG. 1.
Radiation injury across the lifespan is age-dependent; hence, understudied populations (pregnant/lactating, in utero, pediatric, and geriatric) must be considered. Dose-response can change drastically based on age. Stress and damage increase while resolution and repair become less effective with age. As a result, senescence increases, leading to inflammation, tissue damage, immune deficiency, stem cell exhaustion, etc. Created using BioRender.com.
Presentations and discussions from this two-day workshop are summarized in this meeting report. In addition, an outline of the topics addressed during the meeting is shown in Table 1, and the 22 subject matter experts from 20 institutions that presented their research findings are listed in Table 2. The audience included academic investigators, representatives from private industry, global research partners, and government representatives from the NIH, Biomedical Advanced Research and Development Authority (BARDA), the U.S. Food and Drug Administration (FDA), and other federal agencies. Where unpublished data are provided, the presenter’s first initial and last name are shown. The content of this meeting report reflects comments, discussions, and information shared at this workshop and was reviewed for accuracy by all presenters prior to publication.
TABLE 1.
Meeting Sessions and Topic Areas
Keynote Talk:
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Session I: State of the Science for Pregnant/Lactating and In Utero Populations
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Session II: State of the Science for Pediatrics
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Session III: State of the Science for Geriatrics
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Session IV: Modeling and Extrapolations for Drug and Device Development
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Session V: Drug & Biomarker Development Considerations and Concepts of Operation in Understudied Populations Panel Discussion
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TABLE 2.
Workshop Speakers and Areas of Expertise
| Name | Affiliation | Area of expertise |
|---|---|---|
| Antiño Allen, PhD | University of Arkansas for Medical Sciences | Genetic models, neurogenesis, cognitive function, inflammation, oxidative stress |
| Marjan Boerma, PhD | University of Arkansas for Medical Sciences | Radiation tissue injury, cardiovascular system, DEARE, biomarker multi-omics |
| Gilbert Burckart, PharmD | US Food and Drug Administration | Pediatrics, clinical pharmacology, regulatory affairs |
| Deborah Citrin, MD | National Cancer Institute | Radiation oncology, tumors, radiation tissue injury, cell senescence, genitourinary cancers |
| Rachael Clark, MD, PhD | Brigham and Women’s Hospital, Harvard Medical School | Dermatology, human T cell response in healthy skin, skin cancer, inflammatory skin diseases, cutaneous lymphomas |
| Rahul D’Mello, MD, PhD | Oregon Health & Science University | Maternal-fetal medicine, environmental exposures, early fetal and placenta development, obstetrics & gynecology |
| Jonathan Gill, PhD | Federal Emergency Management Agency | Chemical, biological, radiological, and nuclear emerging threat policies, radiological consequence management, nuclear forensics |
| Joga Gobburu, PhD | University of Maryland, Baltimore | Regulatory affairs and drug approval processes, artificial intelligence/machine learning |
| Dionna Green, MD | US Food and Drug Administration | Pediatric therapeutics, ethics, neonatology, rare diseases, pharmacovigilance, international collaboration, regulatory science |
| Joel Greenberger, MD | University of Pittsburgh School of Medicine | Radiation oncology, radiation MCMs, lung cancer, radiation biology, stem cell transplantation, and stem cell plasticity |
| Joshua Hatterschide, PhD | Duke University School of Medicine | 3D cell culture models, placenta-derived trophoblast and decidua organoids, maternal-fetal interface, viral infections |
| Heather Himburg, PhD | Medical College of Wisconsin | Hematology, oncology, normal tissue radiation-induced injury, immune system, tumor radioresistance, DEARE |
| Chad Hrdina, MS, GC-WMD, EMT | Office of the Assistant Secretary for Preparedness and Response | Radiological and nuclear threats MCM requirements, weapons of mass destruction |
| Carol Iddins, MD | Radiation Emergency Assistance Center/Training Site (REAC/TS) | Radiation emergency management, radiological security and safety, disaster medicine |
| Ziad Kazzi, MD, FACMT, FAACT, FAAEM, FACEP | Emory University | Emergency medicine, toxicology, triage and management of poisonings, radiation emergencies |
| Richard Kowalski, PhD | ASELL, LLC | Qualitative/quantitative assessments of radiation exposure, biodosimetry, viral/bacterial diseases, mesenchymal stem cells, bone repair, cancer, gene therapy, translational science |
| Naresh Menon, PhD | ChromoLogic, LLC | Sensor fabrication, instrumentation, novel data analytic methods, wound care, infectious diseases, diagnostics/screening, drug delivery, telehealth |
| Sergei Pigarev, MS | RDInnovation | MCM development, transgenerational oncogenesis, cutaneous and gastrointestinal radiation injuries |
| Artur Plett, PhD | Indiana University School of Medicine | Hematology, oncology, microbiology, immunology, radioprotection and mitigation, bone marrow stem cell, age specific animal models of ARS and DEARE |
| Ellen Quillen, PhD | Wake Forest School of Medicine | Statistical genomics, gene expression, aging biology of bones and muscles, NHP expertise, health span |
| Lauren Richardson, PhD | University of Texas Medical Branch | Obstetrics & gynecology, translational research, ovarian, breast, and lung cancer epithelial-mesenchymal transition, gestational tissues, organ-on-chip technology |
| Perdita Taylor-Zapata, MD | Eunice Kennedy Shriver National Institute of Child Health and Human Development | Obstetric and pediatric pharmacology, drug development, off-patent clinical trials, pharmacoepidemiology, workforce diversity, pediatric adverse effects of medications |
BACKGROUND
Radiation Effects in Understudied Population Cohorts from Chernobyl and Hiroshima/Nagasaki
In August 1945, the U.S. dropped atomic bombs on Hiroshima and Nagasaki, Japan. Although exact numbers are not known, >340,000 and >250,000 total deaths were reported within two to four months after the Hiroshima and Nagasaki bombings, respectively, with 50% of all deaths occurring within the first six days (C. Iddins).2 In the Chernobyl Nuclear Power Plant accident on April 26, 1986, two deaths occurred immediately after the disaster, but ~28 deaths occurred from acute radiation syndrome after three to four months.3 Epidemiological data gathered from studies on these populations can be dissected to provide insight into the impact of radiation exposure on different understudied groups in radiation exposure situations.
Pregnancy.
One of the largest and most comprehensive studies of females after the Japanese atomic bombings was carried out by the Tokyo Red Cross Maternity Hospital in 1947. Researchers studied more than 76,000 pregnancies, looking at major birth defects (2). They noted that from 1950 to 1955, the birth rate decreased by half, but it is unknown if this was attributable to either changes in conception or termination rates. In epidemiology studies carried out in Chernobyl populations, increased levels of spontaneous abortions were noted (3); however, they found no differences in trends in abortion rates between individuals living in contaminated areas and those in non-contaminated areas, suggesting little direct influence of the incident (4, 5). Interestingly, excess female fetal loss following the Chernobyl incident is undisputed (6). Finally, rates of premature delivery (before 37 weeks gestation) did not increase in Chernobyl populations (3).
Lactating females.
Data was collected on radionuclide levels from placentas and breast milk (7), with an average radiation dose from placental cesium during the year after the Chernobyl incident calculated to be 40 to 60 μSv; however, 131I was never detectable in the samples. Researchers also found that the activity concentrations of 137Cs in cow’s milk in many countries increased by two to three orders of magnitude after the incident. Therefore, the risk of exposure to breast milk through nursing was far outweighed by exposure through ingestion of cow’s milk.
In utero.
The American College of Obstetrics and Gynecology “Guidelines for Diagnostic Imaging During Pregnancy and Lactation” suggest that there are no increased risks of anomalies, growth restrictions, or spontaneous abortions for radiation exposures of less than 50 mGy.4 The International Commission on Radiological Protection sets a higher threshold, indicating that more than 100 mGy is needed to cause malformations in newborns (8). An “all-or-none” phenomenon suggests that fetuses irradiated during early pre-implantation may be most susceptible to harmful effects; however, those that survive do not appear to have an increased risk of malformations or growth retardation (9). Once implantation occurs, effects are dependent on radiation dose and time of exposure, where the gestational stage of 8 to 15 weeks shows the highest increase in mental retardation.5 Data on prenatal radiation exposure suggest that central nervous system effects may occur later in life after in utero exposure (10). Furthermore, a 2018 Japanese atomic bomb lifespan study that included ~3,600 individuals exposed in utero and 77,000 children of survivors (11) determined that exposed survivors had an increased risk for malignancy, including those exposed in utero. However, non-exposed offspring from previously exposed parents showed no apparent increased risk of cancer and other diseases. Thus, many unanswered questions remain, and epidemiology studies continue as the children of survivors continue to age.
