NIAID’s history of shared interest between radiation and chemical defense portfolios (A DiCarlo, D Yeung)

There has long been a partnership between US Government funding agencies involved in MCM development for use during a public health emergency. This has included biological pathogens, as well as radiation and chemical injuries. In some instances, government funding has also been provided to address non-emergency exposures (e.g., to radiation or chemicals during cancer treatment, space flight, or in military situations). Since 2005, an annual budget appropriation has been provided to the NIH Office of the Director, executed by NIAID, and shared between NIAID’s efforts to identify MCMs for radiation, ^{Reference Rios, Cassatt and Dicarlo1} (through the RNCP) and chemical (through the CCRP) injuries. ^{Reference Yeung, Harper and Platoff2} Although funding was originally imagined to support areas of science with little overlap, over the years, these programs have identified shared scientific mission spaces, which has resulted in the groups joining together to leverage their investments in several scientific areas. Examples of overlap between the programs are identified as (1) similar considerations for how an emergency response would be undertaken (e.g., post-exposure treatments and response timelines, stockpiling needs, expectations of scarce resources, and the desire to develop MCMs with utility for multiple threats as well as existing, non-MCM clinical indications); (2) need for small and large laboratory models to determine product efficacy and human safety protocols; (3) similar disease states (e.g., acute and chronic concerns such as fibrosis, inflammation, and chronic oxidative stress); (4) the same regulatory requirements for MCM development; and (5) similar organ systems of interest (e.g., lung, skin, bone marrow, and central nervous system).

Considering the funded portfolios of both NIAID programs, it is clear that there are scientific approaches that could be operable in both areas, including biomarker assessment for triage, patient management, prognosis, and efficacy; investigation into molecular pathways to determine druggable targets and identify mechanisms of action of both threat agents and potential MCMs; and classes of treatment investigated, such as anti-apoptotics, anti-inflammatories, anti-oxidants, anti-fibrotics, and cytokines/growth factors. ^{Reference DiCarlo, Horta and Aldrich3}

Both the RNCP and CCRP have had significant achievements in their relevant fields. For example, funding from the RNCP led directly to US FDA approval of 3 drugs to increase survival in patients acutely exposed to myelosuppressive doses of radiation, resulting in hematopoietic Acute Radiation Syndrome (H-ARS) (Neupogen®, ^{Footnote 1} Neulasta®, ^{Footnote 2} and Nplate®, ^{Footnote 3} Amgen). In addition, the RNCP has met with hundreds of companies and has invested in more than 600 compounds to address radiation injuries to several different organ systems. The program has a robust pipeline of funding to address other areas of concern during a radiation emergency, including the development of de-corporation agents that are amenable to mass casualty use, and the identification of biomarkers of radiation injury to triage and guide patient management and predict severity of late radiation-induced health outcomes. ^{Footnote 4} The CCRP, which focuses on discovery research and early development, has supported the development of many animal models, ^{Footnote 5} including those addressing ocular, ^{Footnote 6} hematologic, ^{Reference Beske, Wilhelm and Harvilchuck4} and lung ^{Reference Perry, Neal and Hawks5} injuries arising from SM exposure. The program has also provided guidance and funding to allow the transition of many products to BARDA for further development. Product transitions to date have included galantamine (FDA-approved treatment for Alzheimer’s, under study as a neuroprotective nerve agent MCM) ^{Reference Lane, Carter and Pescrille6} ; midazolam ^{Footnote 7} (epilepsy drug to treat seizures resulting from nerve agent exposure) ^{Reference Lewine, Weber and Gigliotti7} ; tissue plasminogen activator ^{Footnote 8} (drug to break up clots in stroke patients, repurposed to address SM-induced airway blockage; R-107⁵ (a novel nitric oxide donor compound), and a TRPV4 channel blocker ^{Footnote 9} to address inhalational chlorine injuries. Other product transitions include tezampanel and ganaxolone ^{Footnote 10} (as novel anticonvulsant and neuroprotectant for nerve agent toxicity) ^{Reference Barker, Spampanato and McCarren8} ; and INV-102 ^{Footnote 11} (Invirsa, a p53 modulator regulating DNA damage response for SM-induced eye injuries. Lastly, the CCRP works closely with the BARDA chemical countermeasures program to administer in vivo screening programs to identify potential MCM candidates for SM-induced toxicities.

Although the chemical focus of the current meeting was SM, close to 200 chemicals and 12 toxidromes have been identified by the US Department of Homeland Security Probabilistic Analysis for National Threats Hazards and Risks (PANTHR) program, ^{Footnote 12} as high consequence public health chemical threats of concern, including other pulmonary (e.g., chlorine and phosgene), and skin (e.g., nitrogen mustard, Lewisite, arsenicals)- targeted compounds; pharmaceutical-based agents (e.g., synthetic opioids), inhibitors of cellular respiration (e.g., cyanide and hydrogen sulfide); anticoagulants (e.g., brodifacoum and bromadiolone); and neurological-affective compounds (e.g., nerve agents or organophosphate pesticides). Similarly, the scope of work funded by the NIAID RNCP extends beyond just lung and skin models and MCM development, to the support of MCMs for hematopoietic (H-), and gastrointestinal acute radiation syndrome (GI-ARS), cutaneous radiation injuries (CRI, both alone and in combination with other trauma), cardiovascular, and kidney, as well as central nervous system damage. Both NIAID programs also have a primary interest in repurposing products that already have FDA approval/ licensure for another clinical indication. ^{Reference DiCarlo, Cassatt and Dowling9} To streamline this meeting, the programs elected to focus on similarities between radiation exposure and SM-induced injuries to the lung and skin; although arguably, the case could also have been made to include bone marrow myelosuppression. However, to keep the meeting focused and manageable in terms of length, these other areas of study were not included.

BARDA radiation and chemical programs for advanced product development (J Laney, M Homer)

The radiation and chemical programs implemented by the BARDA have historically been responsible for advanced development of candidate MCMs. The programs have recently taken a more systems biology approach to identify therapeutics for both radiation and chemical injuries. For example, the Chemical Countermeasures Program focuses on treating the injury, not the threat agent. Their stated mission is “improving the health outcome for all victims of chemical exposure.” Aspects that are unique to their mission include the fact that there are many agents of concern, and exposure to these chemicals will be unpredictable and localized. In addition, due to the rapid action of many chemical agents and the impracticality to do pre-treatment, centralized stockpiling may not be feasible, hence necessitating forward deployment of treatments. Finally, there is a lack of available diagnostics, making early triage of specific injuries difficult. To address these challenges and ensure that chemical MCMs are available in an emergency, BARDA has developed a strategy that employs a threat-agnostic pipeline (with strong NIAID collaboration) and focuses on repurposing common drugs. In that way, drugs are already in place with end-users who are familiar with their use. Examples of their successful repurposing efforts have included Silverlon® wound dressing (Argentum Pharmaceuticals), which obtained FDA clearance in 2019 for vapor dermal SM injuries ^{Footnote 13} ; and coordinated funding with the Department of Defense and NIH leading to FDA approval of Seizalam (midazolam, Meridian Medical Technologies, Inc.) in 2018 for status epilepticus seizures (including those resulting from nerve agent exposure). Argentum’s Silverlon received FDA’s 510K clearance for radiation dermatitis and cutaneous radiation injury (CRI) subsequent to this workshop but are mentioned here for the sake of completeness.Footnote ¹⁴ Other drugs being considered for repurposing include those to address chlorine-induced lung injuries, opioid-induced respiratory depression, and both SM ocular and inhalational injuries. Finally, BARDA’s chemical program has partnered with the University of Hertfordshire, UK, and first responders across the US to create the second edition of Primary Response Incident Scene Management (PRISM) Guidance for Mass Decontamination. ^{Footnote 15} This valuable resource provides information on operational responses to chemical incidents, with new procedures that remove most contamination, even before first responders arrive while requiring no special equipment, or products.

The BARDA Radiological/ Nuclear Countermeasures Program has followed a similar approach within their portfolio. Awarded contracts, which include approaches addressing radiation-induced vascular injuries, sepsis, and coagulopathy, as well as fibrinolysis, and cell death are the largest part of the program, which also focuses on ischemic injuries and inflammation as key components of irradiation’s bodily impact. The program continues to support both polypharmacy and repurposing approaches, building on a successful track record of working with companies to provide advanced development support and stockpile acquisition of necessary therapeutics for radiation injuries. BARDA funding led to the FDA approval of Leukine® to treat H-ARS (Partner Therapeutics, originally Sanofi), ^{Reference Clayton, Khan-Malek and Dangler10} and had a role in regulatory and procurement-related activities for the other 3 approved H-ARS approaches mentioned above. The program also looks to leverage across other threat areas, including mechanical trauma, thermal burn, and chemical injuries, as well as infectious diseases, to maximize government investments and allow for multi-utility approaches to be pursued. BARDA’s current focus areas include radiation-induced platelet loss, endotheliopathies, and inflammation. BARDA is also tasked with ensuring sustainable supplies of both traditional and next-generation blood products, such as spray-dried blood plasma. ^{Footnote 16} Recently, an interest in enabling technologies like tissue chip platforms led to co-funding of contracts on in vitro micro-physiological systems (MPS) with the National Aeronautics and Space Administration (NASA), given the overlapping missions of irradiation-induced health effects and their mitigation. The NIAID RNCP also supports a contractor with NASA for MPS development.

About the journal selection for a special issue (J James)

The Disaster Medicine and Public Health Preparedness Journal has had a long history of interactions with the CBRN communities, so it was an excellent choice for a journal to reach both radiation and chemical defense agencies and investigators. For example, in 2011, a special issue of the journal was published on nuclear preparedness, and the papers included therein continue to be important resources for researchers, funding, and regulatory agencies. ^{Reference Casagrande, Wills and Kramer11–Reference Coleman, Knebel and Lurie22} There have also been more than 50 manuscripts (as of this writing) published in the journal since 2007, which covered global radiation emergency preparedness, radiation scenario modeling, and other areas of interest. ^{Reference Sander and Randle23–Reference Sawano, Senoo and Yoshida27} Similarly, the journal has reported on findings and discussions surrounding hazardous chemical materials, including but not limited to reports on preparedness, ^{Reference Donahue, Burkle and Blanck28,Reference Schwartz, Sutter and Eisnor29} training exercises, ^{Reference Choi, Jung and Choi30} case studies, ^{Reference Jafari, Saburi and Ghanei31,Reference Sørensen32} and review articles. ^{Reference DeLuca, Chai and Goralnick33,Reference Moradi Majd, Seyedin, Bagheri and Tavakoli34} For these reasons, the meeting organizers began working with the journal at the very beginning of the process, leading to the publication of this meeting report and the special issue in which it resides.

