Inhalation of Low Molecular Weight Heparins as a prophylaxis to prevent SARS-CoV-2 infection

The ongoing Coronavirus disease 2019 (COVID-19) pandemic has placed a high
burden on medical resources as well as cause a large number of deaths.
Moreover, it has led to a severe social and economic disruption and there is
therefore an urgent need for quick and simple prophylactic agents to stop
transmission of the SARS-CoV-2. Strikingly, we have identified low molecular
weight heparins (LMWH) as potent inhibitors of SARS-CoV-2 infections in vitro.
Because the airways are the major route of infection for SARS-CoV-2 we will
investigate whether intranasal application of LMWH will prevent SARS-CoV-2
binding to epithelial cells ex-vivo. To investigate this, we have designed a
study in which healthy volunteers will be exposed to LMWH via nasal inhalation
and epithelial cells will be isolated with nasal brushes. Next we investigate
the binding of SARS-CoV-2 to the epithelial cells.

1. Primary objective:
- SARS-CoV-2 binding (expressed as concentration of virus bound in pg/mL) to
epithelial cells isolated from nasal cavity between intervention and control
groups (every volunteer is his own control group, depending on nostril). A
second control group will consist of twelve clinically admitted patients
receiving therapeutic doses of LMWHs for thrombotic events.
2. Secondary objectives:
- Descriptive characterization of activation markers and cytokine production by
nasal epithelial cells ex vivo after LMWH application

A single center, open label intervention study. Cohort 1 consists of healthy
volunteers (n=12) that serve as their own internal control. Every volunteer
will receive a saline solution (NaCl 0.9%) in the left nostril. Epithelial
cells will be retrieved using a nasal brush from the left nostril.
Subsequently, enoxaparin (Clexane Forte, with a concentration of 150mg/ml) will
be administered in the right nostril after which cells will be isolated after
30minutes.
To ensure adequate delivery to the nasal epithelium we will use a MAD Nasal*
Intranasal Mucosal Atomization Device (made by Telefex medical). This allows us
to nebulise our compounds to allow a good spread through the nasal cavity. We
use a volume of 370µL per volunteer, with 70µL remaining in the MAD device due
to dead space. This applies a volume of 300µL evenly to the nasal epithelium
(corresponding with 45mg of enoxaparin).

We expect that the LMWH exposure will limit virus binding. Moreover, we will
analyse the phenotype of isolated epithelial cells to investigate effect of
LMWH on epithelial activation.

The effect of the regular subcutaneous treatment with LMWH on virus binding
will be assessed in Cohort 2. ,This cohort consists of a total of 12 patients
that are treated with therapeutic doses of LMWH for medical reasons.
Virus-epithelial cell interaction will be analysed from nasal brushes, that are
collected at 4 hours after administration of LMWH to allow for proper
redistribution throughout the body (based on the TMAX of Enoxaparin).
Epithelial cells that are isolated from nasal brushes will be incubated with
SARS-CoV-2 virus and binding of virus to epithelial cells will be measured.
Here we will investigate whether subcutaneous injection of LMWH protect
epithelial cells from SARS-CoV-2 infection. Moreover, we will analyse the
phenotype of isolated epithelial cells to investigate effect of LMWH on
epithelial activation.

Extension of the study (May 2021):
Cohort 1 will be expanded from 12 to 24 volunteers. This choice is made as the
original SOP for applying study medication and withdrawing cells for virus
exposure proved ill-suited for a clinical setting. Therefore material provided
by volunteer 1 to 7 gave largely inconsistent results. Furthermore, the amount
of cells provided by volunteers shows large interpersonal variability,
resulting in some volunteers not providing enough cells for comparative
analysis.
An expansion of volunteers to 24 total will give enough power to compare
results.

Volunteers in cohort 1 will serve as their own internal control. Every
volunteer will receive a total of 300uL saline solution (NaCl 0,9%) in the left
nostril, delivered in three deliveries of 100uL using the MAD Nasal Intranasal
Mucosal Atomization Device at ten minute intervals.
After thirty minutes cells will be isolated using a nasal brush.
Subsequently, 300 uL enoxaparin (Clexane Forte, at 150mg/ml concentration) will
be administered in the right nostril in three deliveries of 100uL at ten minute
intervals. After thirty minutes cells will be isolated using a nasal brush.

Cohort 2 will be changed to include four volunteers instead of twelve clinical
patients. The choice to volunteer is made as within three months of the study
start no clinically admitted patients were eligible or willing to participate
with the study.
Furthermore, those patients who were eligible but decided not to participate
were much older and had multiple co-morbidities, making comparison with cohort
1 problematic.
Therefore, to assure a more comparable study population to cohort 1, cohort 2
will be made up of volunteers, preferably volunteers from cohort 1 will be
asked to participate (after the follow-up period has expired) in cohort 2.
The change in number of volunteers is made to lessen the burden on our
participants. Due to the mechanism of virus-block it is not expected that
subcutaneously administered heparins will have an effect on virus-block.
Therefore it would place an unjust burden on volunteers to have a group size of
12. If no block is observed, participation will stop at 4, if block is
observed, a new amendment will be created to increase the size of this group to
the same size as cohort 2.
Volunteers participating in cohort 2 will receive subcutaneous 1 dose of
therapeutic enoxaparin (100IE/kg) administered abdominally. 4 Hours (Tmax)
after injection cells with be retrieved using a nasal brush comparable to
cohort 1.

370µL enoxaparin solution (Clexane Forte 150mg/ml) and saline (0.9%NaCl)
applied with the MAD Nasal* Intranasal Mucosal Atomization Device will be
administered in the left and right nasal cavity respectively in cohort 1.

Extension of the study (May 2021):
Cohort 2 will receive a single subcutaneous injection of enoxaparin at
therapeutic levels (100IE/kg bodyweight) administered in the abdominal region
in comparable to normal clinical practices.

The intranasal application of enoxaparin is considered safe as no systemic
effects are expected for various reasons. Firstly the LMWH will be neutralised
on the nasal epithelium. Secondly, the applied doses (45 mg) are much lower
than the parenterally administered doses used anticoagulant treatment. Thirdly,
bronchially administered (inhaled) LMWH in much higher doses of up to 2mg/kg
was observed to leave the systemic coagulation unaffected, which was supported
by the lack of detectable anti-Xa activity.1,2

Risks associated with sampling that consists of a nasal brush are also
considered to be minimal.


Extension of the study (May 2021):
Cohort 2 will receive a single dose of therapeutic enoxaparin. Reported
associated risks at therapeutic levels are bleeding associated as well as
prolonged coagulation times. Furthermore a temporary increase of hepatic
enzymes, specifically transaminase increase, has been reported.
Most of these associated risks (such as bleeding) are time-dependent and become
the more risky the longer a patient has to use enoxaparin. Enoxaparin has a
half-life of 5 hours, which means within 24hrs almost all of it will be cleared
from the body.
Furthermore, enoxaparin has an antidote (protamine). Within the AMC there is a
lot of experience working with enoxaparin, as it*s widely used in the clinic as
an anticoagulant. We consider the risks of a single dose of enoxaparin very
low.