Pediatrics.
Children have increased respiratory rates and ventilation volume (12), potentially making them more susceptible to inhaled toxins. Furthermore, their active bone marrow is in a different location than adults, so they take up bone-seeking radionuclides very effectively and efficiently (13). Children are also more susceptible to fallout because they are closer to the ground. All these factors contribute to greater radiation exposure to children in a fallout environment like what occurred after Chernobyl. Among other factors that increase the risk of radioactive exposure are drinking breast and cow’s milk and exposure to dirt while playing. These behaviors make children more susceptible to radiation-induced injuries compared to adults. Following the radiation release from Chernobyl, 6 million people were on the path of contamination within Belarus, Russia and Ukraine (14). Of the ~114,000 evacuees, about 31% (35,000) were children (C. Iddins). Thyroid and effective doses in infants were 8–9 times larger compared to adults, and doses from the ingestion of milk were 20-fold higher in children because of their increased consumption.6
Aging and cancer incidence across exposed populations.
The BEIR VII findings state that although there is an early increase in leukemias (within 10 years after atomic bomb exposure), an aggregate of other solid cancers showed a slow and steady increase in prevalence, beginning at around 10 years after exposure through 40+ years.7 Leukemia risk profiles decrease with age compared to a large spike seen with exposures at younger ages. With the Japanese atomic bomb survivor cohort, 75% of the new cases of breast cancer diagnosed since 1998 were exposed before age 20. Cancers in females exposed at 30 years old had a strong linear dose response (15). Moreover, the Radiation Effects Research Foundation lifespan study found that women have a 2–3 times higher lifetime risk of developing cancer. Other non-cancer diseases in the aged population associated with radiation exposure included cataracts, thyroid nodules and cysts, hyper-parathyroidism, decrease in T-cell mediated and humoral immune response, chromosomal aberrations in lymphocytes, cardiovascular disease, and life-shortening.
Overall, the impacts of radiation exposure differed in the sub-populations after the Hiroshima and Nagasaki bombings and the Chernobyl incident. This could be due to the difference in ionizing radiation delivered in each case. An atomic bomb detonation yields a high external, penetrating dose delivered all at once very acutely, as compared to the chronic, longer-term doses at Chernobyl. In addition, an atomic detonation has gamma and neutron elements, whereas the power plant release was external gamma radiation and internal radionuclide contamination. Nonetheless, similar radiation exposure effects were noted in the populations: Increased incidences of breast and thyroid cancers, fetal loss, and leukemia. Moreover, similar lessons learned included the importance of sheltering in place, when possible, versus evacuation, especially for the elderly. Additionally, acknowledgment of the psychological toll during and following radiation emergencies was critical as the effect on mental health can have a detrimental impact on quality of life. Research gaps that require further investigations include in utero and pediatric epidemiology, consideration of population logistics, communications, and psychological monitoring and assistance.
SESSION I: STATE OF THE SCIENCE FOR PREGNANT/LACTATING AND IN UTERO POPULATIONS
During this session, subject matter experts reviewed available data on humans, reported on novel findings from animal models, discussed potential MCMs for use in these populations, and explored alternative models (e.g., human organoids) to overcome challenges related to radiation research in pregnant, lactating, and in utero individuals. Although the in utero fetus is part of the pediatric population (16), distinct differences exist in pathophysiology and radiation sensitivity compared to postnatal pediatric populations. In utero development in mammals is characterized by active and rapid cell proliferation, migration, and differentiation, making the organism extraordinarily sensitive to the effects of radiation, which can result in teratogenic, carcinogenic, or mutagenic effects (17). The main factors affecting the outcome of in utero exposure include the dose, dose rate, and the gestational stage at which exposure occurs (18). Several in vitro and animal models were discussed to address these understudied populations.
Pregnant/Lactating Women and Radiation Harm
The number of researchers studying in utero radiation exposures is minimal (J. Valentin). Given the limited studies in this area, an alternative is to consider using radiation epidemiology to fill these gaps.8 This has led to consensus on radiation protection, health risks, and industry standards.9 Different diagnostic approaches for triage or treatment, as well as therapeutic methods for rapid response, can also be developed and deployed through this approach. While previous strategies have heavily focused on hematopoietic acute radiation syndrome (H-ARS), it is essential to recognize that radiation exposures lead to multi-organ injuries and to develop MCMs to address these complex issues.
The International Commission on Radiation Protection (ICRP) has published several reports that provide effective, age-specific dose coefficients for inhalation and ingestion of radionuclides, including those for the embryo and fetus (ICRP 71, 72, and 88).10,11,12 However, for external exposures, the ICRP does not account for age-related variations in risk per unit dose. Dose limits and constraints should be optimized based on vulnerable groups to prevent radiation injury across all populations. Additionally, given the multiple reports (ICRP 90, 103) regarding the high radiosensitivity of the mammalian embryo and fetus (19), special regulations for the protection of pregnant women are necessary. Currently, it is impossible to determine whether the ICRP overestimates or underestimates radiation-induced health risks to the embryo and fetus, as supporting data are scarce. One potential source of additional data could be modeling based on medical exposures during radiotherapy and interventional fluoroscopy, with an emphasis on systematic follow-up of side effects (20, 21). Furthermore, animal models of radiation injury can be utilized to investigate different subsyndromes (22, 23) and combined injury (24); however, the relevance to human exposures is not faultless due to significant anatomical and functional differences between small animal models and human fetal-maternal interface tissue.
Placenta-derived Organoids as Model of the Maternal-Fetal Interface
The placenta is a fetal-derived organ that serves as a barrier and conduit between maternal and fetal blood for nutrient and gas exchange (J. Hatterschide). Given this role, placental dysfunction or damage can lead to severe complications for the developing fetus. The placenta consists of both fetal and maternal surfaces. Chorionic villi serve as the primary site of nutrient exchange. These highly vascularized, branched, tree-like structures are anchored to the maternal basal decidua and immersed in maternal blood. The primary cell types of the chorionic villi are syncytiotrophoblasts and cytotrophoblasts (25). Placenta-derived organoid models that recapitulate the maternal-fetal interface have been developed using matched maternal and fetal organoids from full-term human placentas. Although the organoids are matched, each is grown separately and remains unconnected. More than 60 human organoid lines are available from unique placental tissue (26). Trophoblast organoids (TOs) isolated from full-term placental tissue recapitulate first-trimester tissue (26). These TOs have several advantages over existing trophoblast models: 1. They can be continuously propagated, expanded, and cryopreserved while maintaining their TO phenotype; 2. They differentiate into multiple lineages that can be stimulated to enrich selected trophoblast populations using specific cytokine cocktails (27); 3. They can be engineered to alter gene expression. Furthermore, these organoids can be employed to model insults to the placenta, including exposure to infectious agents and drugs (e.g., opioids) and can also be extended to include radiation.
Trophoblast organoids are extremely versatile and provide a comprehensive model to study the maternal-fetal interface. The biobanking of the organoids across clinical outcomes, race, sex (fetal surface), and genetic variations can be a strategy to interrogate the impact of various insults at the maternal-fetal interface across the human population. The major limitations of the model currently are the lack of communication between the niches and the lack of immune cells, which are critical in the context of damage at the maternal-fetal interface. The next steps are to develop organoid-based models of the fetal interface that support communication, such as co-culture, transwell, and perfusion-based models. These models represent an excellent means of testing the impact of radiatiion on these important tissues and also offer the possibility of assessing the effect of different MCMs on radiation-induced damage in a sensitive population.