In summary, all 4 NIAID and BARDA radiation and chemical medical defense programs routinely have contract and grant solicitations for the development of products to treat chemical and radiological injuries, and to ensure that there is a robust pipeline of approaches under consideration to address these threats. They intend to continue to coordinate to leverage government investments in both mission areas and provide guidance to enable multi-utility of approaches across the chemical and radiation spaces. Publication of this meeting report, and the special issue in which it resides, is a step along the way toward ensuring harmonization across the programs administered by these different government agencies.

MCM development under the FDA Animal Rule (A Powell)

There are specialized MCM groups within the FDA. These include the Office of Counterterrorism and Emerging Threats (OCET) (in the Office of the Commissioner); the CounterTerrorism and Emergency Coordination Staff (CTECS) within the Center for Drug Evaluation and Research (CDER); the Preparedness and Response Team in the Center for Biologics Evaluation and Research (CBER), and within the Center for Devices for Radiological Health (CDRH), the MCM group is known as All Hazards Readiness, Response, and Cybersecurity (ARC) (formerly known as Emergency Preparedness/ Operations and Medical Countermeasures EMCM). It is important to note that these groups do not have the regulatory authority over the MCMs; that authority lies with the regulatory review divisions.

In May 2002, the FDA published AR to allow (under very specific circumstances) the approval of new drugs and licensure of biological products when human efficacy studies are not ethical or feasible (21 CFR Parts 314.6000.650 for drugs and 21 CFR Parts 601.90-95 for biological products).Footnote ¹⁷ These regulations allow for product efficacy to be established based on adequate and well-controlled studies in animals “when the results of those animal studies establish that the drug product [or the biological product] is reasonably likely to produce clinical benefit in humans,”Footnote ¹⁸ and safety is evaluated “under preexisting requirements for establishing the safety of a new drug and biological products.”Footnote ¹⁹

For a product to be approved under the AR, all of the following criteria must be met: (1) there is a reasonably well-understood pathophysiological mechanism of the toxicity of the substance and its prevention or substantial reduction by the product; (2) the effect is demonstrated in more than 1 animal species expected to react with a response predictive for humans, unless the effect is demonstrated in a single animal species that represents a sufficiently well-characterized animal model for predicting the response in humans; (3) the animal study endpoint is related to the desired benefit in humans, generally the enhancement of survival or prevention of major morbidity; and (4) the data or information on the kinetics and pharmacodynamics of the product or other relevant data or information, in animals and humans, allows a selection of an effective dose in humans.Footnote ²⁰ The FDA can also consider other information such as published human data.

As of January 2022, 16 products have been approved or licensed under the AR, with indications that can be grouped into 7 broad categories of severe, and life-threatening diseases or conditions resulting from exposure to CBRN agents.Footnote ²¹ In the labeling of these products, 7 have descriptions of animal efficacy studies in 2 species; 8, which also had relevant human efficacy data, have descriptions of efficacy studies in a single animal species; and 1 has descriptions of efficacy studies in more than 2 species. A critically important aspect of product development under the AR is the selection of the animal models in which the efficacy of the investigational product will be tested. The animal models should adequately reflect key elements of the human disease or condition and should be appropriate for use with the investigational product. FDA recommends that sponsors obtain agency concurrence on the animal models that will be used in efficacy testing of their investigational products. Animal model development is a resource intensive process. CDER’s and CBER’s Animal Model Qualification Program (AMQP) was created to support the development of product-independent animal models that will be used for testing multiple products under the AR.Footnote ²² This is a voluntary program, and the use of a qualified model is not required for AR approval. The AMQP provides a formal avenue to obtain FDA subject matter expert feedback on early animal model development. Qualified animal models are made publicly available.

FDA encourages early and ongoing communication, and sponsors should have a thorough understanding of the available guidance documents for developing products under the AR, such as the overarching guidance: ‘Product Development Under the Animal Rule: Guidance for Industry,’Footnote ²³ and any relevant indication-specific guidances. ^{Footnote 24,Footnote 25,Footnote 26} FDA also developed a compliance program for the inspection of nonclinical laboratories conducting AR-specific studies, and it may be helpful for stakeholders to understand how these studies will be inspected.Footnote ²⁷ Regulatory review of product applications is an integrated, multidisciplinary review process under the director of the regulatory review division. A typical CDER review team for an MCM consists of reviewers and team leaders representing chemistry, manufacturing, and controls; pharmacology/ toxicology; clinical medicine; clinical pharmacology; statistics; regulatory project management; and other disciplines, as needed. CDER/ CTECS offers early informational meetings to academic investigators or sponsors of MCMs that will be regulated by CDER, to help them prepare for pre-Investigational New Drug (preIND) interactions with the review division. CTECS does not provide scientific or regulatory advice but does provide general information such as information about the AR, the regulatory review process, contact information for the appropriate review division, and expectations for preIND meetings, as well as meeting packages and useful resources.

Amgen’s regulatory journey to drug approval for Acute Radiation Syndrome (D Andrews)

Amgen’s success with the approval of Neupogen, Neulasta, and Nplate highlights the successful interactions between a sponsor, funding agency, and the FDA. Radiation exposure can result in ARS, where injury severity increases with increasing levels of exposure. ^{Reference Waselenko, MacVittie and Blakely35} In response to the need for an MCM to treat H-ARS, Amgen partnered with NIAID and other agencies, and interacted with FDA to obtain approval for products to treat patients. In 2015, Neupogen (recombinant granulocyte-colony stimulating factor; G-CSF) (the first MCM), was approved by the FDA under the AR, in collaboration with NIAID and included in the National Strategic Stockpile. In the same year, Neulasta, a pegylated form of G-CSF was also approved to treat patients with H-ARS. In 2021, Nplate, a thrombopoietin receptor agonist indicated to treat patients with immune-mediated thrombocytopenia was also approved for the treatment of H-ARS.

These approvals were contingent on the criteria for approval under the AR described above, and it took 11 years for NIAID/ Amgen to develop Neupogen/ Neulasta as MCMs. The NIAID/ Amgen/ FDA interactions played a central role in this achievement, from influential feedback on: (1) animal model development (mouse and nonhuman primate (NHP) studies), (2) long-term patient safety concerns, and (3) pharmacological modeling of adult and pediatric doses in H-ARS. The key discussion points from the FDA joint meeting of the Medical Imaging Drugs Advisory Committee and Oncologic Drugs Advisory Committee leading to approval were: (1) mechanism of injury and repair in irradiated NHP translates to humans with H-ARS; (2) survival benefit in irradiated NHPs administered Neupogen, ^{Reference Farese, Cohen and Katz36} or Neulasta, ^{Reference Hankey, Farese and Blaauw37} translates to a survival benefit in humans; and (3) pharmacokinetic (PK)/ pharmacodynamic (PD) data from irradiated NHPs studies with Neupogen/Neulasta and Amgen’s human patient data can identify an appropriate effective human dose through pharmacologic modeling. The committee was asked to deliberate and vote regarding Neupogen’s clinical benefit in a radiation exposure emergency based on NIAID NHP efficacy study data and Amgen patient safety and efficacy data, resulting in a 17-1 vote in favor.

In 2021, no advisory committee was convened for Nplate approval. In contrast to prior product approvals, Nplate approval required only 5 years, with a more streamlined regulatory path based on the precedent set by Neupogen/ Neulasta. Pivotal NHP studies clearly demonstrated benefit in NHPs administered Nplate as well as comparable pharmacodynamics following exposure. ^{Reference Wong, Chang and Fielden38} In addition to FDA’s AR Guidance, ³⁹ other guidelines that expedited radiation MCM approvals were the Standard for Exchange of Nonclinical Data (SEND) Implementation guide and the compliance program for the inspection of nonclinical laboratories conducting AR-specific studies.Footnote ²⁸

Based on these successful interactions, Amgen’s recommendations for achieving drug approval under the AR regulations are: (1) invest in a well-defined and committed partnership/ collaboration with sponsoring government agencies that have appropriate biology and AR expertise, offer funding opportunities, and share in the mission to develop radiation MCMs for the US population; (2) develop a dedicated and experienced team of cross-functional subject matter experts with appropriate company sponsorship to meet project demands in time and resources; (3) seek early FDA agreement on overall non-clinical strategy especially when animal models are not well established; (4) maintain milestone meetings with the FDA to share progress/ results, design next steps, and ensure alignment on fulfillment of all the requirements of the AR prior to regulatory submission; (5) know and meet expectations under the FDA AR regulations in study designs and when authoring regulatory documents; and (6) publish/ share results to advance animal model development and promote additional drug approvals under the AR.

Development of drug products for treatment of Acute Radiation Syndromes (L Marzella)

For sponsors of SM-focused, or radiation MCMs, it is important to review the regulatory history of products approved for H-ARS and the scientific literature reports of studies of products under development for the treatment of skin and lung radiation syndromes. Also critical is the evaluation of available scientific knowledge for threat agents based on the analysis of fundamental mechanisms of organ injury and dysfunction caused by radiation or SM. Finally, consider the applicability of regulatory strategies for product development and approval adopted by developers of countermeasures for radiation threat agents.

Selection of a pharmacologic target is key. Characterization of the mechanism of action (MOA) of organ injury and dysfunction that is targeted by the MCM candidate is needed for animal efficacy studies. MOA for H-ARS, primarily myelosuppression with acute depletion of myeloid precursors, is well defined; approved MCMs stimulate proliferation, differentiation, and function of myeloid precursor cells. ^{Reference Bunin, Bakke and Green40} Proposed MOAs for skin and lung radiation injuries can be more complex due to the potential for multi-organ dysfunction and a multi-stage clinical course with early acute edema and inflammation, delayed development of necrosis, followed by repair and fibrosis, hence the need for more complicated protocols.

