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Inhalation therapy of AT-H201 in COVID-19 patients 1 AT-H201 constituents collectively are the most potent inhibitors of SARS-CoV-2 infectivity in VERO cells identified and mechanistically act as a chemical vaccine: Human safety data support rapid clinical development as inhaled therapy for COVID-19

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Abstract

The COVID-19 pandemic has infected 4.6 million people and caused over three hundred thousand deaths worldwide. There is no currently available preventative therapy and a vaccine, while under development, is not available. Recently the intravenous nucleotide analogue RNA virus inhibitor, remdesivir, was approved by the FDA under an Emergency Use Authorization for hospital use based on a modest improvement in hospitalization days. Rapid development of more effective therapeutics is needed and one approach is to repurpose other currently available drugs to work against the SARS-CoV-2 virus. AT-H201 was designed to act as a "chemical vaccine" based on a biophysical analysis of the eight separate envelope protein features of SARS-CoV-2 and corresponding host cell surface proteins and glycans which would be expected to be responsible for viral entry. The components of AT-H201, low molecular weight heparin (LMWH) and N-acetyl-cysteine (NAC), bind to the surface of SARS-CoV-2 and therefore would be predicted to inhibit viral infectivity. Here we report inhibition by the components of AT-H201 of SARS-CoV-2 infectivity of VERO cells at concentrations which were not cytotoxic. Incubation of SARS-CoV-2 with LMWH at 125 µM before VERO cell exposure, the mode for testing antibody activity, showed complete inhibition of SARS-CoV-2 infectivity, well below the expected pulmonary delivery dose. For comparison, the concentration of remdesivir needed for a similar level of inhibition was over 600-times higher than is expected to be achieved clinically. N-Acetyl-Cysteine was also effective in inhibiting viral entry at 25 mM, a dose that is 1/50 th the expected clinical dose. Cytoxicity was not seen at these concentrations for either drug. A review of non-clinical and clinical data for inhalation therapy of the components of AT-H201 in adult respiratory distress syndrome (ARDS), asthma, and burn-victim inhalation injury patients identifies 12 clinical studies in over 800 patients tested. The systemic toxicity from inhalation of the components of AT-H201 was analyzed, with a special focus on the anti-coagulant effects of LMWH. Systemic toxicity was minimal and an improvement in lung function was seen. These results, taken together, support the rapid clinical development of AT-H201 for the treatment of pulmonary complications in COVID-19 patients.
Inhalation therapy of AT-H201 in COVID-19 patients
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AT-H201 constituents collectively are the most potent inhibitors of SARS-CoV-2
infectivity in VERO cells identified and mechanistically act as a chemical vaccine:
Human safety data support rapid clinical development as inhaled therapy for COVID-19
Authors: Steven C. Quay*,1 Shu-Chih Chen,1 Janet Rea,1 H. Lawrence Remmel2
Abstract
The COVID-19 pandemic has infected 4.6 million people and caused over three hundred
thousand deaths worldwide. There is no currently available preventative therapy and a vaccine,
while under development, is not available. Recently the intravenous nucleotide analogue RNA
virus inhibitor, remdesivir, was approved by the FDA under an Emergency Use Authorization for
hospital use based on a modest improvement in hospitalization days. Rapid development of more
effective therapeutics is needed and one approach is to repurpose other currently available drugs
to work against the SARS-CoV-2 virus. AT-H201 was designed to act as a “chemical vaccine”
based on a biophysical analysis of the eight separate envelope protein features of SARS-CoV-2
and corresponding host cell surface proteins and glycans which would be expected to be
responsible for viral entry. The components of AT-H201, low molecular weight heparin (LMWH)
and N-acetyl-cysteine (NAC), bind to the surface of SARS-CoV-2 and therefore would be
predicted to inhibit viral infectivity. Here we report inhibition by the components of AT-H201 of
SARS-CoV-2 infectivity of VERO cells at concentrations which were not cytotoxic. Incubation of
SARS-CoV-2 with LMWH at 125 µM before VERO cell exposure, the mode for testing antibody
activity, showed complete inhibition of SARS-CoV-2 infectivity, well below the expected
pulmonary delivery dose. For comparison, the concentration of remdesivir needed for a similar
level of inhibition was over 600-times higher than is expected to be achieved clinically. N-Acetyl-
Cysteine was also effective in inhibiting viral entry at 25 mM, a dose that is 1/50th the expected
clinical dose. Cytoxicity was not seen at these concentrations for either drug. A review of non-
clinical and clinical data for inhalation therapy of the components of AT-H201 in adult respiratory
distress syndrome (ARDS), asthma, and burn-victim inhalation injury patients identifies 12 clinical
studies in over 800 patients tested. The systemic toxicity from inhalation of the components of
AT-H201 was analyzed, with a special focus on the anti-coagulant effects of LMWH. Systemic
toxicity was minimal and an improvement in lung function was seen. These results, taken
together, support the rapid clinical development of AT-H201 for the treatment of pulmonary
complications in COVID-19 patients.
