Toxicology Mechanisms and Methods
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Phosgene: toxicology, animal models, and medical
countermeasures
Stephen T. Hobson, Richard A. Richieri & Missag H. Parseghian
To cite this article: Stephen T. Hobson, Richard A. Richieri & Missag H. Parseghian (2021)
Phosgene: toxicology, animal models, and medical countermeasures, Toxicology Mechanisms
and Methods, 31:4, 293-307, DOI: 10.1080/15376516.2021.1885544
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TOXICOLOGY MECHANISMS AND METHODS
2021, VOL. 31, NO. 4, 293–307
https://doi.org/10.1080/15376516.2021.1885544
REVIEW ARTICLE
Phosgene: toxicology, animal models, and medical countermeasures
Stephen T. Hobsona,b
, Richard A. Richierib and Missag H. Parseghianb
a
Department of Biology and Chemistry, Liberty University, Lynchburg, VA, USA; bRubicon Biotechnology, Irvine, CA, USA
ABSTRACT
ARTICLE HISTORY
Phosgene is a gas crucial to industrial chemical processes with widespread production (1 million
tons/year in the USA, 8.5 million tons/year worldwide). Phosgene’s high toxicity and physical properties resulted in its use as a chemical warfare agent during the First World War with a designation of
CG (‘Choky Gas’). The industrial availability of phosgene makes it a compound of concern as a weapon
of mass destruction by terrorist organizations. The hydrophobicity of phosgene exacerbates its toxicity
often resulting in a delayed toxidrome as the upper airways are moderately irritated; by the time
symptoms appear, significant damage has occurred. As the standard of care for phosgene intoxication
is supportive therapy, a pressing need for effective therapeutics and treatment regimens exists.
Proposed toxicity mechanisms for phosgene based on human and animal exposures are discussed.
Whereas intermediary components in the phosgene intoxication pathways are under continued discussion, generation of reactive oxygen species and oxidative stress is a common factor. As animal models
are required for the study of phosgene and for FDA approval via the Animal Rule; the status of existing models and their adherence to Haber’s Rule is discussed. Finally, we review the continued search
for efficacious therapeutics for phosgene intoxication; and present a rapid post-exposure response
that places exogenous human heat shock protein 72, in the form of a cell-penetrating fusion protein
(Fv-HSP72), into lung tissues to combat apoptosis resulting from oxidative stress. Despite significant
progress, additional work is required to advance effective therapeutics for acute phosgene exposure.
Received 28 October 2020
Revised 25 January 2021
Accepted 1 February 2021
History
Phosgene (carbonyl chloride, COCl2, CAS registry 75-44-5),
the chemical whose name is so inextricably tied to the
trenches of World War I (WWI); it is easy to forget that the
molecule has been around for over 200 years. First
‘photosynthesized’ in 1812 by Cornish chemist John Davy by
exposing a mixture of chlorine and carbon monoxide to sunlight, he bestowed the popular moniker upon his creation by
merging the Greek words for light (phos) and born (gene)
(Davy 1812). It proved useful to the textile industry in the
1880s when German chemist Heinrich Caro and Swiss chemist Alfred Kern treated phosgene with dimethylaniline for the
first step in the synthesis of the brilliant purple dye, Crystal
violet (Reinhardt and Travis 2000). Ironically, a toxic and colorless gas at ambient temperature (BP ¼ 8.3 C https://www.
cdc.gov/niosh/npg/npgd0504.html), was used as a reactant
in the synthesis of a rich dye. But the wide potential of phosgene as a high-volume industrial feed stock chemical was
fully realized in the 20th century in the synthesis of plastics
(polyurethanes, polycarbonates, and polyureas), dyes, pharmaceuticals, and agrochemicals. The majority of phosgene is
currently used in the synthesis of monomers, such as toluene
diisocyanate (TDI), for polyurethane foam. During this process, phosgene is typically prepared on site from the reaction
of carbon monoxide and chlorine with an activated carbon
catalyst rather than sunlight (Holmes et al. 2016). Its ready
CONTACT Stephen T. Hobson
24515, USA.
sthobson@liberty.edu
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
Phosgene; Fv-HSP72; heat
shock protein 72;
pulmonary; chemical
warfare agent; Haber’s rule
availability and high toxicity make it an agent of concern to
the military and to homeland security authorities (Bast and
Glass-Mattie 2015; Baggett and Simpkins 2018).
The destructive potential of phosgene was highlighted by
WWI. Although the first chemical attack during the war was
launched by the Germans at the battle of Ypres, Belgium on
22 April 1915; they used chlorine, a pungent yellow-green
gas that allowed easy visual and olfactory detection. And
yet, the surprise of the attack killed more than 1100 people
and left 7000 injured (Evert 2015). Over the following
months, the German army tested phosgene, a colorless gas.
The phosgene was mixed with chlorine and used on the
approaches to Warsaw in the Eastern Front. The magnitude
of this type of attack was seen on 19 October 1915 when
the Germans released 275 tons of a mixture of one part
phosgene and four parts chlorine at Champagne on the
Western Front from about 14,000 cylinders over 12 km (Ryan
et al. 1996). By the time of the oft-cited attack over 4-5 km
of the front at Ypres on 19 December 1915, the British had
issued gas helmets and the phosgene attack killed only 116
and injured approximately 1000 despite the presence of
25,000 troops in the area (Ryan et al. 1996). Both the physical
and chemical characteristics of phosgene are responsible for
the observed lethality during WWI. Because of its density
(q ¼ 3.5 g/mL, NIOSH (National Institute of Occupational
Safety and Health 1976) https://www.cdc.gov/niosh/nioshtic-
Department of Biology & Chemistry, Liberty University, 1971 University Blvd, Lynchburg, VA
294
S. T. HOBSON ET AL.
2/00052750.html), it tended to collect and persist in low
lying areas (e.g. trenches) during WWI and thus resulted in
the greatest number of chemical warfare agent related fatalities (Bast and Glass-Mattie 2015; Summerhill et al. 2017).
Although the Germans were the first to use it on the battlefield, it became the chemical weapon of choice for the Allies,
predominantly by the French (Ryan et al. 1996). Phosgene is
often cited as being responsible for 85% of fatalities caused
by this class of weapons during WWI (Ministry of Defense
1987); however, authoritative sources dispute that claim
(Ryan et al. 1996). The absolute number of WWI casualties
resulting from exposure to all Chemical Warfare Agents
(CWAs) is difficult to determine due to the lack of analytical
equipment and the presence of multiple injuries to soldiers.
Furthermore, determining which agent is responsible for the
casualty is even more difficult due to the use of CWA mixtures by all armies. It is estimated that of the 518,000 casualties resulting from CWA exposure, between 17,000 and
26,000 resulted in death (Ryan et al. 1996).