Prenatal Radiation Exposure: The State of the Science in Nonhuman Primate (NHP) Models
Many research gaps still exist regarding the detection and consequences of radiation injury to the fetus (R. D’Mello). While basic parameters involving radiation and pregnancy are generally accepted, such as the <50 mGy threshold for “safe” radiation exposure during pregnancy, key questions remain unaddressed around fetal radiation detection and countermeasure safety. Leveraging NHP pregnancy models in combination with cutting-edge clinical and translational assessments could help address these questions. Animal models overcome the limitations of human studies and in vitro experimentation, especially for radiation research. While not perfect, NHPs are the closest animal model to humans, with an abundant overlap of similar characteristics. Among the NHP species, the rhesus macaque is the most studied NHP for reproductive health and pregnancy studies due to its multiple advantages, including similar placental biology, developmental ontogeny, and reproductive physiology. Furthermore, rhesus macaques have been frequently used in studies involving radiation exposure and MCM development (28-30).
The National Primate Research Centers have developed several validated and well-characterized prenatal NHP models and are poised to create a prenatal radiation injury model. Reproductive outcomes of environmental and infectious exposure have been tested in these prenatal NHP models. These models have also been used for translational studies using multi-modal approaches that are not feasible in humans. Clinical assessments such as imaging, behavioral, molecular, and pathology evaluations are also possible in the NHP model. Imaging techniques available to study a prenatal NHP model of radiation exposure include advanced ultrasound with contrast-enhanced evaluation of tissue perfusion, magnetic resonance imaging (MRI) with cutting-edge sequences to non-invasively quantify tissue oxygenation, and positron emission tomography (PET)/computed tomography (CT) scans. In addition, ultrasound capabilities include translating clinical obstetric sonographic measurements and nomograms to rhesus macaques (31). Molecular studies have regularly been utilized with prenatal NHP models and could be leveraged to identify novel biomarkers for detecting and monitoring radiation injury in the fetus. For example, rhesus macaque amniotic fluid proteome can be sampled across gestation by serial collection under ultrasound guidance without compromising the pregnancy (32). A strength of these NHP models is the capability to correlate the previously mentioned imaging and molecular assessments with tissue histological and pathological studies. However, the molecular effects of radiation in an NHP prenatal model need to be correlated with long-term outcomes and behavioral changes. Maternal-infant postnatal assessments can be performed to quantify cognitive development, emotional regulation, sensorimotor development, motor development, physical activity levels, and anthropomorphic measures.
Beyond helping to determine how to best detect and monitor radiation injury to the fetus, pregnant NHP models offer an opportunity to test which radiation MCMs could be safe and efficacious for pregnant/lactating women. Several products are FDA-approved to treat H-ARS, but only small observational studies suggest these medications may be safe in pregnancy. Thus, there is a need for rigorous studies to evaluate the prenatal safety of established and new MCMs, which could be effectively addressed through a pregnant NHP radiation injury model.
Radiation Countermeasures for the Developing Fetus
JP4-039, a mitochondrially-targeted gramicidin S (GS)-nitroxide compound, functions as a free radical scavenger and is hypothesized to act as a fetal radiation mitigator by protecting mitochondrial function in the fetal mouse (J. Greenberger). A model utilizing pregnant female C57BL/6NTac mice exposed to 3 Gy total-body irradiation (TBI) at mid-gestation (E13.5) was developed to test this possible countermeasure (33). JP4-039 was given intravenously or intramuscularly 24 h postirradiation. The effects of TBI on pregnant mice led to a variety of issues in the fetal brain. At E16.5, the fetuses were observed to be ischemic, and MRI imaging revealed hydrocephalus and dilated ventricles, which were confirmed with histological sections. The hydrocephalus was prominent and easily visible as skull dilation in the few irradiated fetuses that survived birth. The observed fetal brain injury in this model is not surprising as the critical organ development of the brain, heart, and central nervous system coincide with the irradiation time point.
The survival rate for non-irradiated fetuses was near 100%, while 3 Gy TBI led to a negligible survival rate (~9%). All three delivery emulsions of JP4-039 resulted in increased survival of irradiated fetuses at weaning (~55–76%). As revealed by 4D in utero MRI imaging, TBI caused cerebral hemorrhage and overaccumulation of cerebrospinal fluid (CSF), whereas the administration of JP4-039 reduced the degree of these abnormalities. Irradiated fetal brains show elevated apoptosis, while JP4-039 treatment decreased radiation-induced apoptosis. Additional experiments showed that the irradiated fetuses could not properly circulate their CSF due to radiation damage to the ciliated ependymal cells, which line the ventricles. JP4-089 targets mitochondria energy production, which helps overcome ciliated cell dysfunction, mitigating fetal death after TBI of the mother. More safety and efficacy studies must be performed to develop JP4-089 as a radiation MCM.
Effects of Paternal Exposure to Gamma Radiation on Lung Tumorigenesis in the Offspring of BALB/c Mice
Maternal influence on in utero effects of radiation is often considered, but the effects of paternal irradiation and its impact on offspring are not well studied (S. Pigarev). This could be problematic due to the increasing number of cancer survivors, especially those of reproductive age, who will possibly have delayed effects of cancer therapies, including radiotherapy. Individuals who survive primary cancer are at a greater risk of developing secondary cancers. Additionally, children born to cancer survivors have a higher risk of diseases and disorders. A transgenerational mouse model was used to confirm the genotoxic/carcinogenic effects of cancer treatment modalities, namely radiation. Studies demonstrated that the offspring of these irradiated mice have a higher sensitivity to carcinogens. The transgenerational model was created using irradiated BALB/c male mice (F0) mated with non-irradiated female BALB/c mice to produce offspring for further testing (F1). TBI (1 Gy) was administered to allow sufficient production of an F1 generation for further experimentation. Deoxyribonucleic acid (DNA) damage in the peripheral blood of the irradiated male mice (F0) was noted at this dose, with a significant increase 4 h postirradiation, persisting at a nonsignificant level for 30 days. Furthermore, 1 Gy TBI induced an increase in micro-nucleated reticulocytes at 48 h and led to telomere shortening in peripheral blood leukocytes 30 days postirradiation, further confirming DNA damage.
To assess susceptibility to secondary cancers, lymph nodes from 1 Gy TBI and control F0 mice were collected, turned into a cell suspension, and re-irradiated with a 2 Gy dose. The cells from 1 Gy TBI mice presented with much higher levels of DNA damage than non-irradiated F0 mice, indicating a higher sensitivity to radiation. Induction of lung tumors was also performed in the F0 TBI mice to confirm the sensitivity to secondary carcinogenesis. The number of lung tumors per animal that developed was significantly higher in the mice that had previously been exposed to radiation. The increased sensitivity to urethane-induced tumorigenesis continued in the F1 generations (male and female) of the irradiated mice compared to F1 mice from non-irradiated parents. Ongoing studies demonstrate that geno-protectants, when given to the F0 generation, reduce cellular DNA damage and sensitivity to carcinogens in the F1 generation, indicating a possible avenue of protection from these transgenerational effects.
SESSION II: STATE OF THE SCIENCE FOR PEDIATRICS
The speakers in this session provided valuable insights into the differences in radiosensitivity and response to MCMs between juvenile and geriatric populations. In addition, pediatric differences in skin, biomarkers, circulating miRNAs, and complete blood counts were also discussed. The findings highlight the importance of considering age, sex, and other demographic factors in radiation research and treatment development.
Juvenile and Geriatric Mice: Differences in Radiosensitivity and Response to MCM
Radiosensitivity and response to MCMs differ between pediatric (3–8 weeks), young adult (3 months), and geriatric (12 and 24 months) C57BL/6 mice (34, 35) (A. Plett). Radio-resistance also changes based on sex. Pediatric female mice are more radiosensitive but become more radioresistant at puberty, then become radiosensitive again during the geriatric stage compared to male mice (36). Investigations into the hematopoietic system showed that younger mice had slower neutrophil recovery when compared to geriatric mice. In geriatric mice, platelet increase contributes to myeloid skewing and neutrophil recovery, resulting in more robust infection and bleeding control and increased radioresistance with age. In addition, geriatric mice showed higher levels of progenitor cells, which are more radioresistant, potentially contributing to observed radioresistance in the geriatric population (34). While the life span of all survivors was negatively affected by radiation, pediatric and adult populations were more impacted, suggesting differential effects due to aging (37).