Keywords: SARS-CoV-2; COVID-19; low molecular weight heparin (LMWH); N-acetyl-cysteine;
VERO cells; AT-H201
Author Affiliations: 1) Atossa Therapeutics, Inc., Seattle, WA; 2) Department of Pathology,
University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
*Corresponding Author: Steven@DrQuay.com; Dr. Steven Quay, Atossa Therapeutics, Inc.,
107 Spring Street, Seattle, WA 98104.
Inhalation therapy of AT-H201 in COVID-19 patients
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INTRODUCTION
The use of existing drugs for rapid deployment in the coronavirus disease-19 (COVID-19)
pandemic has led to the investigation of oral hydroxychloroquine (Gautret et al., 2020; Molina et
al., 2020) as an atypical antiviral drug as well as the recommendation to use subcutaneous
heparin for the systemic coagulopathy characteristic of SARS-CoV-2 infection (Lin et al., 2020),
the causative agent of the COVID-19 pandemic. The success of the pioneering use of inhaled
tobramycin for cystic fibrosis-related infections (Rose et al., 2010) to augment or replace systemic
delivery routes suggested a direct pulmonary delivery approach should be examined for the
COVID-19 pneumonia seen in the majority of hospitalized patients.
In this report, we examine the structural features of SARS-CoV-2, its mode of infectivity and
replication, and the host epithelial and endothelial cell surface features to determine whether two
drugs widely used by oral or parenteral routes of administration would be effective in treating
COVID-19, especially when delivered by inhalation. These drugs are low molecular weight
heparin (H) and N-acetylcysteine (NAC). Clinical trials of H-NAC delivered by inhalation for acute
respiratory distress syndrome (ARDS), burn patients with inhalation injury, and other pulmonary
conditions and diseases have already been conducted and support a recommendation that AT-
H201 be studied in clinical trials of COVID-19 patients infected with severe acute respiratory
syndrome, coronavirus-2 (SARS-CoV-2).
MATERIALS AND METHODS
Low molecular weight heparin (enoxaparin), N-acetyl-cysteine, and hydroxychloroquine were
obtained from Sigma-Aldrich. VERO cells were used by methods previously described (Shen et
al. 2018).
For experiments in which infectivity was being tested, either VERO cells in media or SARS-CoV-
2 viral particles were incubated with potential inhibitors for one hour. Then viral particles were
added to VERO cells and incubation continued for up to seven days. Cytotoxicity and viral-
induced cytopathological effects were examined by light microscopy.
RESULTS AND DISCUSSION
Structural Features of SARS-CoV-2
The SARS-CoV-2 human coronavirus is a 29,903-nucleotide, positive-strand RNA virus that is
associated with a variety of highly prevalent and severe diseases, including SARS and Middle
East respiratory syndrome (MERS).
Mode of Infectivity and Replication
SARS-CoV-2 is the seventh coronavirus to infect humans (Andersen et al., 2020). There are at
least four features that distinguish SARS-CoV-2 from the other coronaviruses and which may be
related to its virulence in humans:
1. A series of six-point mutations in the Spike Protein (SP) receptor binding domain
angiotensin converting enzyme 2 (ACE2) contact residues (residues 455 to 505) that have
partial homology with mutations found in previous coronaviruses and which confer a 10-
to 20-fold increased affinity for the cell entry receptor, ACE2 (Andersen et al., 2020);
2. A polybasic cleavage site (PBCS), not previously found in human coronavirus SP, with a
canonical furin protease peptide sequence (Andersen et al., 2020);
Inhalation therapy of AT-H201 in COVID-19 patients
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3. A main coronavirus protease, called 3C-like protease, which has a slightly higher turnover
rate than previous CoV-2 viral proteases but a >30-fold higher turnover rate than the
human rhinovirus 3C protease (Jin et al., 2020).
4. A transmembrane serine protease, TMPRSS2 that acts on the SP following ACE2 binding
and is necessary to prime cell entry (Lukassen et al., 2020).
An additional feature, shared by all other coronaviruses, is the presence of a viroporin which forms
multimeric, cationic, transmembrane channels reminiscent of model membrane channels such as
alamathicin (Quay and Latorre, 1982; Latorre, Miller, Quay, 1981).
Host Epithelial and Endothelial Cell Surface Features
Human epithelial and endothelial cells have at least three features important for coronavirus
infection:
1. ACE2, the putative primary SARS-CoV-2 viral binding receptor;
2. Cell surface heparan sulfate proteoglycans, identified as pre-ACE2 docking sites which
appear to accelerate ACE2 binding;
3. Furin, a cell surface proprotein convertase as a putative receptor and/or covalent
processor of SARS-CoV-2, and its role in infectivity.
Human coronavirus infectivity and the unique features of SARS-CoV-2 among coronaviruses that
can be targeted with pulmonary inhalation are summarized in Table 1.
