Clinical Manifestations of Sulfur Mustard -Induced Lung Disease

Sulfur mustard gas was first introduced on the battlefields of Belgium during World War I. ^{Reference Darchini-Maragheh and Balali-Mood4} It is estimated that 400 000 of the 1.2 million soldiers exposed required long-term medical care. Widespread use in battle, including arial bombs, were used by Spain and France against the Berbers during the Rif war in the 1920s, and by Italy against the Ethiopians during the Italo-Ethiopian War from 1935 - 1936. ^{Reference Grip and Hart8} In more recent years, sulfur mustard was deployed by the Iraqi military against the Iranian military and civilians, as well as the Iraqi Kurdish minority, during the Iran-Iraq War from 1983 - 1988. ^{Reference Darchini-Maragheh and Balali-Mood4} Sulfur mustard was also used against civilians during the Syrian Civil War from 2013 to 2018. ^{Reference Rimpler-Schmid, Trapp and Leonard1} Although sulfur mustard exposure is acutely fatal in less than 5% of cases, long-term complications associated with immune dysregulation can cause life-threatening illness and debilitating disease among survivors. ^{Reference Balali-Mood and Hefazi9}

The temporal onset and severity of symptoms after sulfur mustard exposure are dependent on the route, total dose, duration of exposure, and proximity to the source, as well as environmental conditions, and usage of personal protective equipment. ^{Reference Malaviya, Abramova and Rancourt10} Exposure may occur through ingestion (e.g., drinking or swimming in contaminated water, eating contaminated food, etc.) and through skin or eye contact and inhalation when sulfur mustard is released as a vapor. The most common acute complications associated with exposure are observed in the eyes, skin, lung, and bone marrow. Heavily exposed patients may also present with gastrointestinal (e.g., nausea, vomiting) and neurological symptoms. Long term immune insufficiency is considered a major cause of increased incidence of infection and cancers in survivors. ^{Reference Darchini-Maragheh and Balali-Mood4}

The most common life-threatening complication of sulfur mustard inhalation is upper respiratory and lower respiratory injury. ^{Reference Darchini-Maragheh and Balali-Mood4} Iranian physicians have extensively characterized molecular, functional, radiographic, and histopathologic sequelae accompanying acute and delayed lung injury among civilians and military exposed during the Iran-Iraq War. ^{Reference Darchini-Maragheh and Balali-Mood4} Briefly, the severity of respiratory effects is dose-dependent and damage may extend from the nasal mucosa to the terminal bronchioles. ^{Reference Balali-Mood and Hefazi9} Upon inhalation, sulfur mustard reacts rapidly with the cells of the upper respiratory tract. As the particles traverse the upper airways, there is dilution due to filtration as the vapor reaches the bronchioles and alveoli. ^{Reference Aliannejad11}

Heavy sulfur mustard exposure in chemical warfare is exceedingly rare. In these cases, mortality due to acute lung injury occurs rapidly, within days. Clinical experience with casualties from Bari Harbor during World War II and Iranian casualties from the Iraq-Iran War indicates acute respiratory symptoms may occur within 4 to 48 hours. ^{Reference Balali-Mood and Hefazi9,Reference Alexander12} Symptoms included productive cough, sore throat, laryngitis, and head congestion; which abate rapidly in most cases. In the severely ill or terminal, symptoms progress to frank hoarseness and aphonia, lower respiratory tract infection, dyspnea, and cyanosis. ^{Reference Balali-Mood and Hefazi9,Reference Alexander12} In those patients with lower respiratory tract infection, death occurs rapidly. ^{Reference Alexander12} These early mortalities are associated with multi-organ failure and acute respiratory distress syndrome (ARDS). ^{Reference Balali-Mood and Hefazi9} Autopsy findings reveal congestion and inflammation, moderate to marked edema, ulceration, and epithelial denudation, as well as small, focal hemorrhages (Figure 1). Cast formation, due to epithelial denudation and fibrin, is observed in the lower trachea with extension into both bifurcations and bronchial ramifications. ^{Reference Alexander12}

Figure 1. Shared pathophysiological mechanisms underlying the development of sulfur mustard (HD) and radiation-induced acute and late lung injuries. Development of threat agnostic, host-directed therapeutics targeting maladaptive systems biology responses to mitigate, and/ or treat acute lung injury, and delayed pulmonary complications of sulfur mustard, radiological, and/ or nuclear exposures can be pursued under the FDA Animal Rule regulatory pathway. This illustration was created with BioRender.com.