Given the age differences noted, MCM efficacy was assessed in these populations. Treatment with pegylated granulocyte colony-stimulating factor (G-CSF), an FDA-approved MCM, proved successful in mice of all ages, depending on the dose used (34). PLX-R18 cells, a placentally-derived cellular therapy, was effective at increasing survival in pediatric (5 weeks), young adult (3 months), and geriatric (12 months) mice but not in 24-month-old geriatric mice. Route of administration should be considered since not all are appropriate for all age groups. For example, pediatric mice are more sensitive to handling stressors and injections. Notably, age and sex can impact the effectiveness of products and amplify the need for tailored approaches in radiation treatment.
Studying Radiation Injury in Living Human Skin Using Skin-grafted Mice
Children are at greater risk after a radiologic incident because they are more likely to encounter exposure to contaminated water and soil arising from nuclear fallout (R. Clark) (38). Children also have a higher risk of developing radiation-induced cancers than adults due to increased numbers of actively dividing cells with open chromatin and a longer life expectancy, with more time to develop cancer after radiation exposures (39).13 Children have a larger surface-to-volume ratio than adults, so skin exposure to toxic and radioactive substances can lead to higher blood levels and greater systemic exposure (40). The skin of younger individuals is also more permeable, has a lower stratum corneum water content, and is more susceptible to water loss. Radiation exposure induces skin barrier defects and increased transepidermal water loss in adults (41). Because of their decreased barrier function, pediatric patients are at an increased risk for dehydration, electrolyte imbalances, infections, inflammation, and skin breakdown. Pediatric patients exposed to a radiologic event will require emollients, wound care, and close monitoring for dehydration, inflammation, and infection. The tendency of children to develop fibrosis after radiation exposure has not been thoroughly studied, but the likelihood of scarring following mechanical injuries to the skin varies with age. Infants younger than six months old have reduced scarring, but older children can develop more marked scarring than adults (42). Radiation induces cellular senescence in the skin, which can contribute to chronic injury by perpetuating inflammation and ROS production (43). Children have fewer pre-existing senescent cells in their skin compared to adults or aged individuals; however, it is not clear how this impacts their risk of developing chronic radiation injury. There is an unmet need for understanding how pediatric skin responds to acute and chronic radiation injury.
Human cutaneous responses to radiation are currently being studied by grafting neonatal foreskin or adult human skin to immunodeficient mice, followed by selective irradiation of the human graft (44). Skin grafts retain viable populations of human immune cells, melanocytes, keratinocytes, and fibroblasts. Human blood vessels persist within the graft and anastomose within the mouse circulation (44). Bulk RNA sequencing of human skin grafts 10 days after 5 Gy irradiation demonstrated upregulation of genes associated with danger sensing (cGAS/STING, inflammasome activation, and type I IFN production), antigen-presenting cell and T-cell activation, and production of inflammatory cytokines (TNFα, IFNg, IL-2, −4, −5). T cells in the skin grafts proliferate and migrate into the irradiated epidermis. TGFβ, a key pro-fibrotic cytokine known to drive radiation-induced fibrosis, increases 10 days after irradiation and remains elevated for three months. S100 genes known to be induced by TGFβ (S100A2, S100A7, S100A7A) and known to drive fibrosis (S100A8, S100A9, S100A12) also increase 10 days after irradiation. There was also evidence of endothelial injury and ROS production 24 h after irradiation, culminating in vascular and pericyte senescence by 10 days postirradiation. Three months after irradiation, there was fibrosis of the skin grafts. This model shows promise for studying the effects of radiation on living pediatric, adult, and aged human skin and for testing radiation countermeasures in vivo.
Biomarkers for Radiation Biodosimetry in Pediatric Models
The Fluorescent Automated Screening Tool for Dosimetry (FAST-DOSE) is a high-throughput biodosimetry platform designed to quickly screen and assess a large number of people in the dose range of 0–10 Gy (H.Turner) (45). The performance of the biomarkers used in this platform has been tested in irradiated adult (12–13 weeks) and juvenile (4 weeks) C57BL/6 mice to determine if age is a confounder. Intracellular protein expression was assessed in all leukocytes, B cells, and T cells at days 1–7, giving mostly dose dependent, linear responses in all age groups; however, an increased B-cell variability was noted in juvenile mice. Boruta machine learning (ML) testing rejected the sex, age, and day variables, giving a strong Receiver Operating Characteristic (ROC) curve for exposure classification and dose reconstruction. In addition, the FAST-DOSE was shown to distinguish between TBI compared with partial-body irradiation (PBI).
Pediatric blood samples from patients with an array of pre-existing conditions involved in acute immune response inflammation were paired with healthy adults to study the effects of comorbidities in the pediatric population. BAX and p53 biomarkers from T cells had equal baseline levels for both populations. Using a human blood ex vivo culture model, biomarker dose responses can distinguish exposed from unexposed, but BAX showed increased scatter in the pediatric population. Again, machine learning rejected age and sex as important predictor variables. It is important to note that sample sizes in these studies were small. Hence, more samples are needed to understand better the cohort variability seen in the pediatric samples.
Are Circulating miRNAs Impacted by Age and Sex? A Study of Clinical Samples
The impact of age and sex on circulating microRNAs (miRNAs) was examined using clinical blood samples from adult donors (age 18+) with a roughly uniform distribution in age (N = 238) (N. Menon). In addition, pediatric blood donors (ages 2–17) were obtained, but the samples were difficult to obtain; therefore, half were obtained from remanent (banked) samples (N = 50) and half from fresh collection (N = 52). Plasma was isolated from the samples, and miRNA was extracted and analyzed by next-generation sequencing. Principal component analysis (PCA) showed batch effects between the adult and pediatric samples. In particular, banked pediatric samples were distinct from both adult and freshly collected pediatric samples. Further analysis correctly assigned the samples based on sample type. As a result, the pediatric samples were analyzed separately.
To better understand miRNA differences between age and sex, expression over time was modeled with a cubic spline curve with a modest number of knots. The model allows the age-sex interaction to be tested using a moderated F-test. A bimodal effect was noted in samples of female adults (18–44 years); however, no differences were seen after 44 years of age. Eleven miRNAs identified as having a statistically significant age-sex interaction effect were linked to immune pathways, consistent with the hypothesis that females of childbearing age have differences in immune response compared to males or older individuals (46). In addition, age and sex differences have been reported in neuronal development (47, 48), supporting the changes seen in 7 miRNAs that showed differences between male and female growth and cognitive development in adolescents (12–17 years of age). Given the immune system’s role in the radiation response (49) and its effects on cognitive development (50), the age-sex effects of miRNA expression in this study highlight the complexity of miRNA interactions and their potential role in radiation biomarker development.
Estimating Radiation Exposure in Children Using a Single Complete Blood Count (CBC) Measurement
After a nuclear detonation in a large metropolitan area, biodosimetry diagnostics will be needed to triage victims (R. Kowalski). Qualitative and quantitative tools can help separate those needing medical attention and determine the amount of radiation absorbed to guide medical decisions. Currently, no FDA-approved biodosimetry device is available. Existing tools for estimating radiation exposure, such as lymphocyte depletion kinetics and chromosome aberration assays, are based heavily on adult responses to radiation and have not been validated with broad populations. ASELL, LLC (https://asell.com/) is developing a qualitative biodosimetry CellRADx algorithm based on the Hematologic Acute Radiation Index (HARI) developed at the Armed Forces Radiobiology Research Institute. HARI was developed using CBCs with differentials from radiation accident victims, healthy adults, and animal models to determine a qualitative dose above or below 2 Gy. Unlike lymphocyte depletion kinetic measurements, which require lymphocyte counts over time, HARI uses blood cell counts from a single time point.
Children have very different radiation responses compared to adults, with a higher propensity for leukemia and higher lymphocyte counts (51). Given these differences, the HARI algorithm performance was tested in the pediatric population. To assess false positive rates of the HARI algorithm, over 15,000 pediatric CBC values were collected from non-irradiated children through the National Health and Nutrition Examination Survey, with attention to age groupings (e.g., <2, 2–12, 13–18 years) and sex. In addition, CBC values were also collected retrospectively from pediatric radiation therapy subjects. The number of lymphocytes is highest in early childhood and decreases with age, while the number of neutrophils is reversed, with the lowest counts starting in early childhood and increasing with age.