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Table 1 SARS-CoV-2: Human Coronavirus Infectivity and Other Features
Host/Viru
s
Feature
Targeted
Structure Function Reference(s)
Virus
Spike Protein
(SP) binding
motif
Polybasic cleavage site is part
of the receptor binding domain
of CoV-
2 and has a basic
amino acid motif containing
nine R/K residues in close
proximity
Allows effective cleavage by
furin and other proteases and
has a role in determining viral
infectivity.
https://www.pnas.org/content/10
6/14/5871
Belouzard et al., 2009
Virus SP-
viral E
protein
interactions
Envelope proteins SP and E
protein are covalently bound
together via three cystine -S-S-
bonds; axial and peripheral E
proteins contain basic, cationic
motifs
Stabilizes the SP and facilitates
SP binding to ACE2; provides a
basis for virus binding to cell
surface heparan sulphate
proteoglycans
https://virologyj.biomedcentral.co
m/articles/10.1186/s12985-019-
1182-0;
Shoeman & Fielding 2019
https://www.ncbi.nlm.nih.gov/pu
bmed/32245806
Chen et al,, 2020
Virus Main
intracellular
protease
A CoV-2 intracel
lular viral
encoded cysteine-class
protease
A cysteine-class viral protease
essential for viral replication by
virtue of processing 11
separate, highly conserved
proteins from the overlapping
polyproteins, pp1a and pp1ab,
required for viral replication and
transcription
https://www.nature.com/articles/
s41586-020-2223-
y_reference.pdf
Virus Coronavirus
viroporin
Small (60 to 120 amino acids),
largely hydrophobic peptides
that form cationic channels in
the host membrane and that
are anchored to the host
membrane laterally with
phospholipid head group-
arginine amino acid residues
While not essential for
replication but their absence
weakens or attenuates the virus
and diminishes its pathogenic
effects
https://virologyj.biomedcentral.co
m/articles/10.1186/s12985-019-
1182-0
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Host/Viru
s
Feature
Targeted
Structure Function Reference(s)
Host
Transmembran
e protease,
serine 2,
TMPRSS2
Responsible for cleavage of
S1/S2 near R residues (R682,
R683, R685, and R667, R797)
Following SP binding to ACE2,
the cleavage of these S1/S2
motifs, primes CoV-
2 for cell
entry
https://www.embopress.org/doi/1
0.15252/embj.20105114
Host
Epithelial ACE2
virus receptor
ACE protein contains seven
extracellular cysteines, one
unusual free cysteine and three
disulfide
cystine residues and
these together contribute to
ACE three-dimensional
structure.
The primary SARS-CoV-2
receptor, found in high
concentration in nasal mucosa,
alveolar cells, and endothelial
cells
https://www.ncbi.nlm.nih.gov/pu
bmed/8755737
Host
Epithelial
heparan
sulphate
proteoglycan
viral recognition
site
Ubiquitous cell surface anionic
polymers
Coronavirus cell surface
recognition site
https://www.ncbi.nlm.nih.gov/pm
c/articles/PMC4942210/
Host
Furin, epithelial
surface viral
processing
enzyme, highly
expressed in
alveoli
Protease with a polybasic
peptide substrate specificity
Activation of the SARS
coronavirus spike protein via
sequential proteolytic cleavage
at two distinct sites by furin
https://www.ncbi.nlm.nih.gov/pu
bmed/32057769;
https://www.pnas.org/content/10
6/14/5871;
http://www.virology.ws/2020/02/
13/furin-cleavage-site-in-the-
sars-cov-2-coronavirus-
glycoprotein/
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Based in part on an understanding of the behavior of polybasic model peptides such as the
honeybee (Apis mellifera) venom, melittin (Quay and Condie, 1983), with respect to membrane
binding and fusion properties, The approved drug heparin, is a polysaccharide with the highest
anionic charge density found in nature, was identified as potential CoV-2 viral entry inhibitor based
in part on an understanding of the behavior of polybasic model peptides such as the honeybee
(Apis mellifera) venom, melittin (Quay and Condie, 1983), with respect to membrane binding and
fusion properties. N-acetyl-cysteine was identified as potential CoV-2 viral inhibitor by the
identification of several key cystine bridge structures that are important for viral cell surface
binding or entry permitted a proposal that and N- acetyl-cysteine might disrupt infectivity by the
known irreversible cleavage of the S1-S2- bonds with the formation of S1-S-NAC and NAC-S-
S2- moieties.
Table 2 lists the proposed mechanisms of action for the components of AT-H201 in inhibiting
SARS-CoV-2 infectivity.
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Table 2 Mechanism of Action for the Components of AT-H201 in the Inhibition of SARS-COV-2 Infectivity
Feature Targeted Structure
Spike Protein (SP) binding motif
Polybasic cleavage site is part of the receptor binding
domain of CoV-
2 and has a basic amino acid motif
containing nine R/K residues in close proximity
Heparin:
preventing SP docking
SP-viral E protein interactions
Envelope proteins SP and E protein are covalently
bound together via three cystine -S-S- bonds; axial and
peripheral E proteins contain basic, cationic motifs
destabilization;
Main intracellular protease
A CoV-2 intracellular viral encoded cysteine-class
protease
NAC: Irreversible enzyme inhibitor
Transmembrane protease,
serine 2, TMPRSS2
Responsible for cleavage of S1/S2 near R residues
(R682, R683, R685, and R667, R797) Heparin: Electrostatic interference with
S1/S2 priming, by creating a fusion motif
Coronavirus viroporin
Small (60 to 120 amino acids), largely hydrophobic
peptides that
form cationic channels in the host
membrane and that are anchored to the host
membrane laterally with phospholipid head group-
arginine amino acid residues
Heparin: Electrostatic interference with
arginine-phospholipid binding a viroporin
collapse
Epithelial ACE2 virus receptor
ACE protein contains seven extracellular cysteines,
one unusual free cysteine and three disulfide cystine
residues and these together contribute to ACE three-
dimensional structure.