It is important that the pathology in the selected animal system model the clinical condition. Natural history studies are necessary to establish the time course and manifestations of the injury caused by various levels of exposure to the threat agent, and the response to various doses of the MCM. CDER has relied on adequate and well-controlled efficacy studies in NHP models for the approval of H-ARS indications, ^{Reference Farese, Cohen and Katz36,Reference Hankey, Farese and Blaauw37} and data from rodent studies have provided key support for efficacy. Rhesus macaque is the standard NHP model for H-ARS and has been under development for lung injury. More recently, the lack of availability of rhesus related to COVID-19 is requiring validation of a cynomolgus macaque model in some instances. Porcine animal models are also under development for CRI.

Efficacy endpoints in animal models for radiation MCMs are primarily survival or reduction in major morbidity. It is advised that the conduct of studies in patients with organ injury and dysfunction caused by similar agents (e.g., chemotherapy) be done to obtain safety, and when possible supportive clinical efficacy data. Survival has been the primary animal efficacy endpoint for H-ARS and is under consideration for radiation-induced lung injury (RILI), but the utility of quantitative measures of lung function or anatomy is under evaluation. ^{Reference Cassatt, Gorovets and Karimi-Shah41} For skin injury, ^{Reference DiCarlo, Bandremer and Hollingsworth42} quantitative assessment of area and depth of skin injury and repair verified by histopathology is recommended as the primary animal efficacy endpoint, while clinically meaningful reductions in severe skin injury or improvement in healing, quality, and durability of repair, bridging to engraftment or reconstruction, and survival should be considered for safety assessment (e.g., in models of combined injury). Endpoints that characterize the recovery of organ injury or dysfunction, such as neutrophil or platelet counts that document recovery from myelosuppression, or positive microbial cultures, are secondary to survival and may be used to support of primary efficacy endpoint, trigger initiation of supportive care, and guide pharmacodynamic (PD) modeling for selection of human dose.

For the successful selection of an effective human dose of an MCM, it is important to conduct dose-ranging studies in animal models. Two approaches commonly used are the PK approach, where comparison of predicted drug exposure in affected humans to the animals receiving a fully effective dose, and the PD approach, where the drug exposure in humans results in a similar magnitude of PD marker in animal models. With these salient points, regulatory strategies for the development of drug products for radiation injuries may be similarly applicable to the development of products for SM-induced injury. A consult of the publicly available FDA drug approval packages and scientific literature will inform the product development pathway.

Repurposing Silverlon® dressings for treatment of cutaneous Sulfur Mustard (SM) injuries (P Antinozzi)

Another successful sponsor-FDA interaction is the repurposing of Silverlon dressing for the management of SM-induced cutaneous injury. Silverlon dressings are sterile, flexible, porous, and non-adherent, as well as knitted nylon plated with elemental silver and silver oxide.Footnote ²⁹ The silver ions provide an antimicrobial barrier for the clinical management of various infected wounds (traumatic, surgical, first/ second degree thermal, and dermal ulcers) and vascular access. With BARDA support, Argentum has explored repurposing Silverlon dressings as an MCM for use in mass casualty incidents involving radiation and vapor SM exposure. The vapor SM indication was pursued as a non-inferiority study (silver sulfadiazine as comparator) against superficial and moderate partial-thickness wounds in Göttingen minipigs. Results of the study were used to support the eventual FDA clearance of Silverlon dressings in July 2019Footnote ³⁰ as the first MCM indicated to manage SM-induced vesicant injuries not requiring skin grafting.Footnote ³¹ Ongoing efforts are geared toward obtaining similar FDA approval of Silverlon dressings as a radiation MCM. Nuclear detonations and dirty bombs presumably produce complex injuries comprised of blast injury, thermal burns, and radiation effects. Since Silverlon dressings are already indicated for traumatic wounds likely to result from blast injury, as well as both first- and second-degree thermal burns, if the product can mitigate CRI, then it may be an effective CRI MCM. As such, a 2-stage regulatory approach was undertaken where: Stage 1 utilized clinical data derived from radiotherapy patients supporting an indication for lower severity radiation injury,Footnote ³² and Stage 2 utilized preclinical data from a Yorkshire swine model to support a higher severity radiation injury indication. Silverlon dressings were granted Breakthrough Device designation in October 2021 for the proposed low and high severity radiation dermatitis indications from FDA’s Center for Devices and Radiological Health (CDRH). As an update, after the meeting on which this report is based, Argentum obtained FDA clearance for the use of Silverlon in radiation dermatitis (e.g., experienced in the clinic following radiotherapy), and CRI.

Regulation of wound dressing devices and considerations for development of medical devices used for Sulfur Mustard and Cutaneous Radiation Injury (A Guan)

Wound dressing devices fall under the product jurisdiction of CDRH and are broadly described under 3 subcategories: (1) solid dressings; (2) gels, creams, and ointments; and (3) wound wash solutions. Wound dressing devices are regulated by the FDA as Class I (generally do not require premarket review and does not contain drugs, biologics, or animal-derived materials), II (generally requires 510(k) premarket review and cleared based on substantial equivalence to predicate devices based on intended use and technological characteristics), or III (premarket approval with reasonable assurance of safety and effectiveness as an intended wound treatment, skin substitute, life-supporting, or sustaining purpose and typically requires clinical data) devices. 510(k) submissions for wound dressings typically do not require animal wound healing study data, unless (1) the device is considered cytotoxic, which may delay the natural wound healing process; (2) a sponsor elects to conduct a wound healing study in lieu of an implantation study to evaluate the local tissue response after application of the device to the wound site; and (3) non-animal testing is not adequate to demonstrate substantial equivalence concerning safety and effectiveness compared to the predicate.

FDA recommends utilization of the swine model for wound healing studies due to anatomical and physiological similarities to humans, and because pigs also close partial-thickness wounds largely through re-epithelialization. While radiation dermatitis (RD) and CRI may present similarly, they are not exclusively interchangeable clinical conditions. CRI patients may experience signs and symptoms that are far more severe, less controllable, and potentially fatal compared to patients with RD. Wound dressing devices indicated for management of the signs and symptoms for RD may be considered as appropriate predicate devices for 510(k) review of a wound dressing intended for the same signs and symptoms in CRI patients, but a CRI indication may need to be supported by additional performance data. More specifically, wound dressing devices indicated for more severe symptoms and co-morbidities associated with CRI (wet desquamation, blisters, ulceration, and hemorrhage, as well as necrosis, and need for skin grafting) may not be appropriate for 510(k) review.

Cutaneous Radiation Injury (CRI) (C Iddins)

CRI is expected to occur in up to a third of individuals exposed in the context of a 10-kiloton detonation in an urban setting like New York City, ^{Reference Barss and Weitz43} where it is estimated that of the 4 million that could be exposed, ∼64 000 could receive a dose of > 6 Gy. Indeed, data from Chernobyl indicate that 54/134 documented cases had CRI as part of their exposure. ^{Reference Barabanova and RCBMEOJFM44} Although CRI alone does not result in mortality (not to be confused with Cutaneous Radiation Syndrome, a much larger and more significant injury), the morbidity with associated pathologies can manifest as disfigurement, recurring wounds and fibrosis, and unrelenting chronic pain requiring constant and lifelong medical interventions. ^{Reference DiCarlo, Bandremer and Hollingsworth42} Therefore, it is critical to understand how to manage this injury in the event of a radiological or nuclear event. Clinical manifestation of CRI results from cellular damage including death of epidermal stem cells, DNA damage, ROS/ RNS, and an inflammatory response cycle. ^{Reference Iddins, DiCarlo and Ervin45} The resulting pathophysiology of this injury can include vascular dilation, increased capillary permeability, microhemorrhage, and platelet consumption. ^{Reference DiCarlo, Bandremer and Hollingsworth42} Neutrophil and lymphocyte infiltrates can result in perivascular edema, cell hypoxia, and cell death. CRI pathophysiology and injuries progress through early “prodrome” inflammatory responses, followed by “latent phase” anti-inflammatory response, accompanied by macrophage activation, and formation of reactive oxygen and nitrogen species, as well as elevated proinflammatory cytokine production. Ultimately, these chronic conditions result in the “manifest illness phase” characterized edema, erythema, and moist desquamation, as well as blistering and possibly, deep ulceration and necrosis. Longer term effects are characterized by fibrosis, poor wound healing, and scarring. These phases and latencies are contingent on the absorbed dose and duration of exposure. ^{Reference Iddins, DiCarlo and Ervin45} At the site of injury, white blood cells, specifically mast cells, are a significant contributor to CRI pathology. Their degranulation and release of heparin and histamine can lead to coagulation and vessel permeability. It is this activation that can further recruit other pro-inflammatory white blood cells like neutrophils, monocytes, antigen-presenting cells, etc. Recruitment of these cells can also lead to sustained TFG-β1 release that can result in fibrosis, which depending on injury severity, can extend into the blood vessels and deeper tissues. ^{Reference Iddins, DiCarlo and Ervin45}

Clinical assessment of CRI severity is done with scenario history including dosimetry or dose reconstruction along with patient history/ physical exam and serial photographs of the injury. ^{Reference Gottlober, Bezold and Weber46} Imaging is another essential tool used to assess radiation injury, including magnetic resonance imaging (MRI), magnetic resonance angiography, and ultrasound. ^{Reference Lefaix and Daburon47–Reference Peter and Gottlöber51} However, the integration of imaging, bio-dosimetry, and clinical picture may provide the best assessment of wound severity. ^{Reference Gottlober, Bezold and Weber46} There are also different grading systems in nature of the injury, including the Radiation Therapy Oncology Group (RTOG) grading system, ^{Reference Cox, Stetz and Pajak52} and METREPOL cutaneous grading system, which uniquely includes body surface area involvement as a quantitative element. ^{Reference Fliedner53} As part of their medical management strategy, The Radiation Emergency Assistance Center and Training Site (REAC/TS) relies on a modification of the 2009 National Council of Radiation Protection and Measurements (NCRP) clinical threshold guidelines.Footnote ³³ REAC/TS has found these clinical grading and dose threshold tables to be informative in a clinical dose estimation even when other dosimetry is available. ^{Reference Gusev, Guskova and Mettler54} Thus, there are standardized tools clinicians have for assessing CRI and their progression.