In the scientific literature today, phosgene’s odor (safety
classification E) has been reported to resemble newly
mowed hay or grass with a threshold for human detection
between 0.5 and 1.5 ppm; however, less than 10% can
detect this odor at this threshold value (Amoore and
Hautala 1983). The NIOSH’s Immediately Dangerous to Life
or Health (IDLH) level for phosgene has been set at 2 ppm
(8 mg/m3) (https://www.cdc.gov/niosh/idlh/75445.html). Thus,
a sizeable majority of humans may be already in immediate
danger as they detect phosgene’s odor. During WWI, the
odor was characterized as ‘musty hay’ or ‘green corn’
(Figure 1). As scientists, we should reflect on that for a
moment. Despite the repeated use of this description in
the literature on phosgene, chances are the reader (as well
as these authors) have not had the opportunity to regularly
smell musty hay, yet it must have been an all too common
experience for the legions of farm boys-turned-dough boys
in the trenches of WWI. With the advent of chemical warfare, the uninitiated had to be quickly trained into a field
chemist, and with that, in the post-WWI era, a chart was
distributed to help soldiers determine the nature of a
chemical attack (Figure 1). Besides the odor, the soldier was
given another description to look out for: ‘Choky-Gas’. And
the CG codename for phosgene is born. An apt term for a
gas which attacks the respiratory system. First aid to be
rendered in the event of phosgene inhalation: ‘Keep quiet
and warm. Give coffee as a stimulant.’ (Figure 1).
Figure 1. Reference and Training Chart. Since most combatants in the trenches lacked a doctorate in chemistry, charts summarizing the litany of poison gasses a
soldier could be exposed to were prepared. This poster was prepared by Lieutenant Colonel Walter P. Burns, a US Army chemical warfare expert. Note the phosgene entry in the 6th row from the top. This chart was issued to Lieutenant William Frederick Nice, 49th Co 5th US Marine Regiment. Image courtesy of the
Veterans History Project of the American Folklife Center at the Library of Congress. William F. Nice (AFC 2001/001/1339), Veterans History Project Collection, Library of
Congress http://memory.loc.gov/diglib/vhp/story/loc.natlib.afc2001001.01339/.
TOXICOLOGY MECHANISMS AND METHODS
Modern risk to society
In 2003, the EPA identified 123 chemical plants in the nation
where a terrorist attack or accident could potentially expose
more than 1 million people to a cloud of toxic gas
(Homeland 2003). This includes high-priority threat agents,
such as phosgene, of which 8.5 million tons were produced
worldwide in 2015 [China 36.64%, Europe 30.67%, North
America
19.83%
https://www.marketresearch.com/GenConsulting-Company-v4078/Global-Phosgene-Outlook10248194/; Retrieved September 25, 2020]. The dangers of
handling phosgene require its manufacture and consumption
within the same plant (Environmental Protection Agency
2003), hence, all facilities producing more than 30 metric
tons per year must be declared to the Organization for the
Prohibition of Chemical Weapons [OPCW, Schedule 3 Annex
on Chemicals. https://www.opcw.org/chemical-weapons-convention/annexes/annex-chemicals/schedule-3;
Retrieved
September 25, 2020]. Unfortunately, we must view such
plants as ground zero in the event of a terrorist attack or
industrial accident.
Although phosgene will react with water to produce
hydrochloric acid and carbon monoxide (t1/2 ¼ 0.26 s)
(Environmental Protection Agency 2003), its low solubility in
water significantly reduces the rate of the reaction and its
rapid hydrolysis mitigates the reaction with the mucus layer
manifesting its presence with moderate irritation in the
upper airways (Borak and Diller 2001). The lack of immediate
symptoms combined with the lack of alarming odor characteristics exacerbates the toxicity of phosgene as many that
are exposed are not immediately aware of their peril (Bast
and Glass-Mattie 2020). Those exposed to a sub-lethal dose
can suffer chronic symptoms causing a long-term burden to
health care systems and the economy. Three years after
phosgene exposure in WWI, 83 British soldiers were examined and despite 53% of the men having no physical lung
abnormalities, they exhibited shortness of breath (70%),
cough with expectoration (54%), a tight feeling in the chest
(25%), sporadic giddiness (14%) and nausea (12%) (Sandall
1922; Bast and Glass-Mattie 2015). These factors have led to
extensive research into the mechanisms of toxicity, development of appropriate animal models for research, and efforts
toward efficient treatments and therapeutics for phosgene exposure.
Mechanisms of toxicity
The primary exposure route for phosgene is inhalation,
although depending on the initial concentration, eye irritation, lacrimation, and upper airway irritation (coughing) has
been reported (Glass et al. 2009; Grainge and Rice 2010).
Observations at the macroscopic level since WWI and at the
microscopic level since the 1970s allow us to understand
phosgene’s interactions with lung tissue.
At the macroscopic level: Decades of studies in mice, rats,
guinea pigs and dogs have generated myriad of Lethal
Concentration (LC) values as seen in Table 23.3 of Bast and
Glass-Mattie (2020, p. 345). Human data has been more
295
challenging to acquire given the sporadic nature of phosgene leakage in the workplace (see Human exposure below).
Categorizing exposure to phosgene according to toxic load
dosages determined by the strict linear application of concentration time has allowed the establishment of exposure
guidelines by the International Program on Chemical Safety
(IPCS) (Table 1). Under the assumption of Haber’s rule, the
toxidrome of phosgene exposure is linearly dependent on
dose and exposure duration, hence, an acute exposure to a
high concentration of CG is no more toxic than a chronic
exposure to a low concentration. At concentrations >30 ppm
x min (>120 mg/m3min), exposure results in pulmonary
edema and a breakdown in the blood-air barrier causing an
increase in fluids (Li and Pauluhn 2019; Bast and Glass-Mattie
2020). This damage results in further accumulation of protein
rich fluids or what is termed “Extra Vascular Lung Water” by
some (Li and Pauluhn 2019). Due to a lack of physiological
cues and symptoms, even at low concentrations, immediate
damage from phosgene in the lower respiratory tract can
remain undetected in what is termed the ‘initial’ phase of
exposure (Bast and Glass-Mattie 2015; Public Health England
2016). Furthermore, clinical recognition of exposure is complicated by an asymptomatic period (Diller 1985). During this
2 24 hour (h) ‘latent’ period, patients appear symptom free
(Russell et al. 2006; Smith et al. 2009). The duration of the
latent phase is inversely proportional to the exposure dose
(Table 1; (Public Health England 2016)). Workers near an
undetectable gas leak exposed to 1 ppm for 2.5 h will have
inhaled a high dose (>150 ppm min; >600 mg/m3min),
resulting in neutrophil infiltration, pulmonary edema and oxidative stress in the lungs (Ghio et al. 1991; Russell et al.
2006). Based on observations from workplace accidents,
inhalation of higher doses (300 ppm min; 1200 mg/
m3min) may also result in mortality (Diller 1985). At higher
doses (500 ppm min; 2000 mg/m3min), pulmonary
edema becomes evident within 3 h with death following
between 12 and 25 h (Li and Pauluhn 2019). This pattern
appears to break down in the event a victim is acutely
exposed (e.g. 2 min) to 300 ppm or more of phosgene (see
yellow box in Table 1), resulting in death within minutes
(Borak and Diller 2001). There are differing views about the
apparent cause of death with one source indicating rapid
occlusion of the pulmonary vasculature likely due to intravascular hemorrhaging (Public Health England 2016) and
another indicating acute overdistension of the right heart
before any pulmonary edema can develop (Bast and GlassMattie 2020). Either scenario may be academic to the dead,
but it should be of concern given the possibility of a terrorist
attack at an industrial plant.
At the microscopic level: Since the mid-20th Century, the
symptoms of toxicity are being dissected at the microscopic
level to understand the macroscopic observations.
Phosgene’s density and low aqueous solubility results in
accumulation in the lower lungs where there is a more
amenable hydrophobic environment (Li and Pauluhn 2019).