NAC: Cystine cleavage with reduced SP
binding affinity
Epithelial heparan sulphate
proteoglycan viral recognition
site
Ubiquitous cell surface anionic polymers Heparin: A soluble decoy substrate for
CoV-2 binding
Furin, epithelial surface viral
processing enzyme, highly
expressed in alveoli
Protease with a polybasic peptide substrate specificity Heparin: in vitro
inhibition of furin
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The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain
undergoes a substantial conformational change upon heparin binding, as predicted (Mycroft-West
et al., 2020). We described here how heparin can bind to the SARS-CoV-2 SP, presumably
through the unique, solvent accessible nine amino acid motif of the polybasic cleavage site, and
that SP undergoes a significant change in secondary protein structure.
SARS-Cov-2 Infectivity in VERO cells and inhibition by AT-H201
In order to test if AT-H201 had antiviral activity against SARS-CoV-2 the standard VERO cells
assay was performed. Test compounds were incubated either with the virus, to simulate antibody-
like surface binding, or with the cells, to simulate either cell surface changes or to act
intracellularly. Serial dilutions were conducted and the cytopathic effect (CPE) was observed by
microscopy. Non-virus related cytotoxicity, if present was also noted.
N-Acetyl-cysteine did not inhibit infectivity when incubated with SARS-CoV-2 but at 25 mM was
able to completely inhibit infectivity. On the other hand, LMWH was effective either when
incubated with the virus or when incubated with the cells. The expected clinical dose of LMWH
and NAC are large multiples of the doses that inhibited SARS-CoV-2 in these in vitro experiments
and compare favorably to the results seen for hydroxychloroquine and remdesivir.
The graph below shows the dose response for heparin in the microscopic cytopathic assay.
Test Compound
Incubation
Conditions
Inhibition of Virus
Infection
Clinical Dose/ In vitro
Effective Dose
Source
LMWH
Both with virus and
with VERO cells
100% at 150 µM>5 This study
NAC
With cells 100% at 25 mM >50 This study
Hydroxychloroquine With cells
100% at 16 µM <0.1 This study
Remdesivir With cells
100% at 100 µM <0.1
Choy K-T, et al. Antiviral
Research, 178 (2020),
104786; Sheahan et al., 2017
0
20
40
60
80
100
120
0 0.5 1 1.5 2 2.5
Percent Cytopathic Effect
Heparin (mg/mL)
Virus Induced Cytopathic Effect
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Finally, in an assay examining the presence of SARS-2 N protein in cells by immunostaining,
pretreatment of either the VERO cells or SARS-CoV-2 with LMWH was capable of preventing all
virus replication. Test conditions and results are shown below.
Previous clinical and non-clinical studies with AT-H201
An extensive body of non-clinical and clinical studies support the use of H-NAC for ARDS,
inhalation injury, idiopathic pulmonary fibrosis, and other pulmonary conditions (Tables 3-5).
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Table 3 Nonclinical/In Vitro Studies
Study Purpose
Study Summary
References
Compare genomic data to
deduce the origin of SARS-CoV-
2 and to
describe notable
features of SARS-CoV-2
genome and how they occurred.
These analyses clearly show that SARS-CoV-2 is not a laboratory construct or
a purposefully manipulated virus.
SARS-CoV-2 appears to be optimized for binding to the human receptor ACE2.
The spike protein of SARS-CoV-2 has a functional polybasic (furin) cleavage
site at the junction of S1 and S2, the two subunits of the spike, through the
insertion of 12 nucleotides. This allows for effective cleavage by furin and other
proteases and aids in determining viral infectivity and host range. A leading
proline was also inserted at this site in SARS-CoV-2 which led to the predicted
acquisition of three O-linked glycans around the cleavage site, a feature unique
to SARS-CoV-2.
https://www.nature.com/articles/s
41591-020-0820-9.pdf
Gain insight into SARS-CoV
biopathology using a SARS-
CoV isolate from a sputum
sample collected from an Italian
patient affected by a respiratory
disease of unknown cause.
The viral strain from this isolate was designated HSR1. Analysis of infected Vero
cells showed virions within cell vesicles and around the cell membrane. The full-
length viral genome sequence was similar to that of the Hong-Kong Hotel M
isolate. Approximately 360 viral genomes were required to generate a PFU.
Heparin (100 mg/mL) inhibited infection of Vero cells by 50%. Overall, the
molecular and biologic characteristics of the strain HSR1 provide evidence that
SARS-CoV forms a fourth genetic coronavirus group with distinct genomic and
biologic features.
https://www.ncbi.nlm.nih.gov/pmc
/articles/PMC3322807/pdf/03-
0683.pdf
Use spectroscopic techniques
with molecular modelling to
measure the interaction
between the SARS-CoV-2
Spike S1 protein receptor
binding domain (SARS-CoV-2
S1 RBD) and heparin.
SARS-CoV-2 S1 RBD binds to heparin which leads to a significant structural
change in the RBD. Basic amino acid residues that make up the heparin binding
domains are solvent accessible on the SARS-CoV-2 S1 RBD surface and form
a continuous patch that allows for heparin binding.
https://www.biorxiv.org/content/1
0.1101/2020.02.29.971093v1
Analyze the spike glycoprotein
of SARS-CoV-2 with its furin-like
cleavage site.