There were concerns that these differences in absolute lymphocyte and neutrophil counts would challenge radiation dose calculations by the HARI algorithm in children. False positive results ranged from 0% to 0.52% across all pediatric ages, less than the rate seen for adults (1.13–1.95%). A slightly higher false positive rate exists in males ages eight and older. Analysis of 30 pediatric radiation treatment subjects showed a high sensitivity of 98%, with a specificity of 75% across ages 4 through 18 years. Of note, the underlying disease state and chemotherapy may confound the results for pediatric radiotherapy subjects. The findings suggest that the algorithm can effectively estimate radiation exposure in children at least 4 years old, aiding in pediatric triage and timely medical intervention.
Biomarkers Predictive of Delayed Radiation Injuries in a Pediatric Animal Model
Radiation exposure from a nuclear incident is expected to lead to multi-organ injury (MOI), where each organ system exhibits unique biomarker signatures that can be measured in urine and plasma (M. Boerma). Some injuries are expected to be acute, and others will be delayed, but early predictive biomarkers can help with disease prognosis and medical interventions. Using PBI delayed effects of acute radiation exposure (DEARE) rat model, radiation injuries to the lung, kidney, and heart were assessed (52). Juvenile rats showed an earlier onset of organ injury than adult rats when exposed to radiation. Plasma was collected on days 14 and 30 to predict metabolic biomarker disruptions. Data were stratified by sex, age, and radiation dose using a PCA analysis; all parameters affected the metabolite expression profile. Using metabolic pathway analysis, common pathways were noted between timepoints; however, they differed significantly by age and sex. Juveniles had major changes to their fatty acids and phospholipids, whereas in adults, the citric acid cycle and amino acid metabolism were perturbed.
To focus on specific organ injuries, such as cardiac fibrosis, samples were collected at 4 months and analyzed to identify biomarkers associated with this disease. Plasma metabolite panels were developed to predict cardiac fibrosis in juvenile rats exposed to radiation. Differences in biomarker responses between juveniles and adults were noted, with studies ongoing to explore urine biomarkers for kidney dysfunction. It is critical to examine biomarker panels by age and sex as these parameters can cause wide variability in the expression levels of metabolites. The findings highlight the potential for predictive biomarkers to improve outcomes for children exposed to radiation.
SESSION III: STATE OF THE SCIENCE FOR GERIATRICS
This session examined the effects of radiation across different life stages using rat models, NHPs, and cellular studies. The potential of radiation mitigators such as lisinopril, growth factors, and senolytics to mitigate radiation effects based on age was also addressed. These findings emphasize the need for age-specific interventions and further research into the biological mechanisms of aging and the radiation response.
Pediatric and Geriatric Rat MCMs and Natural History
Studies comparing the impact of radiation exposure throughout the lifespan were completed in pediatric, adult, and geriatric WAG/RijCmcr rat models, recapitulating the effects of radiation injury in humans (H. Himburg). After PBI, young adult WAG/RijCmcr rats present with a decline in complete blood count, a marker of H-ARS, within a month. Additionally, a decrease in plasma citrulline and damage to intestinal villi, which are typical of the gastrointestinal ARS (GI-ARS), occur in this time frame. Animals that survive H- and GI-ARS manifest DEARE. This is first seen in the lung at 50–100 days postirradiation with immune-mediated radiation pneumonitis. Kidney DEARE then follows at 175–225 days with irreversible renal failure accompanied by labored breathing, weight loss, limited mobility, and general poor body condition. This scenario varies depending on the age at exposure. Pre-pubescent pediatric rats develop lung DEARE significantly earlier than adults at 25–75 days after exposure with lower morbidity. Kidney DEARE is also accelerated in this population but is more lethal than in adults, with 100% mortality reached by approximately 100 days postirradiation. It is yet to be determined what accounts for the accelerated and lower lethality of lung DEARE in pediatric rats. A clue may lie in the observation that the endothelial cell populations present in the lungs of adult and pediatric rats are differentially represented and that different molecular pathways are activated in response to radiation. On the other hand, reproductively senescent geriatric rats, representing 50–60-year-old humans, appear to be resistant to lung DEARE with little histological evidence of lung injury; however, kidney damage is extensive, as measured by the extent of fibrosis, scar tissue, and tubular damage (53).
The efficacy of radiation mitigators may also vary depending on the age at which they are exposed. The angiotensin-converting enzyme (ACE) inhibitor lisinopril mitigates DEARE in adult rats and pediatric animals but has no efficacy in the geriatric population (53). In addition, the long-term effects of pegylated G-CSF are currently being studied. G-CSF-treated rats that survive H-ARS begin to show a higher risk for late renal dysfunction at approximately 180 days, exhibiting higher levels of blood urea nitrogen and increased urine protein to creatinine ratio. Based on these studies, several challenges were discussed, including the difficulty of correlating rodent (2-3 years) and human lifespans and the high cost of lengthy aging studies.
Multimorbidity and Markers of Resilience in Older Adult Macaques after Radiation Exposure
Long-term effects of radiation exposure were studied using older macaques from the Wake Forest Nonhuman Primate Radiation Survivor Cohort, a unique national resource of over 300 aging rhesus monkeys with a 2:1 male to female ratio that received ionizing radiation at 4–5 years of age (E. Quillen). After entering the colony, these animals were monitored at regular intervals for clinical parameters and imaging (e.g., CT, MRI), immune function (flow cytometric analysis and vaccination responses), overall physiological status (comprehensive postmortem pathology evaluation), and genetics (exome sequencing). Compared to non-irradiated controls, the irradiated NHPs have a shorter life and health span, defined as the time of appearance of the first chronic disease. While both groups suffer from similar comorbidities, radiation is associated with higher incidence and number. Differences between control and irradiated subjects are evident 3–4 years after exposure but tend to disappear with advancing age when the controls catch up with the exposed animals. Comorbidities are clustered into three major groups, with the first including diabetes, obesity, and tissue fibrosis in the hepatic system, lungs, kidneys, heart, and GI tract. The second grouping comprises cataracts, periodontitis, testicular atrophy, and hypertension, and the third group reflects weight loss, bone loss, and dysfunction of the female reproductive system. Many of the animals bear a high multimorbidity burden, a paradox that could be explained by survivorship bias, whereby the most resilient subjects were able to withstand ARS and DEARE. The underlying biological differences in resilience to radiation exposure are poorly understood, as is the fundamental aging process. If tissue-specific, interventions would need to be specifically targeted; if universal, it might explain the multimorbidities in older adults, with the hope that single interventions could impact multiple diseases.
Ionizing Radiation-induced Premature Senescence
Ionizing radiation induces cellular senescence and apoptosis, leading to H-ARS, GI-ARS, and other tissue injuries. Late effects include time-dependent accumulation of senescent cells throughout the body, which may trigger tissue fibrosis, bone marrow injury, premature immune aging, bone loss, brain injury, and cardiovascular disease (D. Zhou). As a result, senescent cells could serve as a target to delay the physiological aging process, improve cancer treatment efficacy, and mitigate the effects of radiation. Genetic clearance of senescent cells in mice has long been known to extend their lifespan and delay the onset of age-related chronic pathologies (54, 55). Senolytics and small molecules targeting senescent cells are being tested to see if age-related pathologies and diseases can be treated, extending health-span and lifespan. The senolytic compound ABT263 has been shown to clear senescent cells (56), reverse pulmonary damage (57), and limit the progression of frailty and premature aging (58) after radiation exposure. However, beneficial effects notwithstanding, this drug cannot be adopted as a viable radiation countermeasure because targeting BCL-xL on platelets induces thrombocytopenia in treated animals. This problem can be circumvented by incorporating ABT263 in a BCL-xL proteolysis-targeting chimera (PROTAC), thereby allowing it to exert its senolytic function while reducing its on-target drug toxicity through the degradation of its target molecules (59, 60). Second-generation ABT263 PROTAC compounds capable of targeting BCL-2 in addition to BCL-xL show enhanced senolytic activity in aged mice. By reducing on-target toxicity and improving its senolytic activity, BCL-xL/2-PROTAC drugs have the potential to be developed as a safe first-in-class anti-aging drug and MCMs against radiation. Some of the challenges in advancing this technology include 1. a lack of systemic and chronological examination of senescent cell accumulation induced by radiation and their roles in later effects in young and aged rodent models; 2. a lack of understanding of the role of radiation-induced immune senesce in senescent cells that accumulate after TBI; and 3. limited studies on radiation-induced senescence in nonhuman primates and humans.