Diffusion of phosgene in an aqueous solution is about
8.8 lm; 4–8 times the thickness of all three layers of the air–
blood barrier in the alveoli (Nash and Pattle 1971). Phosgene
LCt100
5200
1300
In addition to exposures listed as ‘Acute’, we categorize any exposure 10 min as an acute exposure. Estimated threshold toxicity values in humans are compared for phosgene inhalation as reported by the
International Program on Chemical Safety (IPCS) obtained from Public Health England (2016).
ppm: parts per million; Conc.: Concentration. Used with permission and modified from Table 23.2 in Bast and Glass-Mattie (2020, p. 344).
a
Effect
Onset of lung damage
Pulmonary edema
LCt1
LCt50
>120
>600
1200
2000
>30
>150
300
500
–
–
–
–
–
–
–
24
30
Minutes
–
–
–
–
–
–
10
5
3
7
–
–
Throat irritation
Ocular irritation
Cough
–
–
–
–
–
–
–
–
–
–
–
81
101.15
607.5
1011.35
2022.7
2104
2427.24
–
–
–
–
20
25
150
250
500
520
600
–
‘Acute’
‘Acute’
‘Acute’
20
5a
150
5a
5a
400
2a
–
12.14
16.18
19.42
4.05
20.23
4.05
202.27
404.54
5.26
1213.62
–
3
4
4.8
1
5
1
50
100
1.3
300
–
Dose
Dose
Time to death (h) ppm min mg/m3 min
Conc. ppm
Conc. mg/m3
Dose
Dose
Duration (min) C T (ppm min) C T (mg/m3 min)
Acute effect
Onset time to pulmonary edema (h)
IPCS acute exposure thresholds
S. T. HOBSON ET AL.
Exposure
Table 1. Concentration dependent toxidrome of phosgene exposure in humans modified from a summary chart by Bast and Glass-Mattie (2020). Below those exposure times listed as ‘Acute’ by the original authors, the
rows are organized according to ascending Dosages (CxT values).
296
is the molecule responsible for the damage that ensues, not
hydrochloric acid resulting from hydrolysis of phosgene with
water (Nash and Pattle 1971). According to one model
detailed in Holmes et al. (2016) and summarized in Figure 2,
upon penetrating lung tissue, it acylates nucleophilic groups
(i.e. amino, hydroxyl, sulfhydryl moieties) present in proteins,
lipids, and nucleic acids. At low phosgene concentrations,
proteins and phosphatidylcholine constituting the alveolar
surfactants are the ones acylated. At >30 ppm min, the disruption of the surfactants and acylation of biomolecules produces a pulmonary edema as the air-blood barrier is
permeabilized to blood plasma during the latent period
(Table 1). Clinical manifestations at this stage include
decreased efficiency in gas exchange with blood cells,
labored breathing, a frothy expectorant and the accumulation of proteins in the bronchoalveolar lavage fluid (BALF)
(Bast and Glass-Mattie 2020). Phosgene also undergoes
homolytic cleavage resulting in the reactive chloro carbamoyl
radical (Arroyo et al. 1993; Holmes et al. 2016). These highly
reactive species can further alter the surfactants in the lungs
through lipid peroxidation, a process that, if left unchecked
can damage the cell membranes of lung and blood tissue.
Chloro carbamoyl radicals can lead to increased oxidation of
glutathione and, with the reduced antioxidant capacity in
the lungs, reactive oxygen (ROS) and reactive nitrogen (RNS)
species are generated. This results in a plethora of reactions
that affect various systems at the molecular level: including
epithelial cells needed for gas exchange (Type I and II
alveoli); neuronal cells innervating the lungs; endothelial and
blood cells (Holmes et al. 2016) (Table 2).
There is some disagreement among researchers regarding
whether cyclooxygenase-2 (COX-2) and inflammation plays a
role in the mechanisms of vasopermeability and pulmonary
edema. Studies in rabbits have demonstrated that pulmonary
edema induced by phosgene intoxication is caused by
increased capillary permeability and not due to any alteration in atrial pressure or other hemodynamic factors (Russell
et al. 2006). That model relies on the phosphorylation of the
JNK/SAPK and p38 MAPK pathways, which increase the
expression of COX-2 and, in turn, prostaglandin E2 which
contributes to inflammation by enhancing edema and
immune cell infiltration. BALF analysis of phosgene intoxicated rats has shown marked increases in prostaglandin E2
(Chen et al. 2013) and cytokine mediators (Sciuto et al.
2003), as well as antioxidant enzymes (Sciuto et al. 2003).
Phosphorylation of the same pathways also increases expression of inducible nitric oxide synthase (iNOS), leading to
vasodilation, oxygen radicals and oxidative stress. Support
for this model comes from the use of ethyl pyruvate, which
inhibits COX-2 and iNOS expression, as wells as phosgeneinduced pulmonary edema (Chen et al. 2013). However,
others point to the fact that there are no changes in COX-2
activity post-phosgene exposure (Guo et al. 1990; Holmes
et al. 2016).
Changing perspectives on the mechanistic details of phosgene intoxication impacts which biomarkers remain relevant
to the scientific community as drug developers screen new
therapeutic compounds. For instance, some have proposed
TOXICOLOGY MECHANISMS AND METHODS
297
Table 2. Biological effects of phosgene.
Biological effect
Lipid peroxidation increase
Animal model
Mice, Rats & Guinea Pigs
Key references
Increased leukotriene production
Rabbit
(Guo et al. 1990)
(Sciuto et al. 1996)
Decrease in intracellular ATP and cAMP
Rat
(Frosolono and Pawlowski 1977; Currie et al. 1987)
(Borak and Diller 2001)
(Currie et al. 1985)
Glutathione redox cycle (gene transcription and protein expression)
Mice
(Sciuto et al. 2005)
Increased glutathione oxidation and reduction of glutathione reductase
Rabbit
(Sciuto et al. 1996)
(Bast and Glass-Mattie 2020)
Stimulates Endothelium-1 (ET-1) induction and its signaling cascade
Rat
(Zhang et al. 2008)
Edema in Type I pneumocytes (alveolar cells)
Rat
(Diller et al. 1985)
(Frosolono and Pawlowski 1977)
Damaged/decreased plasminogen levels
Mice
(Aggarwal et al. 2019)
(Sciuto 1998)
A selection of papers that have helped craft the model described in Holmes et al (2016) and illustrated in Figure 2.
Table 3. Summary of cell-based assays used to study phosgene.
Phosgene dose
Study type
Study rationale and observations
Madden et al, 1991 use human and rat alveolar macrophages to study phosgene’s effects on lung arachidonic acid metabolism.
1 ppm 4 hr
Biomarker Discovery
Rationale: Leukotriene’s are inflammatory molecules produced from arachidonic acid.
Observations: Decrease in leukotriene B4 production; no effect on prostaglandin E2
(rat and human) or peptide leukotrienes C4, D4, and E4 (rat) production.
Gurtner, 1996 used human microvascular endothelial cells and type II alveolar epithelial cells.
300 and 900 ppm
Mechanism of Action
Rationale: Eicosanoid’s are inflammatory molecules produced from arachidonic acid.
Observations: Elevated eicosanoids and leukotrienes. Accumulation of inter-cytosolic calcium
and reactive oxygen species.
Cowan et al, 2005 used human lung small airways cells (SAC) to study changes in a biomarker for phosgene exposure.
0.1–6.4 ppm y 1min
Biomarker Discovery
Rationale: Interleukin (IL)-8 recruits neutrophils to lungs hence causing acute lung injury.