Focus was on a specific furin-like protease recognition pattern in a maturation
site of the S protein of SARS-CoV-2. The coronavirus S-protein is the structural
protein responsible for the crown-like shape of the CoV viral particles. A peculiar
furin-like cleavage site in the Spike protein of SARS-CoV-2, lacking in the other
SARS-like CoVs, was identified.
https://www.ncbi.nlm.nih.gov/pmc
/articles/PMC7114094/pdf/main.p
df
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Study Purpose
Study Summary
References
Determine the role of lactoferrin
(LF) in the entry of SARS
pseudovirus into
HEK293E/ACE2-Myc cells
HSPGs provide the preliminary docking sites on the cell surface and play an
important role in SARS pseudovirus cell entry. LF can block the infection of
SARS pseudovirus by binding to HSPGs, suggesting it may exert a protective
role in host immune defense against SARS-CoV invasion.
https://www.ncbi.nlm.nih.gov/pmc
/articles/PMC3161750/pdf/pone.0
023710.pdf
Investigate ACE2 and
TMPRSS2 expression
levels and their distribution
across cell types in lung tissue
and in cells derived from sub-
segmental bronchial branches.
TMPRSS2 is expressed in both tissues. In the subsegmental bronchial
branches, ACE2 is predominantly expressed in a transient secretory cell type.
These transiently differentiating cells show an enrichment for pathways related
to RHO GTPase function and viral processes suggesting increased vulnerability
for SARS-CoV-2 infection.
https://www.biorxiv.org/content/1
0.1101/2020.03.13.991455v3.full.
pdf
Define the molecular
mechanism that underlies viral
infection with SARS-CoV-2.
SARS-CoV-2 showed a superior plasma membrane fusion capacity compared
to that of SARS-CoV.
The solved X-ray crystal structure of 6-helical bundle core of the HR1 and HR2
domains in the SARS-CoV-2 S protein S2 subunit, revealed that several mutated
amino acid r
esidues in the HR1 domain may be associated with enhanced
interactions with the HR2 domain.
EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-
mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and
15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide,
respectively.
EK1C4 was highly effective against membrane fusion and infection of other
human coronavirus pseudoviruses tested, including SARS-CoV, MERS-CoV,
and SARSr-CoVs, and potently inhibited
the replication of 5 live human
coronaviruses, including SARS-CoV-2.
Intranasal application of EK1C4, before or after challenge with HCoV-OC43,
protected mice from infection suggesting that EK1C4 could be used for
prevention and treatment of infection by the currently circulating SARS-CoV-2
and other emerging SARS-CoVs.
https://www.nature.com/articles/s
41422-020-0305-x
Determine the structure of main
protease (Mpro) from COVID-19
virus and its inhibitors.
Identified a mechanism-based inhibitor, N3, and determined the crystal structure
of COVID-19 virus Mpro in complex with this compound.
Convergence of structure-based ab initio drug design, virtual screening, and
high-throughput screening was efficient to find antiviral leads against COVID-19
virus.
https://www.nature.com/articles/s
41586-020-2223-y_reference.pdf
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Study Purpose
Study Summary
References
Review the main molecular
mechanisms that explain the
antioxidant and reducing activity
of N-acetylcysteine (NAC).
The antioxidant effect of NAC is associated with its role as a reduced precursor
of glutathione (GSH), a direct antioxidant and a substrate of several antioxidant
enzymes.
NAC acts as a direct antioxidant for some oxidant species (NO2 and HOX) with
a significant depletion of endogenous Cys and GSH.
Antioxidant activity of NAC leads to the breakage of thiolated proteins, releasing
free thiols
and reduced proteins.
Disulphide breaking by NAC reduces heavily cross-linked mucus glycoproteins
causing mucolytic activity.
Chemical features of NAC contribute to its efficient disulphide breaking activity.
https://pubmed.ncbi.nlm.nih.gov/2
9742938/
Determine functional
implications of free and
disulfide-
bonded cysteine
residues in human testis ACE.
Human testicular ACE (tACE) is an extracellular protein that contains seven
cysteine residues in a sequential distribution found in the tACE from rabbit and
mouse, and in both domains of all known species of somatic ACE. One of the
cysteines in human tACE, Cys496, is present in the reduced form and is not
involved in catalysis. The other 6 cysteines exist as 3 disulfides which form 3
small loops that contribute to the overall structural stabilization of tACE. These
results may contribute to the current understanding of ACE2 during SARS-CoV-
2 infection.
https://www.ncbi.nlm.nih.gov/pub
med/8755737
Identify the structure of Eastern
equine encephalitis virus
(EEEV) complexed with the
heparan sulfate (HS) analog
heparin.
HS proteoglycan on host cell surfaces has been identified as a host attachment
factor/receptor for EEEV infection. In this study, the first cryoelectron microscopy
structure of the wild-type EEEV has been examined when bound to heparin, an
HS-mimicking polysaccharide. The identified axial and peripheral HS binding
sites on each spike show structural similarity. The discovered HS binding sites
on EEEV may also serve as potential targets for the development of antiviral
agents against this highly infectious alphavirus. These results may contribute to
the understanding of the role of heparin in SARS-Cov-2 infection.
https://www.pnas.org/content/earl
y/2020/04/02/1910670117
Explore the effects of heparin on
Zika Virus (ZIKV) infection in
human neural
progenitor cells
(hNPCs), in particular, virus
replication and induction of
cytopathic effects (CPEs)
resulting in cell death.