While there are similarities of CRI to thermal burns in determining injury severity (e.g., depth and body surface area involvement), unlike thermal burns, CRI severity depends on the type and quality of radiation, and dose rate. ^{Reference Gursky and Hreckovski55} Wounds may be localized or large and could involve deeper tissues and other organs. At higher radiation doses, wounds may have difficulty healing and remaining healed. ^{Reference Iddins, DiCarlo and Ervin45} Case studies demonstrate the pathology and complexity of treatment for CRI. In August 2008, a worker’s hand was exposed while changing out a Co source. ^{Reference John, Koller and Worek60} The individual’s hand was only exposed for seconds and the estimated dose by re-enactment was 6 - 7 Gy. Clinical management included pentoxifylline, topical vitamin E, silver sulfadiazidazine cream, and standard burn therapies. With that regimen, the wound healed completely; however, 2 years later the digit was amputated, and retrospective dosimetry estimated that the actual dose to the finger ranged from 22.5 - 40 Gy. ^{Reference Iddins, DiCarlo and Ervin45} In another case study, a large dorsal wound resulting from a fluoroscopically-induced necrotic lesion was treated with standard burn care in addition to hyperbaric therapy. Ultra-sound and Doppler imaging were performed to assess blood flow at the injury, and surgeons were able to excise the damaged tissue and provide the patient with negative pressure wound therapy. With the excision and extensive wound care, this large wound was able to heal completely. ^{Reference Iddins, DiCarlo and Ervin45} Therefore, REAC/TS recommends that CRI care include protecting the area, providing non-steroidal anti-inflammatories, and pentoxifylline, antihistamines, as well as newer silver-based dressings, emollient moisturizers, and topical steroids as needed. In addition, surgical treatment may also be indicated. While grafts can be successful to treat CRI, they are also at risk for failure with wound recurrence if areas were exposed to doses of radiation resulting in necrosis. Other interventions for CRI can include use of investigational mesenchymal, ^{Reference Benderitter, Caviggioli and Chapel56,Reference Lataillade, Doucet and Bey57} or adipose stem/ stromal cells, ^{Reference Akita, Yoshimoto and Ohtsuru58} as well as adipose-derived stromal vascular fraction, ^{Reference Yu, Zhang and Mo59} and/ or growth factors, potentially utilized with more traditional modalities of dermal constructs, skin grafts, flap, and/ or amputations. ^{Reference Iddins, DiCarlo and Ervin45}

There remains a need for clinical trials repurposing current therapies for similar injuries, for novel MCM research in animal models, and adaptation of bench to bedside advancements of research. CRI can be very complex, recurrent, and may not mimic thermal/ chemical burns. In the event of detonation of an improvised nuclear device, responders will be overwhelmed with these complex, long-term wounds/ injuries. Education and communication will be key in implementing long-term plans to address these injuries.

Sulfur Mustard: the skin and systemic connection (K Lu)

Dermal injury from SM exposure involves epithelial injury and immune activation. Mechanisms involved in SM and nitrogen mustard (NM) effects have been studied since World War II. These efforts led to the development of other analogs as chemotherapy agents (e.g., mechlorethamine, carmustine, uramustine, etc.). SM effects on the skin include blister formation, poor skin healing, and re-epithelization. Injuries that are healed appear to be tinted and shiny indicating underlying fibrosis and limited function, even when closed. ^{Reference John, Koller and Worek60} These injuries can have potential chronic and long-term adverse effects like MSC senescence and squamous cell carcinoma development, both of which are indicative of immune alteration. This immune modulation involves activation of lymphocytes, especially myeloid cell interactions, and activation of macrophages expressing iNOS and TNF-α. SM exposure activates the immune system that is constantly surveying skin and the lumen of surface blood vessels, allowing the connection of this rich microenvironment to circulation. ^{Reference Tacastacas, Chan and Carlson61,Reference Schmidt, Steinritz and Rudolf62}

This activation of the immune system has led scientists to target multiprong, systemic approaches involving myeloid cells present in all organ systems of the body. ^{Reference Schmidt, Steinritz and Rudolf62} One therapeutic under study is high-dose vitamin D, which can induce quiescence of activated macrophages following exposure to these alkylating agents. ^{Reference Hewison63} Data suggests that outside of its normal function in regulating the endocrine system and ensuring musculoskeletal health, vitamin D calms inflammatory macrophages by decreasing iNOS, promoting anti-inflammatory cytokines, and decreasing matrix metalloprotease (MMP) 9, which are all key players in injuries following SM exposure. In an example from a study of treatment with carmustine for lymphoma, a patient experienced a severe skin burning sensation that could have led to his removal from the study; however, administration of vitamin D3 mitigated the skin pain, allowing him to remain on the protocol. ^{Reference Tacastacas, Chan and Carlson61} SM studies using an animal skin model at Battelle have shown that vitamin D3 modulates immune activation and protects from chemical skin injuries - a single dose of vitamin D3 following a dermal exposure to SM rescued 40% of the animals. Administration of 1 dose of vitamin D or the iNOS inhibitor 1400W rescued animals, improved hematological parameters, and promoted wound healing. ^{Reference Au, Meisch and Das64} A correlation was also seen between a decrease in both TNF-α and iNOS with these endpoints. SM/ NM-induced changes in these biomarkers are like those seen in CRI pathologies.

Pre-clinical and clinical studies using vitamin D have been conducted to better understand the relationship between genetic and proteomic changes in SM toxicity and response to possible treatments. Laboratory animal studies including mice, ^{Reference Das, Binko and Traylor65} Yorkshire pigs and rhesus macaques, have explored the impact of vitamin D on damaging exposures to ultraviolet radiation (UV), and SM, as well as NM. Human clinical studies have also explored vitamin D treatments following carmustine, ^{Reference Tacastacas, Chan and Carlson61} or NM exposure. A clinical trial of high-dose vitamin D3 against Valchlor™, an FDA-approved topical NM, in healthy volunteersFootnote ³⁴ involved multiple observations, and biopsies, as well as blood draws. These results showed that most of the pathways involved in the injury and treatment mitigation were related to inflammation, including leukocyte, lymphocyte, and chemokine activation; patients also experience a decrease in erythema, swelling, and pain. The take home of the human studies is that although it seemed that skin has healed, it could break down repeatedly over time, as has been seen for radiation dermal injuries. NIAID funding of Northwestern’s CounterACT Center supports a 3-prong approach for dermal injuries induced by SM/ NM, or chemotherapy agents’ toxicity that involves superficial, mid-dermis, and systemic strategies. These strategies will be used to identify and develop combinatorial treatments to activate repair genes and suppress destructive inflammation, control other downstream processes (neo-angiogenesis and fibrosis), and for topical and systemic intervention for more critically ill patients.

SM lung injury and fibrosis: identifying targets and developing countermeasures (D Laskin)

Acute and chronic lung injuries from SM and NM exposure have been studied in several animal models developed to understand the pathogenesis of toxicity in the lung that would reflect their toxicity in humans. ^{Reference Laskin, Malaviya and Laskin66–Reference Venosa, Gow and Hall70} With these models, several MCMs to target areas of toxicity are being developed. SM and NM exposure can result in pulmonary damage which is the major cause of morbidity and mortality. Pulmonary toxicity following SM inhalation has both an acute and chronic impact and follows similar pathogenesis to that seen in injuries resulting from irradiation. Acute effects are first seen in the upper airway followed by effects in the lower airway (e.g., acute respiratory distress syndrome [ARDS]). Mustard-induced chronic diseases are seen within 10 - 20 years following exposure and are primarily caused by persistent lung inflammation. Asthma, bronchitis, fibrosis, and chronic obstructive pulmonary disease (COPD), as well as bronchiolitis obliterans are some of the chronic diseases that are known to be caused by a single exposure to SM. ^{Reference Laskin, Malaviya and Laskin66}

Both mouse and rat models of SM injury by intratracheal inhalation and NM by intratracheal aerosolization have been developed. ^{Reference Malaviya, Abramova and Rancourt71,Reference Sunil, Vayas and Cervelli72} These animals are exposed to a range of mustard doses and followed from 1 to 28 days post-exposure, with an assessment of lung tissue and lining, bronchoalveolar lavage (BAL) fluid, and immune cells, as well as pulmonary function analyses and live animal MRI/ CT scanning. ^{Reference Murray, Gow and Venosa73} Histopathological effects of SM on the lung include early thickening of the epithelium and airways that become more prominent over time. These effects are pronounced at 16 days, with increased thickening of the alveolar areas, marked presence of immune-inflammatory cells, and increased sloughing of the upper epithelial lining. At 28 days there is congestion, massive amounts of inflammatory cells, and evidence of fibrosis. ^{Reference Laskin, Malaviya and Laskin66,Reference Venosa, Smith and Murray68} SM-induced pulmonary fibrosis is present and confirmed by the presence of collagen deposition. In the pathogenesis of mustard-induced lung injury, there is an increase in extracellular matrix proteins and collagen seen early on. Later pathogenesis of fibrosis is evident, with dysregulated disposition of extracellular matrix proteins and collagen. ^{Reference Laskin, Malaviya and Laskin66,Reference Venosa, Smith and Murray68,Reference Sunil, Patel and Shen69,Reference Venosa, Malaviya and Choi74} These non-clinical results confirm the acute lung injury caused by mustard exposure that progresses to chronic fibrosis, which is also seen in humans.