Observations: Exposures > 0.1 ppm min increase IL-8 expression by 24 h post-exposure
(maximum at 1.6 min ppm). Exposures > 1.6 ppm min result in increased cytotoxicity.
Wijte et al, 2011 used human epithelial lung cells (A549) exposed to phosgene at the air-liquid interface in a CultexV system.
40 ppm min
Model for Drug Screening
Rationale: Identify biological changes to lung cells grown on semi-permeable membranes.
Observations: At 4 hr post-exposure: 1) IL-8 down-regulated (decreased inflammation); 2)
lactate dehydrogenase increased (decreased cell membrane integrity); 3) Alamar Blue
increased (decreased cell viability); 4) glutathione, HO-1 decreased (increased oxidation).
R
R inserts
Olivera et al, 2017 used differentiated human bronchial epithelial cells (16HBE) grown on TranswellV
0–512 ppm min
Model for Drug Screening
Rationale: Identify biological changes to lung cells grown on semi-permeable membranes.
Observations:
Barrier integrity: 64 ppm min phosgene is a threshold dose resulting in a decrease in
transepithelial electric resistance without cell integrity recovery (1–10 hr post-exposure);
Cell viability: 128 ppm min phosgene decreases viability 6 h post-exposure (XTT assay);
Metabolism: 512 ppm min phosgene decreases 14C glutatmine oxidation
(14CO2 emission) and lactate production 6 and 24 hr post-exposure;
that measuring the Extra Vascular Lung Water is more predictive of subsequent severe pulmonary edema than BALF
proteins (Li and Pauluhn 2019). While aspects of each model
are certainly correct, the clearest common denominator for
both models is the formation of ROS leading to oxidative stress.
Cell-based assays for phosgene
Changing perspectives on mechanistic details also confound
the ability to replace in vivo toxicity testing with either
in vitro (cell-based) or in silico approaches (Rim 2020), despite
economic and political pressure to do so for many chemicals
(e.g., USA: Tox21, European Union: REACH). For example, a
recognized limitation of these systems is the inability to
mimic the inflammatory response in the early stages of
exposure and the consequent tissue damage caused by free
radicals (Arroyo et al. 1993; Holmes et al. 2016; Olivera et al.
2017). Within this limitation, a variety of in vitro studies have
examined the effect of phosgene (Table 3).
The complexity of the respiratory system and the technical challenges involved with direct exposure of cells to airborne toxicants has limited the use of in vitro assays to the
elucidation of phosgene’s mechanism of action, identifying
biomarkers of intoxication or for the screening of potential
therapeutics (for a discussion of the latter see Rim 2019).
Recent phosgene studies have tried to utilize a more realistic
alveolar architecture and environment by growing cells on
298
S. T. HOBSON ET AL.
Figure 2. Mechanism proposed by Holmes et al (2016) for phosgene injury. GSH: glutathione; TRPA1: transient receptor potential cation channel: member A1;
NMDA: N-methyl-D-aspartic acid; Met: metabotropic; ET-1: endothelin 1; ETA: endothelin receptor A; ETB: endothelin receptor B; Pmv: pulmonary microvascular
pressure; SOCE: store-operated calcium entry; Rxns: reactions. Used with permission, this figure was published in Toxicology Letters, 244, Holmes et al (2016), p 11,
Copyright Elsevier (2016).
semi-permeable membranes and lowering the insert into a
well so that the basolateral (lower) side contacts the culture
media and the apical (upper) side has an air-liquid interface
(Wijte et al. 2011; Olivera et al. 2017). When cells are grown
to confluency on the insert’s membrane, such studies allow
the added ability to test for cell integrity by measuring the
transepithelial electrical resistance (TEER) established once a
confluent cell barrier forms. Any disruption of the barrier
results in a decrease of the TEER, hence providing a quantitative measure of cell viability. While other observations from
such studies have the potential of being developed into cellbased quality control assays for testing the efficacy of therapeutic agents (Table 3), the use of TEER to measure cell viability is likely to remain in the realm of research given the
challenges of reproducibly validating such a procedure as a
lung barrier integrity assay.
Regardless of the cell type or the architecture, little correlation in toxicity between lung cell-based assays and animal
models were noted with 19 toxicants (Sauer et al. 2013).
Finally, in a comprehensive review on the use of in vitro
assays for chemical warfare agents, the limitations of these
assay have been noted for phosgene by no less than the
National Academies of Sciences, Engineering, and Medicine
(2015): ‘There does not appear to be a particularly good
in vitro model for lung damage associated with chemicals
(for example, phosgene) that are known to increase
pulmonary permeability that results in noncardiogenic pulmonary edema.’ Hence, although continued development of
cell-based assays and exposure systems is necessary for
accelerated development of therapeutics and to further elucidate biomarkers and the mechanism of phosgene toxicity,
in vivo models are still the primary method to screen new
medical countermeasures.
Animal models
While strides have been made in clarifying phosgene’s mechanism of action, capitalizing on it to develop a medical countermeasure has failed so far. A key reason for the lack of
progress is its steep toxicity curve, which was once aptly
characterized as approaching ‘a step function’ (see Figure 2
in Pauluhn 2006 for an excellent illustration of curve steepness). This makes it difficult to determine a consistent LC50
value in an animal model, in turn, affecting the proper determination of a toxic load equation (Concentration [C]Time
[t]) for a toxicologist to rely on when assessing the risk of
exposure to a subject in a given period of time (Figure 3). At
the scientific level, the correct choice of a model for acute
versus chronic exposure is crucial in the development of
therapeutics for phosgene poisoning, given the observation
that the toxicity of phosgene does not linearly follow Haber’s
TOXICOLOGY MECHANISMS AND METHODS
Figure 3. The phenomenon of toxicity for poison gas is mathematically
described by multiplying the concentration (C) of the gas in a cubic meter of
air by the time (t) in minutes (min) that the subject has to breathe the air
before death ensues. Today, we often refer to the product of the equation as
the Toxic Load Dosage (TL). (A) Haber’s Rule is a special case in the continuum
of toxic load modeling where survival depends on a linear relationship between
concentration and time. Mathematically speaking, the equation describing such
a curve assumes the concentration has a toxic load exponent (ne) equal to 1. In
the real world, that means a phosgene release from a chemical plant (represented by the refinery skyline in our schematic) would have similar survival
rates for by-standers during a short-duration, high-concentration exposure
in the immediate aftermath of an explosion at t ¼ 0 min or during a long-duroccurring in surrounding neighborhoods,
ation, low concentration exposure
for example, t ¼ 400 min after the explosion. In our example, we assume the
concentration released is theoretically at the LC50 (C ¼ LC50) hence, the same
and fatalities
are shown in this graphic. (B) When
number of survivors
there is an uneven distribution of survivors in the high and low concentration
zones, despite an LC50 release of gas, then the concentration value in the toxic
load model is modified. If the short-duration, high-concentration exposure is
more toxic and has a greater number of fatalities and the long-duration, lowconcentration zone has far fewer deaths, than the toxic load exponent for C is
ne >1. (C) If the opposite occurs and the acute, high-concentration exposure is
not as toxic compared to a chronic, low-concentration exposure that has
greater lethality, than the ne <1.
Rule (Hobson et al. 2019). At the regulatory level, the FDA’s
requirement for sufficiently well-characterized animal model(s) for predicting the response in humans for new drug
applications filed under the ‘Animal Rule’ has also prompted
research into appropriate model species (Park and Mitchel
2016; Chemical Warfare Toxicology: Volume 2: Management
of Poisoning 2018).