Symptomatic and pathological features of sulfur mustard-induced respiratory illness in long-term survivors is consistent with bronchiolitis obliterans and pulmonary fibrosis. ^{Reference Darchini-Maragheh and Balali-Mood4} The progressive narrowing and scarring of the bronchioles lead to respiratory insufficiency and debilitating diseases that may become life-threatening. In a study by Emad and Rezaian, the primary respiratory complications observed in Iranian combat veterans 10- years after exposure were chronic bronchitis, pulmonary fibrosis, and large airway narrowing, as well as asthma, and bronchiectasis. ^{Reference Emad and Rezaian13} Chronic obstructive pulmonary disease (COPD) was the primary complication observed 16 to 20 years after exposure followed by bronchiectasis, asthma, large airway narrowing, and pulmonary fibrosis. ^{Reference Hefazi, Attaran and Mahmoudi14}

Clinical Manifestations of Radiation-Induced Lung Disease

Clinically symptomatic pulmonary complications following acute total or partial body irradiation occur between the first and sixth month after exposure depending on the overall dose. Lung disease, characterized by acute pneumonitis between 1 to 6 months after exposure and chronic fibrosis months to years later, occur primarily in patients surviving ARS, or alternatively, patients who received the highest doses of radiation to the upper half body.

Total body irradiation (TBI)- induced ARS is characterized by a dose-dependent increase in severity of myelosuppression and gastrointestinal (GI) injury leading to an increased risk for infection, hemorrhage, and death within the first 4 to 6 weeks after exposure. Radiation-induced myelosuppression and GI injury mirror has been reported in sulfur mustard exposed individuals. Four MCMs in the myeloid colony stimulating factor (CSF) class of drugs have been approved by the FDA under the non-traditional Animal Rule (21 CFR 314.600-650 for drugs; 21 CFR 601.90-95 for biologics; effective July 1, 2002) regulatory pathway to treat myelosuppression associated with ARS in adults and children. Empirical observations in humans and controlled studies in animal models demonstrate survival from ARS is possible with minimal to advanced care and cytokine therapies in a medical setting (e.g., CSF, antibiotics). However, ARS survivors are most notably at risk for developing DEARE, debilitating, and/ or life-threatening lung damage within weeks to months after exposure. ^{Reference Baranov, Selidovkin and Butturini15}

DEARE-lung is characterized by acute, life-threatening pneumonitis or alveolitis occurring approximately 1 to 6 months post-exposure to radiation doses of 7.5 Gy or higher, and late fibrosis that may develop several months post-exposure. Fibrosis may progress up to 2 years post-exposure after which it generally stabilizes in the lung, although continued lung volume retraction may be observed up to 5 years after exposure. ^{Reference Boerma, Davis and Jackson16} Clinical signs and symptoms of radiation pneumonitis and/ or fibrosis include abnormal pulmonary function tests (e.g., DLCO, diffusing capacity for carbon monoxide; %FEV, percent change in forced expiratory volume), radiographic changes (e.g., ground glass opacification and increased lung density), and microscopic changes (e.g., increased alveolar wall thickness, mononuclear cell infiltration, proteinaceous alveolar, and interstitial edema, as well as collagen deposition and honey-comb scarring).