Heparin exerted a modest inhibitory effect on ZIKV replication, and prevented
virus-induced cell death of hNPCs.
hNPCs incubated
with heparin (100 mg/ml, 1 h prior to infection with ZIKV) led
to modest decreases (not statistically significant) in infected cell numbers.
Heparin reduced the production of progeny virus by 2- to 3-fold.
Heparin did not exert toxic effects on uninfected cells. It prevented the virus-
induced CPEs of 2 cultures infected separately with 2 ZIKV strains. Heparin
partially uncoupled the virus replication from the virus-induced death of hNPCs.
These results may contribute to the current understanding of the eff
ect of
heparin in SARS-CoV-2 infection.
https://www.ncbi.nlm.nih.gov/pmc
/articles/PMC7113768/pdf/main.p
df
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Inhalation therapy of AT-H201 in COVID-19 patients
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Table 4 Clinical Studies
Disease
Study Design
Treatment
Results
Conclusions
References
IPF
Dose escalation study to
determine the PK and safety and
tolerability of UFH in healthy
volunteers (n=19) and patients
with IPF (n=20).
UFH
Healthy: Inhaled 150,000 IU UFH
significantly increased PTT and anti-factor Xa
activity
indicating threshold dose. Local
alveolar anticoagulant effect up to 72 h;
alveolar half-life ~28 h
IPF:
No acute deleterious effects on
pulmonary function, gas exchange, or
exercise capacity with inhaled threshold dose
(150,000 IU). During chronic treatment, 25%
of threshold dose inhaled every 12 h for 28
days for steady-state anticoagulant activity in
alveolar space: no heparin-
related side
effects; median lung function values,
exercise capacity, and QoL scores
unchanged. Three AEs and one SAE
reported a
nd considered unlikely or no
relation to UFH inhalation.
Inhaled heparin
appears to be safe
and well tolerated in
IPF patients.
https://www.resea
rchgate.net/public
ation/41172444_S
afety_and_Tolera
bility_of_Inhaled_
Heparin_in_Idiopa
thic_Pulmonary_F
ibrosis
IPF
A multicentre, prospective,
randomized, controlled trial
conducted to assess the efficacy
of inhaled NAC monotherapy in
patients with mild to moderate
IPF.
NAC: n=38; 352.4 mg inhaled
twice daily
Control: n=38; no therapy.
Primary endpoint: the change
from baseline in FVC at 48
weeks.
NAC
No significant overall differences between
NAC vs control in the cha
nge in FVC.
NAC therapy associated with stability of FVC
in (i) patients subset with initial FVC <95% of
predicted (n = 49; difference in FVC decline
0.12 L;
P=0.02) and (ii) patients with initial
diffusing capacity of CO <55% of predicted (n
= 21; difference in FVC decline 0.17 L; P =
0.009).
NAC monotherapy
stabilized the serial
decline in FVC in
some patients without
the use of
immunosuppressive
or anti-fibrotic agents.
https://onlinelibrar
y.wiley.com/doi/ep
df/10.1111/j.1440-
1843.2012.02132.
x
Inhalation therapy of AT-H201 in COVID-19 patients
15
Disease
Study Design
Treatment
Results
Conclusions
References
IPF
In this retrospective study,
advanced-stage IPF was defined
as relative decline in FVC of
≥10% within preceding 6 (±2)
months
IPF pretreatment: inhaled NAC
monotherapy
IPF pretreatment: prednisolone
(20 mg/day) + inhaled NAC
IPF pretreatment:
immunosuppressive treatment
with prednisolone (10-20
mg/day) + tacrolimus (2mg/day)
No pretreatment for IPF
Relative declines in FVC <10% =
stable disease (effective group)
Relative declines in FVC >10% =
progressive disease (ineffective
group)
NAC +
Pirfenidone
Pirfenidone stabilized declines in FVC by
10% at 6 months in 8 of 18 cases (44%).
Median changes in FVC at 6 months in
effective group = +120 mL; ineffective group
= -590 mL.
No. of NAC users significantly higher in
effective group (7/8=87.5%) vs ineffective
group (3/10=30%) (p=0.02).
Median change in FVC at 6 months in NAC
group = 0 mL; nonNAC group = -
290 mL.
Median survival period in NAC group =
557±66 days; nonNAC group = 196±57 days
(p=0.03).
Combined NAC
therapy was
correlated with a
favorable outcome.
https://www.jstage
.jst.go.jp/article/int
ernalmedicine/52/
22/52_52.8498/_p
df/-char/en
IPF
Retrospective study of 34
patients with IPF.
NAC inhalation monotherapy in
23 of 34 patients.
Patients inhaled 352.4 mg NAC
twice/day.
No treatment for 11 patients.
Examined clinical courses,
prognosis, lung function (%FVC,
%DLco, %TLC), and changes in
serum markers for interstitial
pneumonia.