Radiation Therapy in Geriatric Populations
Lung impairment is associated with the senescence of alveolar type II epithelial cells (AECIIs), leading to increased macrophage polarization, inflammation, and organ dysfunction (61). Mouse and human gene expression levels were examined to explore the underlying pathways involved in radiation injury. Consistent changes in aging and senescent genes, such as HGF-1, SERPINE1, IL-13RA2, TGF-βR2, TIMP1, and SMAD2, were seen in mice and humans (D. Citrin). In stained irradiated human lung tissue samples, fibrosis and macrophage polarization toward M2 were noted by increased IGF-1, p21, IL-13, CD68, and CD163 levels. Using a fibrosis-prone mouse strain (C57L/J), a fibrosis-resistant strain (C3H/HeN), and a strain with intermediate susceptibility (C57BL/6J), the accumulation of M2 macrophages correlated with the manifestation of fibrosis (62). While murine data support clinical observations, the absence of non-tissue samples (e.g., blood, urine, exhaled breath, etc.) presents challenges. Furthermore, samples from cancer patients are complex to study due to comorbidities, exposure to chemotherapy and other medications, receipt of fractionated radiation, and different dose levels; thus, the observed tissue pathology is a composite, making it difficult to distinguish radiation-related effects from those of treatment.
Clinical observations of radiation effects based on age can be challenging to conduct. While studies show that radiation-induced lung injury is more prevalent in the elderly, baseline organ function is often reduced, which increases the observed toxicity. Additionally, comorbidities, senescence, and prior exposures (e.g., smoking) in the elderly population serve as confounders. Interstitial lung diseases, such as idiopathic pulmonary fibrosis, are characterized by telomere shortening, AECII senescence, increased TGF-β, and myofibroblast activation, features also seen in radiation lung injury. This not only adds to the confounding nature of patient samples but may lead to fatal lung injury after radiation treatment for thoracic malignancies. Persistent lung fibrosis in aged mice is accompanied by a reduced microvascular network (capillary rarefaction), loss of endothelial cell identity, and impaired eNOS expression (64). Therefore, targeting vascular function may be crucial in promoting lung repair in the elderly after radiation exposure.
Differences in Cognitive Outcomes after Radiation Combined Injury
Low-dose radiation can lead to progressive and untreatable cognitive impairment, but the damage is not visible by imaging or histology (A. Allen). To investigate radiation-induced cognitive decline, a traumatic brain radiation injury model using C57BL/6J mice and Moore’s water maze was employed to study both sex and age effects (65). Age was a factor in cognitive decline, with geriatric mice (18–20 months old) performing worse than young (3–4 months old) or middle-aged (10–12 months old) mice. In all instances, radiation combined injury (RCI) leads to greater cognitive decline compared to radiation alone. Females showed deficits in both learning and memory, while males exhibited deficits only in memory. These changes were associated with increased inflammation and RCI neurogenesis in both sexes.
Interestingly, behavioral deficits were not seen after RCI in juvenile mice; these changes were strongly associated with Arc expression, a marker of neuronal activity. Thus, juveniles adapted to the trauma, whereas geriatric mice could not. The differences between the age groups are likely due to neuronal plasticity; however, the available data is mainly focused on adult models and is not available in all age groups. Age-specific studies in the geriatric and pediatric populations are needed.
SESSION IV: MODELING AND EXTRAPOLATIONS FOR DRUG AND DEVICE DEVELOPMENT
This session focused on alternative experimental models (e.g., organs-on-chips), artificial intelligence and machine learning (AI/ML) technology, and extrapolation for drug and device development as mechanisms to predict treatment outcomes and reduce the need for efficacy trials. The session highlighted the complementary nature of these approaches, emphasizing the necessity for further research to accelerate discovery and lower costs, particularly concerning pregnant/lactating, in utero cases, and pediatric data collection.
Feto-Maternal Interface Organ-on-Chip Platforms Provide Insight into Drug Pharmacokinetics (PK) and Efficacy
The level of complexity in studying pre-term birth is extensive, involving two independent systems: the fetus and the mother. Moreover, genetics, nutrition, and environmental exposures can also influence these systems. For the past 30 years, drug, lifestyle, nutritional, and physical interventions have been employed with minimal success in preventing pre-term labor; no biomarkers exist to identify high-risk pregnancies (L. Richardson). Pregnant/lactating women have generally been excluded from clinical drug research and the drug development process owing to legal, ethical, and safety concerns, making them “therapeutic orphans” (66).
Given the delicate nature of pregnancy, therapeutics tend not to focus on the fetus as the patient. To exacerbate the situation, no reliable in vitro or in vivo models are available to study pregnancy, parturition, and preterm birth. Also, only NHP models resemble human pregnancy, and these models can be extremely costly and challenging. Therefore, an organ-on-a-chip (OOC) multi-channel 3D microfluidic cell culture chip was developed to address these gaps. The OOC model simulates the activities, mechanics, and physiological responses of entire organs and organ systems (67). In response to the FDA Modernization Act 2.0,14 OOC models have been used to produce pre-clinical trial data and study PK during pregnancy, considering the complex fetal-maternal interface anatomy. In addition, in silico GastroPlus simulation modeling and OOC platforms can be used together to conduct faster and cheaper pre-clinical trial analyses of drugs (68). The OOC platform is also adaptable for studying the influence of fundamental variables such as fetal sex, maternal race, and ethnicity. This platform enables the study of both healthy and diseased states of pregnancy, preclinical testing of various agents to reduce the risk of adverse pregnancy outcomes, and the reduction of animal models in future studies. Moreover, this platform could allow the radiation research community to understand the impact of irradiation on the fetus and promote a better understanding of drugs that might be administered to pregnant women who have been irradiated.
Leveraging an Existing Pediatric Infrastructure to Address MCMs
The Best Pharmaceuticals for Children Act (BPCA) and the NICHD Pediatric Trials Network (PTN), established between the FDA (on-patent) and NIH (off-patent), aim to leverage existing data for MCM research (P. Taylor-Zapata). Under the BPCA, NIH is authorized to identify and develop off-patent drugs for pediatric use. To do so, NIH can sponsor clinical trials in the off-patent space, pharmacology training, and translational research. Additionally, data extrapolation can also be utilized; however, pediatric data must be available. Generally, if there is no relevant adult data, extrapolation is not possible. Nevertheless, data can be extrapolated using pharmacokinetic, safety, and efficacy data if the disease profile is similar in adults and pediatrics (69). The three overall steps needed for a label change of a drug are: 1. prioritization of medications needing a clinical study; 2. sponsorship for off-patent medications; and 3. submission of the trial data to the FDA for a formal label change.
Since pediatric drug development is neither market- nor passion-driven, supplementation is often necessary to incentivize companies. The NIH BPCA PTN is involved in core and bridging science that has led to advancements in the neonatal and lactation fields. Some innovative study designs include pediatric opportunistic clinical trials, during which PK samples are collected from populations already using the drug to inform dosing. To date, there have been 20 label changes, 51 clinical trials, and 27 FDA clinical study reports achieved. In addition, over 150 drugs are currently under study. The Commonly Used Drugs During Lactation and Infant Exposure (CUDDLE) project underscores a successful PTN study.
Biodefense MCMs were added to NIH BPCA PTN priorities in 2009, with improvements made in a pediatric autoinjector drug delivery system for nerve agent exposure, methods to measure cyanide in the blood (70), and pediatric personal protective equipment (71). Extrapolation and PK studies of organophosphate poisoning and infectious disease exposure were also conducted. The goals of the PTN are to develop partnerships in areas of need and leverage the knowledge gained through these experiences to continue bridging the pediatric gaps in drug development.