Histological studies have shown that SM toxicity is characterized by a persistent macrophage (CD11b⁺) dominant inflammatory response. These inflammatory macrophages are present within 1 day post-exposure, and their size and numbers increase over time. ^{Reference Malaviya, Abramova and Rancourt71} Further mechanistic studies focused on the contribution of distinct subsets of macrophages and the mediators they release on acute lung injury and chronic lung fibrosis induced by SM. These subsets develop from bone marrow precursor cells and are designated as M1 and M2 which develop in response to signals in the tissue microenvironment. ^{Reference Malaviya, Abramova and Rancourt71,Reference Weinberger, Malaviya and Sunil75} Upon exposure, M1 macrophages migrate into the site of injury and are the pro-inflammatory cells that are cytotoxic and release reactive oxygen species (ROS), reactive nitrogen species (RNS), tumor necrosis factor α (TNFα), and other chemokines to promote inflammation and get rid of dead cells, debris, and infectious agents. ^{Reference Laskin, Malaviya and Laskin66} As the inflammatory process progresses, mediators in the micro-environment form anti-inflammatory M2 macrophages that are involved in wound repair and promoting tissue. Dysregulation or excessive activity of either of these subsets of macrophages can complicate the healing process and cause further damage and disease. The levels of each subset of macrophages and their mediators directly correlated with the time course of acute and chronic injury seen in lung tissue. ^{Reference Sunil, Patel and Shen69–Reference Malaviya, Laskin and Laskin77}

Involvement of these macrophages’ subsets in pathogenesis of SM-induced lung fibrosis provides potential targets for mitigating damage. One approach is by targeting M1 macrophages and pro-inflammatory/ cytotoxic mediators specifically, rather than general anti-inflammatory agents. Some possibilities that have proven successful include N-acetylcysteine (NAC), aminoguanidine, 1400W, and pentoxifylline, as well as anti-TNFα antibody. MCMs toward RNS can be effective MCMs for SM-induced injuries as well. Preliminary studies using iNOS knock-out mice have shown protection from mustard toxicity. Administration of aminoguanidine, a specific iNOS inhibitor, further confirmed that blocking this mediator mitigates SM-induced congestion. TNFα is a known pro-inflammatory mediator and is released by stimulated M1 macrophages following SM exposure. Similarly to iNOS, TNFα knockout mice (TNFR1-/-) were protected from mustard toxicity. Other studies have shown that anti-TNFα antibody reduces SM-induced oxidative stress. Small, ventilated animals were used to assess lung elastance and compliance. The lung elasticity or the ability to exhale is reduced following NM exposure and was completely mitigated by an anti-TNFα antibody. BARDA continues to fund work on the anti-TNFα antibody, to advance the approach through the FDA approval process to treat mustard toxicity. ^{Reference Laskin, Malaviya and Laskin66,Reference Sunil, Patel and Shen69–Reference Malaviya, Laskin and Laskin77}

As mentioned above, M2 macrophages accumulate early within the lung. As lung macrophages differentiate to M2 macrophages, following mustard exposure, their morphology changes. For example, M1 macrophages isolated 1 to 3 days after mustard exposure are round and small, and on day 7, there is a mixed population of M1 and M2 macrophages. By 28 days, all macrophages are enlarged, fibrotic, and foamy. Foamy macrophages responding to mustard exposures, both in lung tissue and isolated cells from BAL, are filled with lipids. NM has also been shown to dysregulate macrophage lipid transporters with increased lipid uptake. Obeticholic acid (OCA), FDA-approved for primary biliary cholangitis, restores lipid homeostasis in the liver, and prevents fibrosis. Studies using OCA have shown a decrease in NM-induced dyslipidemia, suppression of M2 macrophage activation, as well as suppression of foam cell formation, reduction of NM-induced histopathological changes in lung tissue, and protection against fibrosis. ^{Reference Venosa, Smith and Murray68} The path forward includes continued testing of the efficacy of OCA as a countermeasure for SM-induced lung injury and fibrosis as well as developing methods for selective delivery of various countermeasures directly to M1 and M2 macrophages.

Evolution of lung injury after SM inhalation (C White)

The primary exposure route of toxic chemicals will determine the characteristics of the injury and signs and symptoms that manifest upon exposure in a lung injury model. For chemical threats like SM, nerve agents, and other toxic industrial chemicals, the inhalation route is the most important for mortality and morbidity. The epidermal route is also common, while exposure by ingestion/ injection is not generally representative of real world scenarios. ^{Reference Willems78} Exposure routes and downstream effects hold true for radiological and nuclear threats as well. SM causes a dose-dependent, multi-system acute injury, with similarities to ARS. The primary organs that are susceptible to acute SM-induced injuries are pulmonary, ocular, and skin. Secondary organs that may show acute SM toxicity are the central nervous system, gastrointestinal tract, liver, and kidney, as well as the heart. SM is also known to cause dysregulation in the immune response, hematologic/ coagulation, and hematopoietic stem cells. Chronic effects in the pulmonary system are the major cause of late disability and morbidity with a high probability of developing pulmonary fibrosis, bronchiolitis obliterans, recurrent pneumonia/bronchitis, and asthma (RADS), as well as COPD, large airway stenosis and/ or, bronchiectasis. ^{Reference Bagheri, Hosseini and Mostafavi79,Reference Arefnasab, Babamahmoodi and Babamahmoodi80} Chronic effects on the skin include hypertrophic scar and keloid formation along with deformity. Ulcerative keratitis is 1 of the major chronic diseases seen in SM-induced ocular injuries. The systemic dysregulation in immune and hematologic/coagulation responses may lead to leukemia and other malignancies. These effects have been seen in patients following the Iran-Iraq war, and in nonclinical studies in rats. ^{Reference Balali-Mood and Hefazi81}

Acute effects of SM inhalation involve injuries to both upper and lower airways. The major effects seen in the upper airway include sloughing and obstruction. Edema is also seen in the lower airways along with inflammation, epithelial destruction, sloughing, and fibrin cast (e.g., pseudo-membranes) formation that can lead to airway obstruction and mortality. The acute effects of SM inhalation exposure utilizing an intubated, anesthetized, ethanolic SM aerosol rat model that was developed by the US Army Medical Research Institute of Chemical Defense, ^{Reference Anderson, Byers and Vesely82} showed that SM inhalation exposure causes dose-dependent mortality due to tissue factor-dependent extra-vascular coagulation and airway thrombosis. Rats were exposed to a range of doses of SM with 24-hour survival ranging from 63 – 20% at lower SM concentrations, but with 100% lethality at higher inhaled dose (4.2 mg/ kg as inhaled intratracheal aerosol). The major cause of mortality following SM inhalation is the formation of fibrin-rich casts that form primarily in the conducting airways, causing choking and breathing difficulties. These lesions, as well as airway obstructions, resolve promptly after administration of the fibrinolytic drug tissue plasminogen activator (tPA) into the airways, demonstrating the crucial role of airway coagulation after acute SM inhalation. Pulmonary artery angiograms in rats following SM inhalation have an approximate 50% distal reduction in pulmonary arteries (pruning) within 24 hours of high doses of inhaled SM when compared to naïve rats, with further evidence of obstructive intravascular thrombosis. ^{Reference McGraw, Rioux and Garlick83}

Studies with this SM aerosol-inhalation rat model have also looked at effects upto 6 months to study late pulmonary disease and fibrosis. Results have shown that SM causes progressive hypoxemia and mortality in a SM dose-dependent manner over 6-months. Rats exposed to inhaled SM have decreased oxygen delivery to the tissues and poor weight gain that correlate to changes in mortality over 180 days. ^{Reference Anderson, Byers and Vesely82}

Lung function is also affected by SM, with mixed obstructive and restrictive lung physiology observed. Lung compliance steadily declined, while fibrosis was also demonstrated by increase in tissue damping. ^{Reference McGraw, Dysart and Hendry-Hofer84} Bronchiolitis obliterans was present in the lesions obtained by airway microdissection, and there was a dose-dependent increase in airway collagen that correlated with mortality. ^{Reference McGraw, Dysart and Hendry-Hofer84} Results with interstitial collagen showed that SM exposure also caused parenchymal lung fibrosis, a form of interstitial lung disease that is typically irreversible. Pulmonary fibrosis was seen in SM-exposed rats in a dose- and time- dependent manner. Lung pathology showed evidence of patchy, heterogeneous areas of fibrosis that were especially predominant in sub-pleural region. ^{Reference McGraw, Dysart and Hendry-Hofer84} The coagulation pathway is also critical in fibrosis in early and late SM inhalation models. In the acute chemical inhalation models evaluated, Tissue Factor initiates the extrinsic coagulation pathway very early, ^{Reference Rancourt, Veress and Guo85–Reference Rancourt, Veress and Ahmad87} and in both acute and late models, the plasminogen activator inhibitor (PAI-1) enzyme inhibits fibrinolysis. ^{Reference McGraw, Dysart and Hendry-Hofer84,Reference Rancourt, Veress and Ahmad87,Reference Rancourt, Ahmad and Veress88} Furthermore, in chronic survival models after SM inhalation, protein levels of transforming growth factor (TGF) β1,and platelet-derived growth factor (PDGF)-A/B, as well as PAI-1 (the latter associated with coagulation in both humans and in rat models) significantly increased in lung homogenate, and BALF, as well as plasma, and lung. These results suggest that pro-fibrotic pathways are involved in rats and human SM-induced lung parenchymal and airway fibrosis. ^{Reference McGraw, Dysart and Hendry-Hofer84}

eNAMPT: A medical countermeasure target in radiation and chemical-induced inflammatory injuries (J Garcia)

Exposure to either ionizing radiation or toxic chemicals (such as SM) results in tissue injury and the release of multiple Damage-Associated Molecular Pattern (DAMP) proteins, which bind pathogen recognition receptors (PRRs) to initiate or amplify activation of inflammatory pathways that result in organ injury. eNAMPT or extracellular nicotinamide phosphoribosyltransferase, is a key DAMP that binds with high affinity to Toll-like receptor (TLR) 4, a critical PRR. eNAMPT-mediated TLR4 signaling results in NF-kB-dependent expression of pro-inflammatory and profibrotic genes/ proteins. ^{Reference Camp, Ceco and Evenoski89} NAMPT is a cytozyme that also exhibits intracellular enzymatic activity and is involved in regulation of NAD metabolism (iNAMPT); however, eNAMPT secretion and activation of the evolutionarily-conserved eNAMPT/ TLR4 signaling pathway occurs in multiple human pathologies including bacterial infection, ^{Reference Bime, Casanova and Oita90–Reference Bime, Casanova and Camp92} trauma, ^{Reference Bime, Casanova and Nikolich-Zugich91,Reference Bime, Casanova and Camp92} and COVID-19 infection. ^{Reference Bime, Casanova and Nikolich-Zugich91} It has been strongly implicated in pre-clinical models of both radiation- and SM- induced lung injury. ^{Reference Garcia, Casanova and Valera93,Reference Garcia, Casanova and Kempf94} Released from epithelial cells, endothelial cells, and leukocytes to regulate innate immune responses following lung injury, ^{Reference Quijada, Bermudez and Kempf95,Reference Ye, Simon and Maloney96} eNAMPT acts as a DAMP in the development of vascular and fibrotic injuries. ^{Reference Quijada, Bermudez and Kempf95} Human subjects with chest trauma, ARDS, sepsis, and COVID-19 pneumonitis, as well as radiation pneumonitis, all exhibit elevated plasma levels of eNAMPT compared to healthy controls. ^{Reference Bime, Casanova and Oita90–Reference Bime, Casanova and Camp92}