Historically, Haber’s Rule has been a guiding hypothesis
when interpreting toxicity results in an animal model (Miller
299
et al. 2000). Mathematically predicting the physiological
effects of a toxin on an organism in proportion to the toxin’s
concentration and the organism’s duration of exposure was
first proposed by Warren (Warren 1900) regarding the toxicity of salt solutions that kill the planktonic crustacean,
Daphnia magna. However, it is Fritz Haber whose name
graces the iconic formula C t, based on work he did exposing cats to phosgene (Haber 1924). Ironically, Haber was not
an adherent to the idea that C t applied linearly to all
intoxications; he appreciated the importance of the time factor, particularly for chronic exposures, where metabolism and
detoxification (toxicodynamics and toxicokinetics) would
begin to influence the toxic load (Rozman and Doull 2000).
To improve the accuracy of the dose response equation, ten
Berge et al. (ten Berge et al. 1986) introduced the ‘toxic load
exponent’ (ne) for the concentration parameter (Figure 3). An
exponent for the time parameter (p) improves toxic load
modeling further, particularly for chronic studies; however, if
p < 1 the toxicity relies more on concentration than exposure
duration, and conversely if p > 1 the toxic effect may not be
linear over an extended period of time. In a survey of 21
inhaled toxicants, 14 had an estimated p < 0.45 and only 3
had p > 1 (Belkebir et al. 2011). Those with a p > 1 did not
exceed p ¼ 1.25. While the survey did not cover phosgene,
the results suggest our primary focus here on the concentration parameter with Toxic Load ¼ Cne t is not unreasonable. An excellent discussion of all the power curves
generated by the equation Cne tp is presented in (Miller
et al. 2000).
Improved survival after exposing rodents to the LC50 of a
toxic inhalant is commonly used to screen drug efficacy in
early preclinical development. Inaccurate LC50 values, particularly when studying acute phosgene exposures, can result in
costly disruptions. This experience led the authors to develop
a more robust nose-only inhalation model for rats to study
the effects of short-term exposures (Hobson et al. 2019).
Earlier systems have provided whole body and nose-only
exposures, however, the LC50 data has been mixed. In 1990,
(Zwart et al. 1990) intoxicated rats and mice with phosgene
in custom made horizontal glass cylinders for whole-body
exposure and reported LC50 values much higher at a 10 min
exposure than for longer ones at 30 and 50 min (Figure
4(A–C)). A similar result was obtained in rats using a noseonly inhalation system, albeit with a ne closer to 1 (Pauluhn
2006) (Figure 4(D)). The ne < 1 determination from such
data sets can lead to an underestimation of phosgene toxicity in an acute exposure study, as was the case when the
authors of this article ran a similar analysis in rats [compare
our rat survival curves to Pauluhn’s in Figure 2 of (Hobson
et al. 2019)]. Variation in phosgene toxicity between small
and large mammals has been noted since the 1940s [see
Figure 2 in (Boyland et al. 1946)]. Whole body exposure studies in guinea pigs (Figure 4(E)), rats and mice (data not
shown), provided ne values of 0.87, 0.65 and 0.73, respectively (Boyland et al. 1946). This was not the case for dogs. In
Table 4 of Boyland et al. (1946), the authors report an amalgamation of LCt50 results from several studies, including their
own (Figure 4(F)). The ne > 1 seen in dogs is more reflective
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S. T. HOBSON ET AL.
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
Figure 4. Determining the correct toxic load exponent (ne) is crucial to ensure the level of risk is correctly assessed in the event of an acute exposure. Plotting
exposure time versus the LC50 on a logarithmic scale allows for regression analysis to determine ne (Weinrich et al. 2008). Using the method of Sweeney et al.
(2015), both time and LC50 values are converted to Log10 before plotting (Sweeney et al. 2015). In these studies, concentration (C) is the more independent variable than the time (t) it takes to produce a given effect, hence, C is typically plotted on the x-axis and the negative slope is the ne value (Miller et al. 2000). (A–C)
Rodent results reported by Zwart et al. (1990) show phosgene does not adhere to Haber’s Rule; with ne <1 their data suggest short-duration, high-concentration
exposures are not as toxic as long-duration, low concentration ones. (D) Pauluhn (2006) conducted studies in rats using nose-only inhalation and obtained a lower
LC50 at 10 min compared to Zwart et al. (1990). This brought the results closer in line to Haber’s Rule (ne ¼1). (E) Early small animal studies using whole body exposures of rodents and guinea pigs appeared to confirm phosgene’s lower acute toxicity (Boyland et al. 1946). Readings taken at 0.5 and 0.25 min appear as negative
log time. (F) This is not the case for dogs also immersed in the same type of whole-body chamber. With ne >1 their data suggest acute, high-concentration exposures more toxic than chronic low concentration ones. (G–H) Using an improved procedure for nose-only inhalation in rats (Hobson et al. 2019), a lower LC50 was
obtained for 10 min exposures that align better with the realistic toxicity seen during phosgene exposure. Modifying the tables by replacing this value with the
10 min values in the Zwart et al. (1990) and Pauluhn (2006) data sets suggests ne >1 and, thus, more toxic during acute, high concentration exposures than previously thought. Replaced values denoted by red star and arrow (compare with C,D).
of the toxicity seen with phosgene in our rat studies using a
more robust system.
While researchers have generally determined phosgene’s
LC50 by varying the exposure concentration for a specific
length of time, Plahovinsak et al (2015) took a different
approach by varying the exposure time male mice inhaled an
8 ppm (32.36 mg/m3) dosage from a nose-only inhalation
tower. One of their key results further illustrate phosgene’s
steep toxicity curve. 50% mortality occurs at 48 hours when
mice are intoxicated with 8 ppm for 26.9 min, but that LC50 is
reached in 24 hours when intoxication occurs for 28.3 min
(for both p < 0.001); a mere difference in exposure of
1.4 minutes! They report a 48 hour LCt50 of 215.2 ppm min
(8 ppm 26.9 min) and a 24 hour LCt50 of 226.4 ppm min
(8 ppm 28.3 min); the latter LCt50 translating to 915.8 mg/
m3min (32.36 mg/m328.3 min). This result highlights differences seen from one laboratory to the next. For example,
Plahovinsak et al (2015) achieve 50% mortality in 24 hours
when male mice are exposed to 32.36 mg/m3 of phosgene
for 30 min, yet Zwart et al. (1990) expose male mice to
more than double that concentration (76 mg/m3) for 30 min
(Figure 4(A)) and they achieve 50% mortality in 14 days.
Our nose-only inhalation system is designed to rapidly
deliver the target concentration of phosgene in a step function rather than gradually increasing to the target, thus, recreating a more likely acute, high-concentration exposure
following an accident or explosion (Hobson et al. 2019). The
exact concentration of phosgene delivered to the rats is verified via Fourier Transform Infrared spectrometry, a level of
sophistication not used by any of the other researchers in
Figure 4. Equally important, we acclimated our rats to the
confining environment of the exposure tubes for 10 min on
two consecutive days prior to exposure in order to minimize
any confounding effects caused by stress proteins and to
better recreate the calm demeanor and early-stage behavior
of victims at ground zero. For a 10 min exposure to a series
of phosgene concentrations, a significantly lower LC50 in rats
was calculated by Probit analysis (129.2 mg/m3) along with
tighter 95% confidence limits (CL) of [109.2 145.7 mg/m3].