Extrapolation of Efficacy to Pediatrics: Conceptual Framework for MCMs
Many drug development efforts fail, including MCMs developed under the Animal Rule. To improve this success rate, AI/ML can be used to mine existing biomarker data to determine predictors of mortality. Once specific areas of interest are identified, they can be perturbed in silico to assess their impact on overall survival (J. Gobburu). A five-step machine learning process could be applied to these situations: 1. conduct a pharmacology or natural history study, 2. develop a disease-drug (treatment) model, 3. simulate blood cell measurements and lab results, including radiation dose and volume effects (e.g., PBI), 4. predict survival rates after treatments for multiple scenarios, and 5. decide if drug development should continue (i.e., if the predicted survival rate is >70%). AI/ML is not a substitute for understanding the mechanism of action or biomarker discovery; however, it can serve as a tool to expand that knowledge beyond the limits of conventional analysis. The relationships may be more complicated than linear or hyperbolic mathematical relationships, and AI/ML can help analyze the data in multiple dimensions. AI/ML can assist with pediatric data extrapolation, but success can vary depending on available data. If enough toxicity, PK, efficacy, and safety data are accessible, performing further efficacy studies could be avoided.
SESSION V: DRUG AND BIOMARKER DEVELOPMENT CONSIDERATIONS AND CONCEPTS OF OPERATION IN UNDERSTUDIED POPULATIONS PANEL DISCUSSION
During this final session, representatives from the FDA (D. Green and G. Burckart), the Administration for Strategic Preparedness and Response (ASPR) (C. Hrdina), the Federal Emergency Management Agency (FEMA) (J. Gill) and Emory University (Z. Kazzi) discussed federal disaster preparedness and response, regulatory approval of radiation MCMs, use of biomarkers to guide in disaster medicine, modeling of emergency scenarios, and anticipated concept of operations (CONOPS) during a radiological or nuclear (radio-nuclear) incident with particular attention to understudied populations.
Special Population Considerations
In emergency planning and response, consideration of pediatric and geriatric populations, as well as other specialized groups including pregnant or lactating women, those in utero, neonates, and individuals with intellectual and physical disabilities, is necessary to ensure a comprehensive and equitable approach. Behavioral aspects must be considered for both pediatric and geriatric groups. Infants and young children have developmentally appropriate increased hand-to-mouth tendencies. In geriatric populations, individuals with dementia may exhibit repetitive hand behaviors that can lead to hand-to-mouth actions. Of significant interest for both pediatric and geriatric populations are the mental health impacts and the psychological stress these incidents generate. Importantly, there is a significant need to include other specialized populations, such as pregnant and lactating women, in clinical research, as well as individuals with developmental and physical disabilities. The absence of these populations in research results in a lack of understanding regarding treatment effects and safety standards. The issue of thalidomide use during pregnancy serves as a prime example of how failure to study drugs in pregnancy can lead to tragic outcomes and highlights the need for expanded FDA regulations.
Service Considerations
Much can be learned about the fragility of populations by comparing medical care during everyday times and emergency incidents. Encounters with pediatric or geriatric patients during routine medical care typically occur in a controlled setting. In contrast, if these individuals become separated from their caregivers during an emergency, complications may arise due to ineffective communication and developmental state (e.g., young children) or other factors (e.g., dementia). Moreover, communication challenges may exist for individuals who speak languages other than English. Informed consent (e.g., assent for children and older individuals with dementia) presents an ethical dilemma that can result from communication difficulties, separation from caregivers, and the inability to follow up. Many geriatric victims lack local assistance, and their needs for rehabilitation or nursing services may continue beyond the incident. Similarly, children who experience the loss of one or more caregivers during an acute incident must find safe and permanent support. Planning for these situations during non-emergency scenarios can help create pathways for medical management during and after a radio-nuclear incident.
Scarce Resource Concerns
In a resource-limited setting, young and elderly populations alike will be triaged based on severity (75). The 1986 Emergency Medical Treatment & Labor Act was instituted to “ensure public access to emergency services regardless of ability to pay.” Hospitals with an emergency department must, by law, evaluate and stabilize any patient with a medical emergency and accept or transfer patients as needed. Through grant programs, the U.S. Government has collaborated with healthcare facilities and physicians/health scientists to develop capabilities for detecting radiation exposures and making informed treatment decisions. Modeling can help inform triage after radiation exposure, regardless of resource capacity, by estimating needs across the age spectrum.
Ethics
The ethical framework varies between pregnant/lactating, pediatric, or geriatric populations; while other understudied populations may be able to consent, children cannot legally consent. The federal regulations that govern research involving children have two basic elements: 1. the Common Rule, codified by DHHS Subpart A of Title 45, Part 46 of the Code of Federal Regulations (45 CFR 46), which provides a robust set of protections for research subjects, and 2. 21 CFR part 50, subpart D, which ensures clinical investigations do not pose greater than minimal risk and present the prospect of a direct benefit to the subjects, which includes additional safe-guards for children.
Regulatory
The FDA seeks alternative methods (e.g., human tissue chips) to leverage information beyond the traditional laboratory or clinical setting, including collecting information on pediatric and geriatric individuals. Traditional clinical research is not always feasible; therefore, technologies and methods that use data to inform on safety and effectiveness are of interest. The FDA Drug Development Tool (DDT) Qualification Program,15 established in 2016 as part of the 21st Century Cures Act,16 is tasked with maturing these technologies for drug development use. In 2023, NIAID sponsored a workshop on Advanced Technologies in Radiation Research, which covered cutting-edge approaches being developed and used in radiation research (76), and the FDA has also published a website on alternative methods.17 During advanced drug development, a drug must first show prospective benefits and safety and efficacy in adults before including other specialized groups (i.e., children and pregnant women). As a result, caution is often taken when conducting studies on these populations due to risks, concerns for potential adverse effects, and worries about possible litigation. Nonetheless, one case law analysis, commissioned in April 2024 by the National Academies of Sciences, Engineering, and Medicine, evaluated lawsuits since 1962 resulting from the use of products in pregnant/lactating women and did not find any claims of liability in these populations who participated in clinical trials.18 Even with copious knowledge about a disease and a drug’s mechanism of action, there might still be limited information about a treatment’s effectiveness and safety because there haven’t been enough clinical trials with the product. Hence, extrapolation is a valuable research method that uses data from well-studied groups, such as adults, to make informed predictions about other, less-studied groups. This technique can help identify potential risks and benefits without gathering new data from these understudied populations, making the research process more efficient. To use extrapolation effectively, researchers must understand the similarities between the different populations and predict how they might respond to treatments. The FDA has guidelines that allow researchers to start pediatric studies without first completing research in adults.
MCMs
Another area that requires more study is the development of pediatric MCMs. Gardner et al. (77) conducted a literature review and gap analysis on the lack of clinical guidance for emergency use of radiation MCMs and Strategic National Stockpile (SNS) dispensing to the pediatric population. The high-priority gaps include a lack of clinical guidelines and instructions to the parents/guardians for MCM treatment of H-ARS using cytokines, guidance for inhaled/ingested radionuclides, and MCM for these scenarios. There are also no pre-established protocols for the assessment and management of radiation injuries in children after radiological emergencies. A 2024 review considered 24 MCMs and their potential use in children (78). Of those studied, only 62% had FDA approval for specific radio-nuclear threats. While NCRP Report 161 (Management of Persons Contaminated with Radionuclides) contains recommendations for managing radioactive material contamination in different populations, these recommendations are not approved; therefore, they are often not mentioned in papers or guidelines.19 In 2023, the World Health Organization (WHO) published a Stockpile Guidance Document, which mentioned using decorporation agents such as Prussian blue and DTPA on understudied populations. Prussian blue was found to be very safe in children, and data were available for a few pregnant women who also took the product.20 In the context of radiation exposure MCM approvals, the FDA has extended the approval of specific hematopoietic treatments (filgrastim, pegfilgrastim, sargramo-stim, romiplostim) from adults to pediatric groups. This decision was based on extensive data showing the use of these growth factors in pediatric populations. As for Prussian blue radionuclide decorporation, a pediatric formulation is not yet available; however, efforts have been made to establish appropriate dosing for both pediatric and geriatric use. In 2011, BARDA awarded a contract to Heyltex Corp, the company supplying Prussian Blue to the U.S. Government for cesium decorporation, to develop a pediatric formulation. Unfortunately, this development faced technical challenges and stalled. Despite the availability of H-ARS MCMs with a pediatric indication, MCMs for other subsyndromes of radiation are needed. In addition, MCM efficacy can change depending on age; therefore, geriatric and pediatric populations may require a different dose. As these are developed, we need to ensure that the development, testing, and dissemination of effective MCMs continue for understudied populations.