Based upon pre-clinical studies utilizing the eNAMPT - neutralizing, humanized immunotherapeutic ALT-100 mAb, eNAMPT expression is markedly elevated, and contributes significantly to the severity of acute, sub-acute, and chronic inflammatory lung injury. ^{Reference Garcia, Casanova and Valera93–Reference Quijada, Bermudez and Kempf95,Reference Bermudez, Sammani and Song97,Reference Sammani, Bermudez and Kempf98} For example, the anti-inflammatory ALT-100 mAb has proved highly protective in a rodent model of trauma-induced ARDS, ^{Reference Bermudez, Sammani and Song97} in an ARDS porcine model of septic shock with ventilator-induced lung injury, ^{Reference Bermudez, Sammani and Song97,Reference Sammani, Bermudez and Kempf98} and in a murine model of whole thoracic lung injury (WTLI)- induced radiation pneumonitis. ^{Reference Quijada, Bermudez and Kempf95} In each case, animals receiving the ALT-100 mAb exhibited reduced phosphorylation of NFkB, inflammatory indices, and histologic evidence of inflammatory injury compared to untreated animals, suggesting that blockade of eNAMPT/ TLR4 signaling alters disease pathophysiology. In pre-clinical WTLI models of murine and NHP lung fibrosis, eNAMPT lung tissue and blood expression at 12 weeks was markedly increased and mice receiving ALT-100 mAb exhibited reduced severity of lung fibrosis compared to untreated WTLI mice. ^{Reference Garcia, Casanova and Kempf94} These studies highlighting the potential role of eNAMPT in sustaining chronic inflammation and fibrosis were supported by transcriptional analysis of RNA-sequencing data from lungs of irradiated animals. ^{Reference Garcia, Casanova and Kempf94}

The utility of the eNAMPT-neutralizing mAb was also assessed in a partial body irradiation (PBI) murine model (6.75 Gy, C57BL/6 mice) with 5 percent bone marrow (PBI, 5% BM) sparing with specific examination of both survival and multi-injury progression. Compared to IgG-treated controls, PBI-exposed mice receiving the ALT-100 mAb given 24 hours post-PBI exhibited dose-dependent increased survival at 21 days post-exposure, reduced circulating cytokine levels, and importantly, reduced histopathological severity scores of lung, liver, kidney, and small intestine tissue injury.

Given commonalities in radiation- and SM-induced inflammatory lung injury, a rat model of SM exposure was used to query the role of eNAMPT in SM-induced pathology and inflammation. In limited studies of acute and sub-acute SM exposure models, increased eNAMPT and NOX4 expression in rat tissues was observed; ALT-100 given 2 hours post-SM exposure did not improve survival although a higher dose of ALT-100 mAb did yield a survival benefit compared to the SM-exposed group. In a model of chronic SM exposure, ALT-100 mitigated SM exposure-induced mortality, similar to ALT-100 results in radiation-induced lung fibrosis, and attenuated multiple inflammatory and fibrosis markers. Together these data are consistent with the premise that eNAMPT is a key DAMP involved in radiation- and SM- induced lung injury and fibrosis with ALT-100 mAb a potential MCM to mitigate both radiation and SM-induced lung injuries.

Radiation- and SM- induced skin injuries, models, biomarkers, and regulatory strategies (P Antinozzi)

Argentum Medical has worked with research partners to develop the appropriate animal models, experimental approaches, and regulatory strategy to obtain FDA approval for Silverlon. The silver-nylon dressing has been shown to be safe and effective for the treatment of burn wounds in several animal species including rats, hairless guinea pigs, and Göttingen minipig models (of partial thickness, full thickness, thermal, and chemical burns). The company’s regulatory strategy included 4 necessary milestones. The first was a Good Laboratory Practice (GLP) safety evaluation of Silverlon in a Göttingen minipig model of deep dermal wounds. The study showed wound healing with the dressing and no toxicity on the local or systemic level. ^{Reference Barillo, Croutch and Barillo99} The second milestone was to determine the benefit of Silverlon in Yorkshire pigs after skin injury induced by ionizing radiation. The radiation source, a beta-particle emitting Strontium-90 (⁹⁰Sr) device designed by J Daniel Bourland PhD at Wake Forest School of Medicine, was employed for the task. ⁹⁰Sr is an isotope in fallout from nuclear weapons and nuclear accidents. The ⁹⁰Sr irradiator allows for reproducible and uniform dose delivery in a confined area, has an adjustable total dose, and can create multiple irradiated targets per animal. ^{Reference Burnett, Gabard and Robinson100} The third milestone, based on Argentum’s clinical activities, was a trial studying the ability of Silverlon to reduce radiation dermatitis in breast cancer patients. The 30-patient safety study was conducted at the University of Rochester to support Argentum’s FDA submission. Trial investigators generated an unbiased, standardized computed skin toxicity score to document severity of radiation dermatitis in the patients.Footnote ³⁵ The fourth milestone involved rescoring study images with clinical grading to develop software for the human clinical trial. Over 1000 images were evaluated by blinded dermatologists using the RTOG scale. In-person and computational scoring was found to be closely aligned and 98% of images received scores within 1 point between pairs of scorers. This database of images supported the development of a computational scoring method with an unbiased approach.

Skin models help define mechanism of action of SM (J Laskin)

The SM l alkylating agent and vesicant causes blistering on the skin and mucous membranes. The Rutgers University CounterACT Research Center of Excellence studies these target tissues to identify FDA-approved products that could be repurposed to treat SM injury. The Rutgers Center’s goals also include drug formulation for optimal delivery to target organs (e.g., nanomedicines, controlled release, foams, etc.), elucidation of mechanisms of SM-induced toxicity and tissue repair, and development of rapid screening assays. In terms of drug delivery, the program formulates nanomedicines. Skin models are used to investigate mechanisms of SM damage, screen drugs, and assess product efficacy. Models include the Göttingen minipig, Guinea pig, human ex vivo full thickness skin constructs, and mouse ear and dorsal skin vesicant models. Göttingen minipig skin exposed to SM causes progressive epidermal damage, with complete epidermal degradation at later time points. ^{Reference Laskin, Wahler and Croutch101} Eschar formation occurs by day 9, with epithelial cell regrowth and wound healing evident by day 14. Data generated in ex vivo human full thickness skin constructs and mouse skin models support that the initial SM response includes DNA damage, inhibition of cell growth, and apoptosis. Reactions with DNA form mono- and bi- functional adducts that interfere with cell cycle progression and new proteins can be synthesized when DNA is cross-linked. ^{Reference Inturi, Tewari-Singh and Agarwal102} Like SM, NM also inhibits cell cycle progression by modulating cell cycle proteins as shown in vitro with epithelial cells. NM has been shown to induce acetylation, ubiquitination, and cross-linking to p53 to elicit DNA damage in human keratinocytes. There are multiple and dynamic interactions that are occurring on a cellular level upon exposure to mustards. The DNA damage response pathway could help identify key proteins involved in the process of DNA repair and cell proliferation that occurs in response to a mustard insult.

Radiation skin injuries in the clinic: what we know, urgent gaps, and translation of MCMs (J Wolf)

Similarities in animal models of CRI described throughout the workshop can help address gaps in the field to improve the management of skin reactions seen in radiotherapy patients. Radiation dermatitis occurs in ∼ 95% of patients receiving radiotherapy, with up to 30% of those skin reactions being severe. Acute reactions can present with mild erythema, followed by dried desquamation, then moist desquamation, and finally followed by ulceration. Chronic effects can occur weeks to years later. ^{Reference Iacovelli, Torrente and Ciuffreda103} At doses above 45 Gy, late and chronic radiation effects can include fibrosis, telangiectasia, and dermal atrophy. Reductions in these skin reactions may be possible by using intensity-modulated radiotherapy; however, radiation skin injury is still a concern. Lack of standardization of treatment and clinical rating of severity of radiation skin injury are also factors. ^{Reference Ryan104}

Known mechanisms of radiation-induced skin injury include loss of basal keratinocytes, stem cells and Langerhans cells, impairment of the epidermal barrier with increased trans-epidermal water loss (TEWL), and endothelial cell/fibroblast damage, as well as stimulation of resident and circulating immune cells, chronic waves of inflammation, ROS, and antioxidant imbalance. To address the multitude of effects, extensive research has led to many therapeutic approaches; however, no effective agent has been identified to prevent the onset of injury. There is evidence of the benefit of antioxidants; however, findings are inconsistent, but topical steroids work well due to their broad-spectrum activity of anti-inflammation and immunosuppression. Treatment of the described skin reactions to reduce their progression is key but treatment for symptoms such as pain, itching, and burning is necessary as well.

Recently links have been made between the skin microbiome and radiation dermatitis. In 2021, Ramadan et al. ^{Reference Ramadan, Hetta and Saleh105} showed that a predominance of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus are associated with delayed healing of radiation dermatitis. Also shown was a tendency toward radiation dermatitis and delayed healing in patients with a raised proteobacteria/ firmicutes ratio. This suggests that microbiome profiling could inform treatment plans by indicating the progression of radiation skin injury. ^{Reference Ramadan, Hetta and Saleh105} There are notable similarities between SM and radiation skin injury. Both stressors cause damage to basal keratinocytes leading to erythema, blisters, hyperpigmentation, and pain, as well as itching, burning sensations, and desquamation. While wounds may heal, both injuries can produce chronic effects on pigmentation, telangiectasia, and pain. These similarities imply that potentially, treatments that work for 1 could be translated to help the other. To improve radiation skin injury treatment options, current gaps in the field must first be addressed. ^{Reference DiCarlo, Bandremer and Hollingsworth42} These gaps include lack of consensus and standardization of effective treatment for the management of radiation skin injury, need for improved means of objective and quantitative severity measure of injury for all skin types, and identification and utilization of predictive markers and factors to identify individuals at high risk for severe skin reactions. A systematic review and Delphi Consensus Survey, conducted by the Multinational Association of Supportive Care in Cancer (MASCC) Onco-dermatology Study Group has evaluated standards of care in radiation dermatitis with the help of an expert panel. Their results indicate a lack of standardization for effective treatment or combination of treatments. Therefore, although there are clearly still gaps in understanding the underlying biology of radiation and SM-induced damage, there are several approaches under study to reduce the severity of these skin injuries, with many promising research findings.