NAC
14 patients continued NAC inhalation ≥1
year.
Average period of NAC inhalation = 29.9
months (12-
88 months).
5 patients continued treatment ≥2 years.
Death most frequent cause for stopping NAC
inhalation, followed by ADRs.
Of 9 patients who continued NAC inhalation
<1 year, AEs kept 4 patients from continuing.
Clinical course and changes in respiratory
test results showed various patterns.
No significant differences in survival curves
between treated and not treated.
Acute exacerbation in 4 of 14 patient (28.6%)
receiving NAC inhalation.
Compared results just before initiating NAC
inhalation, Δ%FVC and Δ%DLco in treated
patients were -4.7% and -2.9% 1 year later
and -4.0% and -5.8% 2 years later,
When studying the
efficacy of treatment
for mild IPF, need to
consider clinical
information from 6
months and 1 year
before treatment,
instead of only one
time point before
initiating treatment,
and to evaluate
changes over a longer
time period.
https://www.jstage
.jst.go.jp/article/int
ernalmedicine/49/
21/49_21_2289/_
pdf/-char/en
Inhalation therapy of AT-H201 in COVID-19 patients
16
Disease
Study Design
Treatment
Results
Conclusions
References
respectively. In patients without treatment,
Δ%FVC and Δ%DLco were -3.5% and +5.3%
1 year later and +0.2% and +1.0% 2 years
later, respectively.
EIB
Double-blind, randomized,
crossover design.
13 asthmatic subjects studied on
7 different experiment days.
EIB assessed by FEV1 before
and immediately after exercise.
On 5 different experiment days,
subjects were pretreated with 4
ml of aerosolized UFH (80,000
units=7.5 mg/kg), placebo, or 3
different doses of enoxaparin
(0.5 mg/kg, 1 mg/kg, 2 mg/kg);
exercise challenge was
performed 45 min later.
Bronchial provocation with
methacholine performed in 5
subjects, on 2 additional days
after pretreatment, and treated
with placebo or enoxaparin (2
mg/kg). Plasma antifactor Xa
was tested.
LMWH
(enoxapari
n)
UFH
Maximal decreases in FEV1 (mean ± SE)
after exercise = 30±4% and 29±5% on control
and placebo days.
Exercise-
induced decreases in FEV1
inhibited by 31% with UFH (change in FEV1
= 20±4%); and by 28%, 38%, and 48% by
enoxaparin at doses of 0.5 mg/kg (change in
FEV1=21±5%), 1 mg/kg (change in
FEV1=18±5%), and 2 mg/kg (change in
FEV1=15±3%), respectively (p< 0.05).
Inhibitory effect of 0.5 mg/kg enoxaparin was
comparable to UFH (7.5 mg/kg); 2 mg/kg
enoxaparin was the most potent.
Inhaled enoxaparin (2 mg/kg) had no effect
on methacholine-induced
bronchoconstriction and did not change the
plasma antifactor Xa activity.
Inhaled enoxaparin
prevents EIB in a
dose-dependent
manner, and its
antiasthmatic activity
is independent of its
effect on plasma
antifactor Xa activity.
https://www.atsjou
rnals.org/doi/pdf/1
0.1164/ajrccm.16
0.2.9812076
Coagul-
opathy
In this dose escalation study
design, determine anticoagulant
action of inhaled LMWH on
release of TFPI and on antifactor
Xa activity, Heptest coagulation
times, APTT, and TCT.
LMWH given to 20 healthy
volunteers each at 4 weeks
intervals by inhalation.
Group 1: 3000 IU LMWH
Group 2: 9000 IU LMWH
LMWH
APTT and TCT did not change after
inhalation of any dose of LMWH.
Group 1: No change in all parameters.
Group 2
: Heptest coagulation times were
prolonged: 18.7 ± 2.0 s before inhalation to
26.1 ± 5.2 s 6 h inhalation and 20.5 ± 1.9 s 24
h after inhalation. Other parameters
remained unaffected.
Group 3: Antifactor
Xa activity and plasma
LMWH increased 6 h after inhalation,
returned to normal values after 24 h. TFPI
The data demonstrate
a resorption of LMW-
heparin by
intrapulmonary route
in man. The
dose to
produce antifactor Xa
levels, prolongations
of Heptest
coagulation values
and in releasing TFPI
is about ten-fold
https://journals.lw
w.com/bloodcoag
ulation/abstract/19
96/06000/anticoa
gulant_effects_an
d_tissue_factor_p
athway.8.aspx
Inhalation therapy of AT-H201 in COVID-19 patients
17
Disease
Study Design
Treatment
Results
Conclusions
References
Group 3: 27 000 IU LMWH
Group 4: 54 000 IU LMWH
antigen increased from 74.1 ± 13.9 to 80.5 ±
14.2 ng/ml 3 h after inhalation. TFPI activity
remained unchanged. Heptest coagulation
values were prolonged to 42 ± 7.6 s after 6 h
and returned to normal within 72 h after
inhalation.
Group 4: Antifactor Xa activity
increased to
0.343 ± 0.196 U/ml after 6 h and normalized
after 72 h. The protamine assay detected 0.2
± 0.18 U LMWH/ml after 6 h, TFPI antigen
i
ncreased to 103 ± 17.9 ng/ml, and TFPI
activity to 1.14 ± 0.23 U 3 h after inhalation.