In comparison, wider limits were reported by Zwart et al.
(1990) [LC50: 334 mg/m3; 95% CL: 306–363 mg/m3] and
Pauluhn (2006) [LC50: 253.3 mg/m3; 95% CL: 194–331 mg/m3].
Exposing another set of rats (n ¼ 9) to the calculated LC50 of
129.2 mg/m3 affirmed the toxicity of this dosage in terms of
24-h survival and lung/body weight ratios (Hobson et al.
TOXICOLOGY MECHANISMS AND METHODS
2019). Replacing this value for the 10 min results reported by
Zwart et al. (Figure 4(G)) and Pauluhn (Figure 4(H)), provides
a ne > 1 and in agreement with a model of greater toxicity
during short-term high-concentration exposures. Our LCt50
value of 1292 mg/m3min corresponds to the expected
range of 1000 2000 mg/m3min seen for phosgene in
many experimental animals and the accepted LCt50 in
humans (2000 mg/m3min in Table 1) as determined by
the IPCS (Public Health England 2016). Such a result should
not be surprising. We note that the Acute Exposure
Guideline Levels (AEGL) from the Environmental Protection
Agency (2002) for phosgene states (pg. 16) ‘Haber’s law has
been shown to be valid for phosgene within certain limits.’
Picking models that will approximate human intoxication
requires more than accounting for an acute or chronic duration. One must account for physiological similarities as well.
The need to power screening studies so they are statistically
significant will continue to require the use of rodents; however, rodents are obligate nasal breathers and exhibit physiological differences from humans and dogs, which breath
both from the nasal or oral cavity. As an example, Pauluhn
(2006) attributes the increased survival times in rats and,
hence, greater LC50 values obtained for 10 min phosgene
exposures to ‘an initial and concentration-dependent
increase in the apnea time, accompanied by decreased
respiratory minute volumes.’ This reflexive behavior in rats
for acute exposures is believed to be caused by phosgene’s
stimulation of the vagal C-fibers innervating the lower airways and controlling spontaneous breathing. Evidence of
phosgene’s interaction with the vagal nerves is seen in dogs
exposed to doses > 7000 mg/m3. There is a cessation of respiration with the lungs deflated; a phenomenon that does
not occur when the vagus nerve is cut prior to phosgene
exposure (Li and Pauluhn 2014). Interference from this reflex
in rodent models may subside for exposures greater than
10 min. For more physiologically relevant phosgene studies,
it has been reported that the dog (beagle) more closely
matches the pathophysiology of humans post-exposure (Li
and Pauluhn 2019). With these data in mind, the agreement
between our ne in rats using part of Pauluhn’s 2006 data (ne
¼ 1.17) and the ne determined for dogs by Boyland et al. in
1946 (ne ¼ 1.13) (Figure 4(F & H)) suggests that our robust
nose-only inhalation system correctly predicts a greater toxicity when a subject is exposed to a short-duration, high concentration dose of phosgene than a long-duration, low
concentration. The dog is an expensive model to be utilized
in late stages of preclinical drug development; however, in
the early stages, our acute exposure system provides an
alternative option (Hobson et al. 2019).
Human exposure
Perhaps the most important gaps in our knowledge regarding phosgene involves human exposure. For example, determination of the exact concentration and exposure time for
casualties exposed to phosgene during WWI is complicated
by the nature of the exposure and the lack of analytical/diagnostic equipment in use (vide supra). Since phosgene’s use
301
as a CWA, a number of industrial accidents have occurred
with surprisingly few deaths. The largest incident in which
phosgene is implicated is the Union Carbide Bhopal accident
in which 2500 people died (Ryan et al. 1996). Both the
cause and the main component of the leak (methyl isocyanate) makes it improbable that phosgene was involved.
Further investigation into Union Carbide revealed allegations
that workers were used to ‘sniff out the sources of phosgene
leaks’ in plants located in Bhopal and West Virginia (Ryan
et al. 1996). More recently, in 2010 at a DuPont facility in
Belle, WV, a faulty hose that was transferring pressurized
liquid phosgene ruptured exposing three workers, one of
whom died (Johnson 2011). Finally, from the 1920s to the
1950s, there was an extensive offensive and defensive CWA
research program at Porton Down in the United Kingdom;
during this time, both US and UK military volunteers were
exposed to various levels of CWAs including phosgene
(Isenberg 2001).
Table 1 consolidates observations of human exposure incidents described by W.F. Diller and colleagues; however, a
perusal of the NIOSH’s webpage for the phosgene IDLH finds
other historic human exposure data. Some observations support the IPCS’s threshold toxicity values (Table 1), such as a
30 min exposure to 17 ppm (510 ppm min) being lethal
(Diller 1978). Some observations are contradictory, such as a
30 min exposure to 5 ppm (150 ppm min) being ‘probably’
lethal (Jacobs 1967). And some lack sufficient information:
‘brief exposure to 50 ppm may be rapidly fatal’ (Henderson
and Haggard 1943). How long is ‘brief’?
Given the paucity of human data and the results from
some animal data suggesting that phosgene may not follow
Haber’s rule under all circumstances, it may be time to refine
the IPCS guidelines that are currently based on a linear relationship of dose and exposure duration. Before any such
action is taken, we suggest further experimentation with a
single species that closely mimics the pathophysiology of
humans, most likely a non-human primate. In large-scale simulations, phosgene can be released from the center of a
large sealed room blanketed with state-of-the-art detectors
and in which multiple test subjects have been positioned at
varying distances from the center. This format allows investigation of acute and chronic intoxication scenarios to determine the overall pattern of lethality (as seen schematically in
Figure 3) and, hence, derivation of robust power curves that
mathematically allow us to calculate precise exposure thresholds. Experimentation on such a scale is not for testing therapeutics. The experimental strategy may be elaborate and of
concern regarding the use of so many animals, yet the alternative of waiting for a large-scale release of phosgene to
study the pattern of lethality in humans is not a better strategy.
Development of medical countermeasures
Despite its industrial prevalence, there is no FDA-licensed
therapeutic treatment for toxic phosgene exposure (Sciuto
and Hurt 2004; Russell et al. 2006; Smith et al. 2009; Holmes
et al. 2016; Summerhill et al. 2017). While current
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hypothesized mechanisms of action de-emphasize the role of
inflammation (Holmes et al. 2016), previous attempts have
focused on the inflammatory processes that underlie edema,
such as curbing neutrophil infiltration using cyclophosphamide, the 5-lipoxygenase inhibitor AA861, or the microtubular poison colchicine; all have been unsuccessful (Ghio et al.
1991). The acid-resistant protease inhibitor, ulinastatin, suppresses neutrophil accumulation, and it has shown efficacy
in small animal studies, but its exact mechanism of action
remains unclear (Shen et al. 2014). The efficacy of ibuprofen,
given prophylactically 30 minutes (min) prior to phosgene
exposure, for the reduction of observed lung edema in rats
has been reported (Sciuto et al. 1996), although such a pretreatment strategy is unrealistic given the nature of ‘nowarning’ chemical exposure events, like those expected on
the battlefield, a terrorist attack, or workplace accident.
The use of a nebulized b2 adrenergic receptor agonist, salbutamol, has shown a deleterious effect on arterial oxygenation (Grainge et al. 2009). Neither treatment with nebulized
nor intravenous steroids improves survival (Smith et al. 2009;
Grainge and Rice 2010). And draining the lungs with the
diuretic furosemide also did not increase survival (Grainge
et al. 2010).