Airway thrombosis as the leading cause of acute respiratory failure after SM inhalation (L Veress)

Airway thrombosis, characterized by the presence of fibrin airway casts composed primarily of white blood cells and fibrin networks, forms inside the airway lumen in response to conditions such as smoke inhalation, and airway burns, as well as asthma, H1N1 influenza, COVID-19 infection, and sickle cell disease. It also forms in response to pulmonary embolism, pulmonary lymphoma, and chemical inhalation. ^{Reference Veress, O’Neill and Hendry-Hofer106} These airway casts obstruct the airway and have a high mortality rate of 30 - 60%. This blockage impacts oxygenation and ventilation significantly, causing asphyxiation. There is currently no standard of care but surgical interventions include manual removal via serial bronchoscopy, lobectomy, thoracic duct ligation, and Fontan fenestration, as well as heart transplant and heart function optimization. Pharmaceutical interventions include tPA, heparin, azithromycin, and sildenafil/ tadalafil. ^{Reference Veress, O’Neill and Hendry-Hofer106} A retrospective analysis of SM-exposed patients showed that 23% presented with airway casts. Of the patients with casts, 50% died and 20% of the survivors required emergent tracheostomy due to sudden airway occlusion by casts. ^{Reference Willems78} A case from World War I involved a patient with a 1-hour SM exposure who presented with vomiting and skin erythema within hours and eventually developed oral diphtheric necrosis. On day 7, the patient was in significant respiratory distress, and on day 11, the patient died. An autopsy revealed the larynx contained a large membrane of fibrin cast and smaller bronchi and bronchioles dilated and filled with fibrin and red cells. ^{Reference Martin and Weller107}

A model of airway thrombosis from SM inhalation was developed in male Sprague-Dawley rats in which SM vapor was administered, with multiple endpoints evaluated. The highest dose of SM (4.0 mg/ kg) had a 20% survival rate, and the lowest dose (3.7 mg/ kg) had a 62.5% survival rate. In the first few hours after exposure, there was no decrease in oxygen saturation; however, by hour 12, there was a dose-dependent drop. Rats developed fibrin-rich airway casts at 12 to 24 hours. The development of airway casts was dose-dependent at 12 hours, with the lower lobes developing the most severe obstruction across all doses. Notably, at the highest dose, the trachea began to develop significant obstruction. Overall, cast obstruction was dose-dependent, with higher doses leading to higher scores.Footnote ³⁶ A female Yorkshire swine model was also developed due to their similar lung branching patterns to humans. This model allowed for the use of bronchoscopy to monitor real-time obstruction and used an intubated, anesthetized system, and similar endpoints to the rat model; study researchers developed a novel scoring system that allows for consideration of multiple endpoints. The score correlates with a dose-dependent decrease in oxygen saturation, with high doses producing both a high decrease and score and low doses producing both a low decrease score. There is hope that this scoring system can also be used for trigger-to-treat and early euthanasia criteria.Footnote ³⁷ In the rat model, intravascular thrombosis formed in the smaller vessels, indicating endothelial activation. In both models, D-dimer and thrombin-antithrombin complexes were elevated. In summary, airway thrombosis/ airway fibrin casts can occur in various lung injury states, can cause significant respiratory compromise, and can be life-threatening. SM exposure causes acute mortality due to airway fibrin cast obstruction in humans and animals. To mimic a human response, rat and swine models have been developed for SM inhalation. The swine model specifically is ready for the development of rescue drugs, with appropriate human-relevant endpoints including bronchoscopy, monitoring, and serial biomarkers.

Radiation and Pulmonary Vascular Dysfunction: mechanisms and consequences (S Chatterjee)

Any radiation involving the thorax triggered inflammation in the lung tissue. This leads to radiation pneumonitis/ fibrosis that alters pulmonary function. Radiation-induced inflammation collapses alveoli and obliterates the pulmonary alveolar-vascular interface by the growth of connective tissue, interfering with the facilitation of gas exchanges done by the interface. The endothelial layer acts as the converging site for inflammation, meaning an activated endothelium is a prerequisite for inflammation. Neutrophils and other immune cells in the blood will only attach to endothelial cells when the endothelial layer is activated and produces cellular adhesion molecules and selectins. The average adult human has over 1000 m ^{Reference Yeung, Harper and Platoff2} of endothelial surface, ^{Reference Feletou108} meaning that even a minor activation of the endothelium will have major ramifications. Radiation has several immediate effects on the human body, including DNA damage, protein modification, and lipid peroxidation, as well as cell death. It also has delayed effects, including a chronic inflammatory response. Studies were undertaken to determine whether low dose irradiation (< 1 Gy) would amplify inflammation and drive a pro-oxidant/ pro-inflammatory environment. One protocol looked at the intercellular adhesion molecule (ICAM-1). Under normal circumstances, ICAM-1 expression is low on the surface of endothelial cells. In response to a stimulus, these cells are activated, and ICAM-1 expression increases, facilitating the sticking of ICAM-1 molecule to the endothelial layer. ^{Reference Patel, Colangelo and Reiner109}

Another molecule studied was NLRP3 inflammasome that in healthy cells shows low expression. The NLRP3 inflammasome can be activated by oxidative stress, leading to caspase 1 activation, IL-1 β formation, and cell death by pyroptosis. It is now considered an amplification factor, leading to a cascade of inflammation once expressed. Two models were created to study these effects. One was a reductionist approach using a flow chamber to recreate endothelium as it would be in vivo experiencing radiation. The other model used an integrative approach, using human, precision-cut lung slices, and allowing the organ to be studied in its native architecture. A dose-dependent increase of both ICAM-1 and NLRP3 expression was induced by radiation over 24 hours post-exposure, which continued beyond 24 hours. ^{Reference Chatterjee, Pietrofesa and Park110} Notably, NLRP3 expression at even low levels of radiation stimuli was comparable to the NLRP3 expression at high levels of pathological stimuli. These studies show that even low doses of radiation can increase inflammation and suggests a long-term effect on the endothelium. More studies are being done to understand the conditions under which these inflammation signals are retained for 72 hours and beyond, as well as examine the presence of oxidative stress markers.

Oxidative stress: an intersection between radiation and SM lung injury (B Day)

SM and 2-chloroethyl ethyl sulfide (CEES) are alkylating, cross-linking and blistering agents, that can cause serious, and widespread injury to lungs (airway obstruction, edema, and hemorrhaging), skin (blistering and inflammation) and eyes (blindness). AEOL-10150 is a catalytic antioxidant meso-porphyrin with a combination of superoxide dismutase (SOD) and catalase activities that has been shown to diminish SM and CEES-induced lung injury. ^{Reference McClintock, Till and Smith111} In a rat model, anesthetized animals were nose-cone exposed CEES for 15 minutes, and AEOL-10150 was administered subcutaneously starting 1 hour after. ^{Reference O’Neill, White and Veress112} Rats exposed to CEES had elevated markers of lung edema and oxidative stress attenuated by the antioxidant. Similarly, in a SM model where rats were anesthetized, intubated, and exposed to SM, AEOL-10150 given after SM exposure increased survival at 48 hours (36% for SM alone, 73-88% survival with AEOL-10150). ^{Reference McElroy, Min and Huang113} Lung airway obstructions created by the SM were also reduced in the AEOL-10150 treated rats, and the product mitigated SM-induced lung oxidative stress.

Similarly, laboratory models to study radiation injury included a hemithorax rat exposure and a fractionated radiation-induced lung injury. ^{Reference Rabbani, Salahuddin and Yarmolenko114} In the hemithorax model, rats were exposed to a single dose (28 Gy) to the right hemithorax, and AEOL-10150 was administered with a mini-osmotic pump 1 day after exposure for 10 weeks at doses of 1, 10, and 30 mg/ kg/ day. ^{Reference Rabbani, Batinic-Haberle and Anscher115} AEOL-10150 mitigated radiation-induced lung injury and fibrosis as well as radiation-induced lung oxidative stress. In the fractionated irradiation model, daily fractions of 8 Gy were administered to the right hemithorax for 5 consecutive days (40 Gy total) to adult female Fisher-344 rats. In the treatment arm, AEOL10150 was administered subcutaneously 15 minutes prior to irradiation, and continued for 30 days post-irradiation. AEOL-10150 mitigated radiation-induced lung injury, radiation-induced lung fibrosis, and radiation-induced lung oxidative stress. These data suggest common oxidative stress mechanisms in many threat agents and indicate antioxidants could be developed as a MCM to combat these lung toxidromes.

Recapitulation of human pulmonary response in small and large animal models of DEARE-Lung After TBI with bone marrow sparing (L Jackson)

The lung is a late responding tissue, which upon irradiation manifests clinical signs and symptoms months and years post-exposure. The early period following irradiation is marked by tremendous activity that finally culminates in the clinical manifestation. RILI manifests as 2 specific sequelae, pneumonitis, and fibrosis. In the clinic, radiation pneumonitis occurs 1- 6 months after exposure and is marked by alveolar wall thickening, edema, and inflammation, with persistent cough and/ or lung failure. Radiation fibrosis develops after months to years with collagen deposition, scarring, and lung retraction leading to inefficient gas exchange and respiratory distress. ^{Reference Kong, Ten Haken and Eisbruch116} The ability to dissociate these 2 pathologies suggests that they are not inextricably linked. These conditions are recapitulated in various mouse models of RILI, when MCMs are shown to mitigate fibrosis without impacting the pneumonitis phase and vice versa. ^{Reference Dabjan, Buck and Jackson117} Development of MCMs under the AR requires researchers to link the pathophysiological outcome of small and large animal models with clinical manifestation of the disease, and establish that the outcome in animal models is sufficiently representative of the anticipated responses in the human population. The goal is to develop and validate models that link in temporal onset, dose response, and pathology to non- NHPs, and humans.