It is not uncommon for chronic lung fibrosis to present in the absence of acute pneumonitis, and the ability to dissociate fibrosis from pneumonitis through pharmacological intervention suggests these are 2 separate disease conditions. ^{Reference Movsas, Raffin and Epstein17,Reference Dabjan, Buck and Jackson18} As such, therapeutically targeting the mechanism of action to improve survival from radiation pneumonitis may require a different approach than mitigation and/ or prevention of debilitating lung injury arising from chronic, progressive fibrosis.

As at the time of writing this paper, there are no FDA approved MCMs to prevent, mitigate, and/ or treat sulfur mustard or radiation-induced lung injury. The lack of MCMs poses a major unmet medical need for which several academic researchers, early-stage biotech, and clinical-stage pharmaceutical companies are seeking to address through the development of novel therapeutics and repurposing (i.e., label expansion) of commercially available products.

Regulatory Considerations for Development of Early to Late-Stage Therapeutics as MCM for Sulfur-Mustard and Radiation-Induced Lung Disease

In the United States, MCMs may be approved by the FDA under a non-traditional regulatory pathway known as the Animal Rule (“AR”). The pathway permits licensure of MCMs when it is neither ethical nor feasible to conduct clinical trials in humans. Under the AR, MCMs may be approved based on demonstrated efficacy in adequate and well-controlled studies: (1) with appropriate randomization and blinding, (2) in 1 or more animal models that recapitulate the disease in humans, (3) with robust safety data in humans, and (4) sufficient pharmacokinetic (PK)/ pharmacodynamic (PD) data to allow for adequate therapeutic dose selection in humans. When these criteria are satisfactorily met, regulators reasonably assume demonstrated efficacy in animal models will translate to clinically meaningful benefit in humans.

To support product approval under the AR, therapeutic efficacy studies are run in trials like Phase 1 – 3 trials conducted in humans, but in surrogate animal models. Successfully meeting the criteria for approval can be arduous and time consuming with an average development time of 5-7 years for therapeutics with prior commercial approvals, and even longer for novel therapeutics not previously approved by any regulatory agency for any indication (Figure 2).

Figure 2. High-level overview of the medical countermeasure development process under the FDA Animal Rule regulatory pathway. Efficacy data in animal models demonstrating a significant improvement in mortality and/ or major morbidity as the primary endpoint must be supported by a robust database of safety in humans and sufficient PK/ PD data through PK - PD modeling and simulations to justify therapeutic dose selection in humans. This illustration was created with BioRender.com.

Generally, early development work to identify appropriate dose/ dose-schedule selection and elucidate the therapeutic mechanism of action against the disease caused by the challenge agent (e.g., chemicals, radiation) is performed in small animal models such as the mouse, rat, guinea pig, or rabbit. Model selection is based on the desired clinically meaningful endpoint for efficacy, usually major morbidity (e.g., debilitating lung disease) or mortality. Advanced development of chemical and radiation countermeasures are generally performed in swine or non-human primates (NHP). ^{Reference Fairhall, Jugg and Read19–Reference Weber25} PK/ PD studies are conducted under Good Laboratory Practice (GLP) regulations using validated assays in healthy and challenged animals to establish the best dose and schedule for proof-of-concept efficacy evaluation in subsequent Phase 2, randomized, blinded, and placebo-controlled trials. Finally, the pivotal or registrational study is conducted in an adequate and well-controlled model to confirm the earlier evidence that a MCM is safe and effective. Approved MCMs for treatment of myelosuppression associated with ARS such as Neupogen®, Neulasta®, Nplate®, and Leukine® are considered current standard of care. Therefore, drug developers should include 1 of the approved MCMs in the registrational study design.

Animal Models of Sulfur-Mustard Induced Lung Disease

It would be highly unethical, and unfeasible, to evaluate MCM efficacy in humans due to the risk for debilitating morbidity, and mortality associated with chemical and radiological/ nuclear exposures. Therefore, it is intended that efficacy evaluation of MCMs is conducted in nonclinical trials in animal models in lieu of clinical trial in humans. As animal models serve as a human surrogate in these trials, the pathophysiological mechanism and natural history of disease progression and clinical manifestation must mirror that observed in humans. Registrational studies are required to be adequate and well-controlled, with appropriate randomization and blinding, and sufficiently powered to assess a clinically meaningful benefit against the primary endpoint (e.g., mortality/ morbidity).