In an ambitious screening program using whole body
exposure of outbred CD-1 male mice to 32-40.5 mg/m3 (810 ppm) phosgene for 20 min, a wide array of compounds
was administered at a series of doses post-exposure. These
included: antioxidants; an ETA receptor blocker; NMDA and
muscarinic receptor antagonists; a radioprotectant; an ACE
inhibitor; an antihistamine; an AMPK activator; and inhibitors
for PDE4, PDE5, 5-lipoxygenase, a couple for GABA transaminase and three TRP channel inhibitors (Holmes et al.
2016). Conclusions of this wide screen indicated that neuromodulation and interruption of vascular tone/permeability
pathways (PDEs, angiotensin, and endothelin) are promising
leads. However, limiting ROS formation, as seen with some
of the antioxidants tested, may be key in preventing mortality following phosgene exposure (Holmes et al. 2016).
Targeting oxidative stress and apoptotic pathways
In separate studies, inducible nitric oxide synthase (NOS2,
iNOS) inhibitors have shown efficacy inhibiting phosgene
induced acute lung injury with inbred C57BL mice (Filipczak
et al. 2015); meanwhile, angiopoietin-1 transfection and
induction has shown similar results in rats (He et al. 2014).
Answers as to why such strategies work in phosgene exposure models can be found not in pulmonary research, but in
neurology. Using an epileptic seizure model in rats, researchers have shown that sustained seizures activate nuclear factor-kappa B (NF-jB), a transcription factor with multiple
roles, including the upregulation of NOS2, which can lead to
the biosynthesis of peroxynitrite from nitric oxide when in
an environment rich with ROS such as the superoxide radical
(O2 ) (Chang et al. 2014). As noted above, phosgene
depletes the lung of glutathione, the main defense against
reactive oxygen species (Bast and Glass-Mattie 2020).
Peroxynitrite and ROS generation in the lungs in turn
activate caspase cascades that ultimately lead to apoptosis.
However, angiopoietin-1 suppresses the NF-jB transcription
pathway in phosgene induced acute lung injury (He et al.
2014). The suppression of NOS2 and NF-jB can involve multiple intermediaries along a metabolic pathway, and yet
there is a more direct suppression mechanism now under
investigation in our laboratories, Heat Shock Protein 72
(HSP72) (Parseghian et al. 2016).
HSP72 is a pleiotropic agent with multiple anti-stress,
anti-apoptotic roles and has been investigated as a cytoprotectant in a wide variety of tissues, including in the pulmonary system (Parseghian et al. 2016). HSP72 is known to
inhibit three apoptotic pathways that can occur with traumatic injury to any tissue: (1) During ATP-dependent apoptosis, mitochondria release cytochrome C, triggering the
construction of a molecular machine known as an apoptosome. Apoptosomes cleave procaspase-9 proteins into their
active caspase-9 form, which go about triggering a cascade
of cellular destruction leading to cell death. HSP72 binds the
apoptosome, preventing conversion of procaspase-9 into its
active caspase-9 form (Apoptosome) (Beere et al. 2000). (2)
During ATP-independent apoptosis, mitochondria release
apoptosis inducing factor (AIF). Sufficient AIF in the cell cytoplasm can also trigger cell death. Unlike the apoptosome,
this apoptotic process triggered by AIF does not require ATP,
therefore it occurs even under low energy conditions. HSP72
binds AIF, inhibiting this ATP- and Caspase-independent
apoptotic pathway. (Cande et al. 2002). (3) Most importantly,
in the context of this discussion, HSP72 has also been shown
to directly bind NF-jB and prevent its release from sequestration with IjB in the cytoplasm (Zheng et al. 2008). If
released from the NF-jB: IjB complex, NF-jB can translocate
to the nucleus and upregulate NOS2, leading eventually to
apoptosis caused by oxidative stress (Figure 5). HSP72 induction is one of the body’s responses to ROS synthesis caused
by oxidative stress (Madamanchi et al. 2001); however, in certain cases, an ROS can actually impair HSP72 expression
through RNA interference with microRNA intermediaries
et al. 2012). NF-jB also plays a role
(Adachi et al. 2009; Spiro
in the induction of inflammatory pathways. In fact, while the
use of angiopoietin-1 helps attenuate inflammation in phosgene injured lungs (He et al. 2014), another of the body’s
natural responses is the induction of HSP72 via IL-6 (Levada
et al. 2018).
Based on acute lung injury studies, the induction process
for HSP72 requires 12 h after the initial insult to the lungs,
an unacceptably long period of time in the event of phosgene exposure, although it remains at a level above baseline
for up to 72 h (Villar et al. 1993). We have been developing a
strategy that rapidly delivers human HSP72 into lung tissue
using the scFv fragment of a proprietary cell-penetrating
antibody, mAb 3E10, as an intracellular transport system for
protein therapeutics (Weisbart et al. 2000; Hansen et al.
2007; Weisbart et al. 2015). The 3E10 monoclonal is a wellcharacterized antibody with a unique cell penetration pathway (Hansen et al. 2007) that has been found to be safe in
an FDA approved Phase I clinical trial (Spertini et al. 1999).
3E10 binds DNA allowing its penetration through a specific
TOXICOLOGY MECHANISMS AND METHODS
303
(A)
(B)
Figure 5. (A) Transcription factor NF-jB is activated and triggers a cell death cascade upon phosphorylation of IjB by its kinase, IjK, under conditions of neural
cell stress. (B) Immunoprecipitation data indicates Hsp72 blocks IjK’s access to IjB, preventing NF-jB’s translocation to the nucleus (Zheng et al. 2008). Image
from Parseghian et al. (2016) courtesy of the Annals of the New York Academy of Sciences/John Wiley & Sons.
nucleoside salvage channel found in most cells, known as
the equilibrative nucleoside transporter 2 (ENT2) (Lu et al.
2004). This 100 kD fusion of a humanized 3E10 to human
HSP72, formerly known as Fv-Hsp70 (Zhan et al. 2010), and
now known as Fv-HSP72, targets extracellular DNA, to deliver
HSP72 to damaged lung cells. Nucleoside targets are abundant, stable and quite accessible during tissue damage
where there is cell necrosis (Chen et al. 1990; Parseghian and
Luhrs 2006; Weisbart et al. 2015). Selectivity of this agent
in vivo is based on the simple concept that tissues undergoing significant cell injury possess a high concentration of
extracellular DNA. Salvaging of the DNA by surrounding cells,
through the ENT2 channel, provides 3E10 the opportunity to
enter those energy-deficient cells still hanging on to life in a
diffusion-driven process that does not depend on ATP-fueled
endocytosis.
Rapid post-exposure response
Any successful therapeutic to treat phosgene exposure
requires the following equally important technical characteristics: (1) efficient storage, distribution and drug administration, (2) efficacy when delivered post-exposure, (3) efficacy in
the broadest population possible, and (4) rapid delivery and
localization to the lungs. Let us briefly ponder each of these
concepts in the context of the HSP72 approach
just discussed.