Several species have been used to model RILI (e.g., rodents, ^{Reference Jackson, Vujaskovic and Down118} hamsters, ^{Reference Breuer, Tochner and Conner119} and rabbits, ^{Reference Rubin, Finkelstein and Siemann120} as well as pigs, ^{Reference Hopewell, Rezvani and Moustafa121} dogs, ^{Reference Poulson, Vujaskovic and Gillette122} NHP, ^{Reference MacVittie, Farese and Parker123} etc.) demonstrating that animal models need to closely resemble the human lung response to radiation, pathogenesis, and relevant clinical endpoints. Typically, survival is the primary endpoint, ^{Reference Van Dyk, Keane and Kan124} while organ function, and imaging, as well as histopathology serve as secondary endpoints for major morbidities. Primary study outcome and secondary endpoints are considered to determine if the strain is appropriate as a model. Even among the mouse strains, development of RILI variation is documented for survival times, lung weights, and airway congestion. ^{Reference Jackson, Vujaskovic and Down118} For instance, C57L/J, CBA/J, and C3H/HeJ strains are characterized to be pneumonitis prone; C57BL/6 strain is fibrosis prone, while the BALB/6 strain is both pneumonitis prone, and has a DNA repair defect. ^{Reference Jackson, Vujaskovic and Down118} Using 6 strains and radiological imaging to look at lung damage, pathobiology of RILI following WTLI demonstrated significant variations based on survival, lung weights, and breathing frequencies well as lung density, and volume. ^{Reference Jackson, Vujaskovic and Down125} From this work and given the threshold dose, 95% incidence dose and temporal onset, ^{Reference Jackson, Vujaskovic and Down118} the use of C57L/J strain is recommended to best translate murine findings to higher species like NHPs, and humans. ^{Reference Jackson, Xu and Nguyen126,Reference Garofalo, Bennett and Farese127}

Based on FDA recommendations, there has been a shift in preferred models from WTLI to PBI (5% bone marrow sparing), to recapitulate a multiorgan impact and administration of growth factors more accurately on the efficacy of lung-MCMs in NHPs and C57L/J for RILI. Medical management/ supportive care was scaled from the NHP model to the rodent model; this was determined in coordination with NIAID, the industry partner, and FDA. One major difference in the supportive care regimen is that dexamethasone is not preferred for the mouse, although it is often used in the NHP as a trigger to treat modality (L Jackson). In summary, the mouse model, with certain preferred strains and exposure geometries, continues to be a good simulation of many aspects of RILI seen in larger animals and humans, and thus, remains an option for preclinical development on lung MCMs for radiation injuries.

Pivotal, GLP non-inferiority study to evaluate Silverlon dressings against superficial and moderate, partial-thickness SM Vapor wounds in Göttingen minipigs (C Croutch)

Silverlon obtained a 510(k) FDA indication for a burn dressing product in 2013. Since 2003, Silverlon has been widely used by the US Army in combat settings in Afghanistan and Iran. ^{Reference Cancio, Horvath and Barillo128,Reference Barillo, Cancio and Stack129} Silverlon was tested serendipitously on a patient with a SM wound following sulfamylon debridement, and in the 8 weeks post-treatment, the wound healed. From this success, pilot studies were created to develop a model for SM burns in hairless guinea pigs (HGP) and Göttingen minipigs. ^{Reference Barillo, Croutch and Reid130} SM wounds were created – a superficial and a moderate dermal wound depending on the length of exposure – and animals were observed for 48 hours. Endpoints included histopathology, modified Draize scores, TEWL, and ultrasound, as well as digital images of wounds, and colorimetry, among other endpoints. To achieve consistent results, a histopathology scoring matrix was established, and a method was developed so that bandages remained moist, and in place during healing; debridement was standardized with 2 methods (laser and sulfamylon). Based on these data, FDA recommended that only the minipig model was needed for GLP studies, as it is more robust and produces more consistent results than the hairless guinea pig. Histopathological scores informed group sizes for GLP study, and these scores were the primary endpoint. The FDA also recommended a ‘non-inferiority’ study rather than an efficacy model and preferred a saline wet-to-wet debridement instead of those originally proposed. The GLP objective that was decided through consultation with the FDA was to assess non-inferiority of Silverlon when compared to gauze with 1% silver sulfadiazine (SSD) for the treatment of SM-induced dermal lesions. The primary endpoint was the histopathology composite score of dermal wounds obtained at the end of the study. Study outcomes showed that Silverlon outperformed SSD in every treatment arm and more wounds healed with the Silverlon bandages. Therefore, Silverlon was found to not be inferior to the SSD treatment, and was in fact better, leading to an FDA approval of the device in 2019.Footnote ³⁸

Based off the success from these studies, recommendations to implement during the development process are: (1) include a strong regulatory group as part of the team, (2) engage in frequent and clear regulatory communications, (3) refine the product label early in development, (4) include a clinician with experience in human injury and working knowledge of the animal model, (5) select animal models and study designs that reflect the human injury, and (6) generate as much preliminary data as possible, to help the pivotal GLP study succeed.

Dealing with characteristics outside of your control: genetics to animal behavior (M Doyle-Eisele)

Outside of the inherent challenges that arise from using animal models, there are other challenges that overlap across institutes and organizations, specifically overarching issues such as regulatory, and test system, as well as clinical translation, assays, and supportive care challenges. Starting with regulatory issues; a contract research organization, such as Lovelace Biomedical Research Institute, conducts a large variety of studies that are highly regulated. While regulatory challenges are typically discussed in terms of what is needed for submission, other challenges exist. For instance, occupational safety (safety of facility, technical staff, and animals a CRO works with) can lead to procedural or protocol changes and this can differ by facility. In addition, the number of study animals based off statistical analyses may be unrealistic. Also, utilization of purpose bred versus wild-caught animals can result in varying outcomes; restrictions on specific species, despite being the best model that characterizes the human condition, can also make study implementation challenging. Another potential hurdle is facility-specific Institutional Animal Care and Use Committee’s (IACUC) requirements that add a distinct layer of complication by changes in policy that can impact anything from a simplistic housing requirement to complex supportive care modalities.

Test system challenges can arise due to issues with animal availability, animal species consideration, and logistical issues such as shipping delays. For example, in 2013 there were considerable delays in Göttingen minipig research due to increased toxicology and radiation studies, confounded by decreased breeding across the US. In another instance, a shift from rhesus to cynomolgus macaques for MCM testing for radiation indications has been necessitated due to the severe scarcity of the rhesus monkeys, but it is unclear what this will mean for current work and approved models; particularly because variations in species (physical or genetic variability) can result in unexpected and non-reproducible results. ^{Reference Farese, Drouet and Herodin131}

While the objective of clinical development is to achieve reproducible and biologically relevant data, the elements that go into this (test system, challenge material, test article, and endpoints) can have variability, which creates unanticipated challenges during development. ^{Reference Seyhan132} Clinical translation challenges include: (1) manifestation of injury cannot always be directly monitored in animals, (2) endpoints are not always practical, (3) animals cannot comply with dosing instructions, and (4) inadequate infrastructure and trained workforces. Assay challenges include pivotal immunological endpoints that are missing or unavailable, lack of availability of assay reagents, and batch-to-batch variability. When comparing human to animal endpoints that are critical for clinical translation, there is a gap between expectations and what is feasible and practical.

Supportive care challenges include occupational safety for staff, placebo treatments (if no standard of care is approved), IACUCs mandating analgesia, and monitoring protocols that can alter outcomes, as well as standards of care that are not readily available to research institutes because of clinical need. The ultimate goals of pre-clinical studies are to accurately model the desired biological effect of a drug in animals, to predict treatment outcomes in patients, and characterize all toxicities associated with a drug to predict adverse events in humans. Therefore, animal models must be carefully designed to mimic what happens in humans while balancing feasibility.

Advances in translational large animal lung injury SM models for mass casualty (V Bebarta)

One of the primary approaches to responding to any chemical injury is to focus on the toxidrome. Toxidrome is defined as the syndromic effect of a toxin, and a threat agnostic approach is to treat the injury, not the threat. ^{Reference Bebarta133} Given that scenario, the current research focus is on oxidative stress and how it impacts mammals (largely pigs), as assessed by lung inflammatory cell accumulation, and increased TNF-α, as well as other pro-inflammatory cytokines, lung matrix metalloproteinases, and DNA damage. Several animal species have been studied in SM research: large animals include dogs, sheep, NHPs, and pigs. Among these, swine is the best model to use because of the similarities to the human condition with regard to drug dosing, anatomy, and physiology (cardiac, pulmonary, and immunology). Furthermore, their clinical similarities to humans, ^{Reference Fairhall, Jugg and Read134} including a similar airway size, allows for use of the same devices as those used clinically, and the ability to perform serial blood draws/ large tissue samples. Currently, swine are used in other chemical MCM development, such as against SM pulmonary, and eye/cutaneous injuries. They are generally accepted by the FDA for these indications. One main drawback to the model is that swine are hypercoagulable compared to humans. ^{Reference Bebarta, Garrett and Boudreau135}

An effective animal model must consider the preexposure, exposure, and post-exposure phases. In the pre-exposure phase, several factors are important such as handler expertise, animal weight, and subspecies, as well as vendor, prep work conducted, and anesthesia. Other factors include air/ oxygen used, and ventilation. During exposure, it is important to consider dose variability, total dose in bag/ plenum, and duration of exposure, as well as ventilation. In the post-exposure phase, it is important to consider off-gassing, recovery of the animal, and husbandry, as well as monitoring. Key outcomes measured include PK and general toxicology; clinical, behavior, and physiological; airway cast; system inflammation, blood cell counts, lung edema, and injury markers, as well as survival. Euthanasia criteria are crucial for rigor and reproducibility. Scenarios for real chemical responses have involved consideration of SM mass causality events for the Colorado/ Mountain plains region.Footnote ³⁹ In the event of SM exposure, most patients will present with mild symptoms, although some may have severe concerns. Early decontamination is critical - supportive care should work for most patients in acute care; however, trauma evaluations are also needed. Field drugs need to be used within 1 hour post-exposure to be effective, and developers need to keep in mind the importance of untrained personnel being able to deploy/ use the developed drugs.