The most common animal models used to interrogate the effect of chemical agents and/ or radiation on the lungs and screen promising MCM candidates for therapeutic efficacy are the mouse, rat, rabbit, and minipig, as well as swine (farm pig), canine, and NHP. Species/ strain and model selection is dependent on disease specificity and primary efficacy endpoint for evaluation. The rat, guinea pig, swine, and NHP are the species of choice for evaluation of MCMs to mitigate and/ or treat lung damage caused by sulfur mustard vapor exposure. Sulfur mustard is a highly reactive vesicating agent. Studies have shown spontaneously breathing swine exposed to sulfur mustard vapor in air at a dose of 100 - 150 mg/kg develop hypoxic lung injury with respiratory acidosis and necrosis and erosion of the tracheal epithelium within 6 hours post-challenge. ^{Reference Fairhall, Jugg and Read19} To avoid acute morbidity/ mortality resulting from nasopharynx toxicity (e.g., edema and necrosis), sulfur mustard is delivered via intratracheal installation into the lower respiratory tract to induce acute lung injury in the experimental setting. This artifact of the animal model impacts the ability to assess MCM efficacy against sulfur mustard-induced lung injury commonly observed in humans. Intratracheal administration allows deposition into the lower respiratory tract and avoids this common laboratory pitfall.

A sulfur mustard exposure system for delivering controlled/ accurate, and quantifiable concentrations to the lower respiratory tract of the male Sprague Dawley rat was developed by Perry, Yeung, and colleagues. ^{Reference Perry, Neal and Hawks26} In the Perry study, animals (n = 4 – 5/ group) were exposed to sulfur mustard vapor at a target dose of 0.30 mg/kg to 3.20 mg/kg. Study endpoints included overall survival on day 28, clinical exam, pulmonary function tests, and histopathology. Mortality (28- day) was observed at doses of 0.45 mg/kg or higher. In the Perry study, there were 2 waves of major morbidity/ mortality – an acute phase within the first 3 days post-challenge characterized by ALI (e.g., alveolar edema, alveolar epithelial necrosis, and alveolar hemorrhage, etc.), and a late phase approximately 22 - 28 days post-challenge, generally characterized by alveolar, septal/ interstitial, and pleural fibrosis more widespread than observed at earlier time points. ^{Reference Perry, Neal and Hawks26} The Probit-estimated lethal dose for 50% of animals within the first 29 days was 0.80 mg/kg (95% confidence interval: 0.42, 1.18). Features of acute respiratory illness following inhalational exposure in the rodent model include a decrease in lung compliance and increase in resistance, fibrin cast formation within the bronchial tree, epithelial necrosis, and apoptotic cell death, as well as hemorrhage and necrosis of lymphoid tissue. ^{Reference Perry, Neal and Hawks26}

Mortality and clinical pathology in Perry’s model were consistent with that reported by Malaviya. ^{Reference Malaviya, Abramova and Rancourt10} Microscopic exam of acute lung damage after sulfur mustard vapor inhalation revealed ulceration of proximal bronchioles and perivascular and peribronchiolar edema and proteinaceous alveolar exudate with entrapped inflammatory cells. ^{Reference Malaviya, Abramova and Rancourt10} Limited studies have extended the follow-up time beyond 28 days in experimental models. Mishra et al. demonstrated sulfur mustard inhalation in Fisher 344 (F344) rats and cynomolgus macaques (i.e., NHP) induced acute and chronic inflammation, similar to that observed in humans. ^{Reference Aghanouri, Ghanei and Aslani27–Reference Mishra, Rir-sima-ah and Grotendorst30} Mishra followed cynomolgus macaques up to 60 days post- sulfur mustard inhalation. Those studies revealed an increase in Interleukin-17 (IL-17) positive cells in the areas of lung inflammation and fibrosis 60 days after sulfur mustard exposure. ^{Reference Mishra, Rir-sima-ah and Grotendorst30}