All tests were normal after 24 h. Heptest
coagulation values increased to 77.5 ± 11.8 s
6 h after inhalation and normalized after 144
h. The area under the activity time curve and
of the Heptest assay increased with
increasing doses (r = 0.677 and r = 0.571),
respectively. The calculated elimination half-
life of the aXa-effect was 7.5 h.
higher than after
subcutaneous
administration.
Lung
function
and
coagula-
tion
Determine the safety of
increasing doses of inhaled
heparin
on lung function and
coagulation in healthy volunteers
UFH
Lung function variables were not affected by
inhaled UFH. No significant changes in the
AUC for APTT. Inhaled UFH resulted in
significant, dose-
dependent changes in the
AUC for anti-Xa activity. AUC for TFPI did not
change significantly. Inhaled UFH did not
cause any change in the
AUC for WBCT, platelets, vWF or CRP. No
spontaneous, prolonged, or excessive
bleeding, or breathing difficulties were
reported after UFH inhalation.
Inhalation of ≤32,000
IU of UFH as a lower-
respiratory tract dose
is safe.
https://erj.ersjourn
als.com/content/1
9/4/606
Burn,
ARDS,
risk of
ARDS
A systematic review including
critically ill patients with lung
injury and treated with nebulized
anticoagulants.
Heparin+N
AC
Eleven publications in this review were based
on human trials with patients who had
inhalation trauma, ARDS, or increased risk
for ARDS.
Results from patients
with lung injury who
received nebulized
heparin in were
variable and systemic
side effects were of
concern.
http://atm.amegro
ups.com/article/vi
ew/16388/html
Inhalation therapy of AT-H201 in COVID-19 patients
18
Disease
Study Design
Treatment
Results
Conclusions
References
Inhalatio
n injury
A retrospective study to
determine the efficacy and safety
of nebulized heparin in
mechanically ventilated adults
with IHI.
Heparin
Primary outcome: duration of mechanical
ventilation.
Secondary outcomes: lung injury score,
ventilator-free days during the first 28 days,
28-
day mortality, hospitalization length,
ventilator-
associated pneumonia incidence,
bronchoscopy incidence, and bleeding
events.
Nebulized heparin
was safe and did not
result in an increase in
bleeding events.
https://academic.o
up.com/jbcr/article
-
abstract/38/1/45/4
568904?redirecte
dFrom=fulltext
Acute
lung
injury
Systematic review that included
clinical trials on the efficacy and
safety of nebulized
anticoagulants in the treatment of
acute lung injury in humans.
Heparin
Nebulized heparin improved survival of
patients with smoke inhalation-induced ALI.
In criticall
y ill patients on mechanical
ventilation more than 2 days, nebulization of
heparin was associated with a higher number
of days that patients were ventilator-free.
Nebulization of heparin had an effect on
systemic coagulation,
but did not cause
systemic bleedings.
Limited data show
that nebulized heparin
may be beneficial and
safe for patients with
acute lung injury.
https://ccforum.bio
medcentral.com/a
rticles/10.1186/cc
11325
Other
pulmonar
y
condition
The development of novel
coronavirus vaccines is
underway at pharmaceutical and
biotechnology companies and
research organisations around
the world. Some of these
endeavors are listed on this web
site.
https://www.clinic
altrialsarena.com/
analysis/coronavir
us-mers-cov-
drugs/
In addition to the nonclinical and clinical studies in the literature, a recent anecdotal report
(personal communication) revealed that a single COVID-19 patient on mechanical ventilation has
been given nebulized heparin and within 24 hours a “good response” was reported. No other
details are available.
CONCLUSION
Based on the in vitro activity of AT-H201 components in potently inhibiting SARS-CoV-2 infectivity
in VERO cells, a standard model for testing potential pharmaceuticals as anti-viral agents together
with the demonstrated non-clinical animal and clinical studies of the drug combination the
development of this treatment by the inhalation/nebulized route should be encouraged.
Inhalation therapy of AT-H201 in COVID-19 patients
20
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Background Chloroquine and hydroxychloroquine have been found to be efficient on SARS-CoV-2, and reported to be efficient in Chinese COV-19 patients. We evaluate the role of hydroxychloroquine on respiratory viral loads. Patients and methods French Confirmed COVID-19 patients were included in a single arm protocol from early March to March 16th, to receive 600mg of hydroxychloroquine daily and their viral load in nasopharyngeal swabs was tested daily in a hospital setting. Depending on their clinical presentation, azithromycin was added to the treatment. Untreated patients from another center and cases refusing the protocol were included as negative controls. Presence and absence of virus at Day6-post inclusion was considered the end point. Results Six patients were asymptomatic, 22 had upper respiratory tract infection symptoms and eight had lower respiratory tract infection symptoms. Twenty cases were treated in this study and showed a significant reduction of the viral carriage at D6-post inclusion compared to controls, and much lower average carrying duration than reported of untreated patients in the literature. Azithromycin added to hydroxychloroquine was significantly more efficient for virus elimination. Conclusion Despite its small sample size our survey shows that hydroxychloroquine treatment is significantly associated with viral load reduction/disappearance in COVID-19 patients and its effect is reinforced by azithromycin.