Legislation and Incentivization
Congressional mandates and increased funding for pediatric research have successfully boosted FDA pediatric studies. A similar approach could enhance industry-funded research for these and other specialized populations. For example, the Pediatric Research Equity Act (PREA) authorizes the FDA to require drug companies to study their products in children under certain circumstances.21 PREA is based on the sponsor’s focus on adult indications. If there is a pediatric equivalent, the company must study it in children. However, this is driven by what sponsors are developing for adults, not by the specific needs of children. This legislation has led to over a thousand pediatric label changes. The BPCA, passed in 2018, also provides incentives for pediatric testing and post-marketing requirements, significantly advancing pediatric studies. Unfortunately, similar policies and incentives for older participants are lacking. However, in 2021, the FDA sponsored a workshop on the Inclusion of Older Adults in Clinical Research (79). The report highlighted the parallels between pediatric and geriatric patients, emphasizing the importance of adequately enrolling older adults in phase II and III clinical trials to confirm dosage, safety, adverse effects, and effectiveness. The ASPR also frequently uses contract authorities through the SNS procurement process or through work supported by the BARDA to encourage studies in understudied populations. Post-marketing requirements, closely coordinated with the FDA, are also included. All these authorities and legislation could be valuable in incentivizing studies for understudied populations and strengthening MCM and other mitigation strategies for future radio-nuclear incidents.
DISCUSSION
The discussions following each session are summarized here. Key gaps identified include the lack of open-access data to support AI/ML modeling, the presence of knowledge silos, insufficient data for new drug entities, and the need for improved access and advocacy for understudied populations. Addressing these gaps will accelerate drug discovery and reduce costs.
Alternative Preclinical Models of Special Populations
Advanced understanding of radiation effects on physiology requires novel experimental models and outside-the-box thinking. It is important to study data from animal studies and technologies used for other insults (e.g., environmental exposures) to develop in silico models for radiation exposure. Similarly, single-cell RNA sequence analysis can help extrapolate findings to radiation injury. While pregnant and in utero animal models exist and can be used to investigate radiation effects during the early stages of pregnancy, irradiating these models presents significant challenges and ethical dilemmas. Therefore, alternative models like organoids, OOC, and AI/ML models may offer flexible and less costly approaches.
OOC platforms can be genetically modified using techniques like CRISPR and siRNA to knock out specific maternal genes for studying immune or downstream effects. Additionally, similar to OOC infection models used to test treatment effectiveness (80), radiation can potentially be used as a stressor, and MCMs can be screened for efficacy. Utilizing the fetal-maternal OOC model may be possible to explore the effects of variability due to the sex of the fetus, maternal race, and ethnicity, since a biological sample bank is being developed. Oversimplistic OOC models can be minimized by incorporating all relevant cell types and engaging different research areas to avoid creating scientific gaps.
AI/ML was discussed as helping study biomarker data for indications like ARS since biomarkers may be in the causal pathway of the diseases (81). However, a limitation is that biomarkers rarely translate across species, highlighting the need for wide access to federally funded data to support data-intensive AI/ML strategies. The NIH’s Data Management and Sharing (DMS) policy, effective January 25, 2023, aims to promote the sharing of scientific data, accelerating biomedical research discovery by enabling validation of research results, providing access to high-value datasets, and promoting data reuse for future studies.22 While this policy positively impacts data availability, incorporating historical data can be time-consuming and complex due to incompatible data structures.
Biomarker Variations
The reasons for variations in biomarkers observed in adolescents and those of childbearing age were discussed. While hormonal changes during these periods could provide an explanation, it remains unclear. The quality of miRNA and metabolites was also mentioned, particularly the detailed and user-independent protocols that minimize batch effects and the design of instruments to address field variability. Differences in skin radiation doses were observed because skin biomarkers change based on exposure levels. Inflammatory signaling emerges at lower doses (1–2 Gy), TGFβ and immune signaling at higher doses, and hypoxia and vascular signaling appear at doses over 5 Gy. These differences could aid in distinguishing between TBI and PBI, but more research is needed.
Radiation Effects on the Immune System
Radiation-induced inflammation is a significant concern. In the aging, irradiated Wake Forest NHP cohort, chronic inflammation is associated with harmful changes in plasma lipid and lipoprotein concentrations, lipoprotein composition, and glycation which may contribute to the progression of atherosclerosis and diabetes (82, 83). Despite decreased subcutaneous fat, the remaining fat in these NHPs was highly insulin-resistant, absent of pancreatic damage. Various immune metrics are being tracked and explored in NHPs to understand immune resilience, including the delayed effects of chronic inflammation. Macrophages also play a significant role in inflammatory and immune responses, potentially due to myeloid progenitor cell skewing or Damage-Associated Molecular Pattern (DAMP) release. Myeloid cell production increases following pegfilgrastim treatment of irradiated juvenile animals. In clinical settings, myeloid leukemia has been found in pegfilgrastim-treated pediatric patients; however, filgrastim and pegfilgrastim treatments failed to show lymphoid skewing in NHPs. T cell numbers increase in the skin after irradiation and a decline in circulating T cells, suggesting lymphocytes may leave circulation and move into the skin. Moreover, non-recirculating resident memory T cells that populate human barrier tissues, such as the skin, are radio-resistant and may show enhanced immune functions after irradiation (84).
Non-steroidal anti-inflammatory drugs (NSAIDs) and other anti-inflammatory drugs may mitigate immunological radiation effects; however, they are only indicated for short-term use in clinical settings since long-term NSAID use can weaken the immune system. Some research is aimed at understanding the impact of NSAIDs on space radiation and therapeutic radiation, particularly concerning cognitive effects. Additionally, antioxidants are being explored because reducing inflammation could mediate M1 and M2 microglia polarization, which is crucial for repair (85). More studies are needed to understand the underlying mechanisms of the immunological responses to radiation and any differences in specific populations.
New Molecular Entities
PREA addresses data acquisition for new molecular entities.23 Advocacy is needed to ensure that understudied populations are considered from the beginning of the drug development process. Managing company reluctance to engage in post-market data collection is another challenge. While the NIH BPCA PTN does not work directly With pharmaceutical companies, it extrapolates relevant data and submits it to the FDA, which then works with companies to change the label. Collaborating with companies in non-competitive areas, such as training and trial design, could advance pediatric drug development. Continuous improvement in these processes should always be sought.
CONCLUSIONS
Understudied populations that include pregnant/lactating, in utero, pediatric, and geriatric individuals present multiple challenges and opportunities in developing appropriate MCMs for radiation injury. In utero and pregnant/lactating populations present distinct pathophysiology and heightened radiation sensitivity, which varies by gestational stage. As research continues, it is crucial to develop traditional and alternative models of the maternal-fetal interface, which can be used to study various insults, including radiation. Extrapolating data from these studies and other sources is essential for understanding and mitigating the effects of radiation exposure in pregnant/lactating and in utero populations. Radiation studies in pediatric and geriatric populations have provided significant insights into radiosensitivity, response to MCMs, skin characteristics, biomarkers, and cognitive outcomes; however, research gaps still exist. In addition, tailored radiation protection guidelines and MCMs are needed due to unique physiological characteristics and varied susceptibility to radiation-induced injuries. The development of age-specific biomarkers and biodosimetry tools to enhance radiation response and treatment outcomes in these populations is paramount. Continued radiation research is essential to understand population differences and ensure that all vulnerable groups receive the most effective and appropriate care.
ACKNOWLEDGMENTS
The authors would like to express their deep appreciation to all the speakers listed in Table 2, who provided valuable insights during the workshop and discussions. We also extend our gratitude to the NIAID Meet staff for ensuring a successful workshop. Additionally, we thank our NIAID colleagues (David Cassatt, Carmen Rios, and Tom Winters) who reviewed the manuscript before submission. The information contained herein is a summary of the presentations and discussions in the workshop and not necessarily those of the NIAID, NIA, NICHD, or NCI.
Footnotes
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