Swine are another common model for interrogating the effects of sulfur-mustard on the lower respiratory tract and MCM evaluation. The swine model presents several advantages to the rodent model for sulfur-mustard induced lung toxicity. In the swine, physiological measurements are nearly identical to that in humans. The temporal progression to disease development in the swine model is more protracted compared to the rat, and cardiopulmonary and respiratory endpoints can be non-invasively evaluated longitudinally to monitor the therapeutic effect of candidate MCMs on clinical progression of disease. However, in this model, there are natural variations in cardiopulmonary output, which should be taken into consideration during data evaluation.

Animal Models of Radiation-Induced Lung Disease

For radiation-induced lung injury, the most common experimental models are the rodent (e.g., mouse, rat) and the NHP. Species and strain differences in pulmonary response to radiation are well-characterized. ^{Reference Dabjan, Buck and Jackson18} The most widely accepted small animal species/ strains for MCM development are the C57L/J mouse (i.e., leaden coat color, The Jackson Laboratory) and the WAG/ RijCmcr rat, a strain proprietary to the Medical College of Wisconsin. ^{Reference DiCarlo, Jackson and Shah31–Reference Satyamitra, Cassatt and Marzella33} For advanced research and development, including registrational studies, the Chinese-bred rhesus macaque has generally been considered the most suitable species as it most closely resembles the anticipated pulmonary response in humans. ^{Reference MacVittie, Farese and Parker34} However, the 2020 global outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes Coronavirus Disease 2019 (COVID-19) led to a severe reduction in the availability of NHP due to China’s ban on wildlife exports, and redirection of animals to vaccine and therapeutic research. ³⁵ As a result, the development of a cynomolgus macaque model of radiation-induced lung injury has become a national priority to advance product development programs and is underway at this time.

Federal agencies, including the FDA, have weighed in on the most suitable radiation geometry for induction of DEARE in the lung. While several models have been used including whole thorax lung irradiation and total body irradiation with a “top-off” dose, the most widely accepted model is partial body irradiation with 2.5% - 5% bone marrow sparing in mice and the NHP, respectively. ^{Reference Satyamitra, Cassatt and Marzella33} This model more closely mirrors the human scenario in which animals progressing through ARS develop DEARE-associated multiorgan dysfunction/ failure weeks to months after exposure. Thus, these models allow efficacy evaluation in a disease course most likely observed in humans and in the context of evolving multi-organ injury. One challenge, however, is the high rate of lethality from ARS in these models. As a result, studies must be sufficiently powered to evaluate all-cause mortality and/ or major morbidity at the study endpoint, generally 180 days post-exposure in mice and NHP. Some studies extend follow-up beyond 180-days (e.g., 220 days) to evaluate the durability of response.

In both the rodent and NHP models, the primary clinically relevant endpoint has traditionally been overall survival. ^{Reference MacVittie, Farese and Parker34,Reference MacVittie, Farese and Parker36} In the NHP, secondary endpoints include respiratory function (e.g., non-sedated respiratory rate), radiographic imaging (e.g., computed tomography), dexamethasone courses, and microscopic abnormalities on hematoxylin and eosin (H&E)- stained tissue sections and semi-quantitative analysis of lung fibrosis (e.g., Masson’s trichrome-stained tissue sections). ^{Reference MacVittie, Farese and Parker34,Reference MacVittie, Farese and Parker36} Exploratory endpoints often include onset, duration, recovery from Grade 3 and Grade 4 neutropenia, and thrombocytopenia, as well as severe acute anemia, clinical chemistry (e.g., renal function), coagulopathy (e.g., fibrinogen, D-dimer), and histological abnormalities in other organs (e.g., gastrointestinal tract, spleen, kidneys, liver). ^{Reference MacVittie, Farese and Parker34,Reference MacVittie, Farese and Parker36}