IL-10 in the serum and BALF. These results were statistically
significant even compared to those rats receiving MSC without any HSP70 transduction (Jin et al. 2020). While they verify the HSP72 approach, it is hard to imagine the utility of
stockpiling millions of vials of MSCs in liquid nitrogen tanks
throughout the country for distribution to hospitals that can
deliver the product intra-tracheally to victims after an exposure event. The authors of the study point out that 90% of
MSCs can undergo apoptosis after transplantation, coupled
with poor targeting of the cells to the areas of greatest
injury, and only a small number of MSCs are going to reach
the damaged cells. Development of the Fv-HSP72 fusion protein allows reformulation into existing modes of delivery,
such as inhalers, that can be deployed close to phosgene
vulnerable sites. In that case, one must consider the option
of delivering a therapeutic dose intranasally (IN) versus intratracheally (IT) and weigh the amount of drug needed for
either administration. In mice, IN delivery of volumes less
than 35 lL have been reported to be distributed primarily to
the upper respiratory tract, whereas a 50 lL volume was predominantly deposited in the lower respiratory tract, based
on studies tracking radiolabeled colloids (Southam et al.
2002; Turner et al. 2011). Our laboratory is exploring dry
powder formulations that can be placed into inhalers, stored
at room temperature and distributed to paramedics, front
line troops, police/fire units for rapid deployment.
Efficacy postexposure
Efficient storage, distribution and drug administration
Recently, independent laboratory confirmation that delivery
of HSP72 to the lungs is a viable option comes from a group
that virally transduced mesenchymal stem cells (MSC) to
overexpress HSP70 before intra-tracheally delivering 1 106
cells/50 lL into the lungs of phosgene intoxicated Sprague
Dawley rats (Jin et al. 2020). The results of the study showed
that exogenous HSP70 production in these MSCs can significantly reduce damage associated with phosgene, including
(1) the lung wet/dry ratio; (2) protein and cell content in the
BALF; and (3) the levels of inflammatory cytokine TNFa. The
same strategy also increased the anti-inflammatory cytokine
A prophylactic is unrealistic if responding to an accident or
act of terrorism. Case in point, there are small molecules that
can be provided to induce HSP72, such as geranylgeranyl
acetone (GGA), which requires lag times varying from 8 to
24 h in in vivo models, depending on the organ being targeted (Zhang et al. 2009). Based on the exposure models discussed above, the victim may be fully symptomatic or dead
within that time frame. The Fv-HSP72 strategy is not prophylactic and has no lag time. Immediate activity has already
been proven in vivo with cerebral and myocardial infarction
models in which delivery of the Fv-HSP72 postreperfusion
has reduced the infarct volume by 68% in the brain (Zhan
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et al. 2010) and reduced cardiomyocyte death by 43% in the
heart (Tanimoto et al. 2017), respectively. Early proof-of-concept data has been obtained in rats using the acute phosgene exposure model developed by our team (Hobson et al.
2019) followed by intravenous (IV) delivery of Fv-HSP72
30 min post-exposure (unpublished results). We are now in
the process of developing a formulation that delivers the
therapeutic agent directly into the lungs for testing in our
acute exposure model.
Efficacy in the broadest population
Induction of many HSP70 protein family members is attenuated with aging (Fargnoli et al. 1990; Pardue et al. 1992;
Heydari et al. 1993), dampening the effectiveness of any
small molecule strategy in older individuals. The Fv-HSP72
approach rapidly rescues lung tissue by placing therapeutic
doses of exogenous human HSP72 into cells at risk regardless of the age of the patient.
Rapid delivery and localization to the lungs
A small molecule inducer of HSP72 can lack specificity and
may suffer from a double-edged problem. Without targeting
specific cells, product potency can be diminished unless
excess material is delivered to the lungs. Conversely, excessive material delivered to the lungs can result in dangerous
side effects. For a small molecule that induces gene expression, the need to evaluate unintended induction of other
genes is critical. Thus, finding an optimal therapeutic window
can often be challenging. On the other hand, with Fv-HSP72,
we are targeting a cytoprotectant to the alveolar and bronchial cells that interface with the air; we are not delivering
our biologic to the systemic circulation or going beyond the
cells lining lungs and airways. Fv-HSP72 is designed to congregate in regions of traumatic injury (Hansen et al. 2006),
which it should be able to do in the lungs when it is delivered by dry powder or by aerosol. A further advantage is the
use of an scFv fragment and not the entire 3E10 antibody. If
an intact antibody were to be delivered to the lungs, one
must consider the work conducted by the Sakagami lab
which determined a relatively low rate of IgG transport
across the pulmonary epithelial barrier (80 ng/h in rats) using
the FcRn system (Sakagami et al. 2006). Such an absorption
rate, if allometrically scaled by body weight to a 70 kg
human would equate to 3 lg/h, far below what may be
delivered to a patient minutes after exposure. Furthermore,
there are an estimated 25 million alveolar macrophages and
their Fc receptors which can act as an IgG sink in the lungs
(Sakagami and Gumbleton 2006). Since Fv-HSP72 does not
possess the Fc portion of the 3E10 antibody, it should largely
remain in the lungs.
countermeasures that would allow for a clear FDA approval
pathway. Questions have arisen even about the applicability
of Haber’s Rule with or without modifications to determine
accurately phosgene’s toxicity at various exposure concentrations and times. Establishing a consensus will be critical to
allowing more testing of potential countermeasures under
the Animal Rule. To that end, our laboratory has developed a
robust model for acute, short-term, high-concentration inhalation of phosgene that is likely to occur at ground zero of
an industrial accident or terrorist attack. Furthermore, working with our collaborators, we believe the rapid intracellular
delivery of exogenous Fv-HSP72 can protect cells from
stress-based injury and this simple solution avoids the lag
times needed for endogenous HSP72 induction post-phosgene exposure. Although comprehensive toxicity studies of
Fv-HSP72 in all potential vulnerable populations (i. e. pediatric, geriatric, pregnant), have not been completed, it is not
anticipated that the addition of exogenous human HSP72 is
going to have a deleterious effect. A recent maximum tolerated dose study of 3 Fv-HSP72 variants did not find any
signs of gross toxicity in either males or female Sprague
Dawley rats (unpublished results).
Finally, one may ask what is the existing standard of care
any new therapy that is going to seek approval have to compete against? Ironically, despite the potential role of oxidative stress and inflammation in phosgene-induced acute lung
injury, clinical protocols currently advise providing victims
with supplemental oxygen, which appears to be the only
current treatment that results in improved survival, improved
arterial oxygenation and reduced lung edema (Russell et al.
2006; Grainge et al. 2010). That and “Keep quiet and warm.
Give coffee as a stimulant.”
Acknowledgements
The authors wish to thank Dr. Robert P. Casillas and Dr. Glenn T.
Reynolds for excellent and sage advice during a technical reading of
the manuscript.
Disclosure statement
MHP, RAR, and STH are compensated by Rubicon Biotechnology. MHP
and RAR are co-owners of Rubicon.
Funding
The acute phosgene exposure model developed by Rubicon and our
preliminary Fv-HSP72 studies in lung were supported by the National
Institute of Environmental Health Sciences (NIEHS) of the National
Institutes of Health (NIH) under CounterACT Award Number
R21ES024028. The content is solely the responsibility of the authors and
does not necessarily represent the official views of the NIH.
Future needs
Over 200 years after it was first synthesized, one of the most
toxic, yet critically important industrial reagents, has no
standard efficacy testing model for potential therapeutic
ORCID
Stephen T. Hobson
Missag H. Parseghian
http://orcid.org/0000-0001-6634-6264
http://orcid.org/0000-0001-6712-6925
TOXICOLOGY MECHANISMS AND METHODS
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