4 DISCUSSION
Some studies have already reported the association between treatment
with systemically administered heparin and lower mortality in patients
admitted to hospital with COVID-19 (Ayerbe, Risco et al., 2020; Tang,
Bai et al., 2020), assumed to be a consequence of the known
anticoagulant effect. Coagulopathies have caused major problems in late
stage COVID-19 disease (Kollias, Kyriakoulis et al., 2020) and the use
of heparin in the treatment of COVID-19 has become part of the standard
care for patients in ICU. However, the dose and type of heparin (UFH or
LMWH) delivered either by the sub-cutaneous or intravenous route varies
across the world. Regarding standard systemic usage, the therapeutic
range for UFH is typically 0.3 - 0.7 IU/ml (~2 - 4
μg/ml) (Hirsh, Anand et al., 2001), with peak dosing concentrations
reaching ~10 - 20 μg/ml. It is reassuring to see that
the ND50 of 12.5 μg/ml falls within the peak dosing
range suggesting that at least partial antiviral effects could be
expected in this setting.
In the UK, in addition to systemic heparin use, a novel approach of
delivering UFH via nebulisation directly to lungs of COVID-19 patients
is undergoing evaluation in a clinical trial (by the ACCORD clinical
trials platform (https://accord-trial.org/), seeking to gain benefit
from the additional known anti-inflammatory activity of heparin.
Nebulisation allows for the targeting of lung tissue directly and
therefore impact upon the local hyperinflammatory response and alveolar
coagulation resulting from SARS-CoV-2 viral load in the lung. During the
UK trial UFH (Workhardt) will be administered at 25,000 IU (125 mg)
every 6 h to patients. The efficiency of nebulising UFH through a high
efficiency mesh nebuliser is estimated to be about 20 %, thus the
delivered dose to the lung is ~ 25mg. Assuming the
normal human airway surface fluids are in the range 10 - 60 ml
(Frohlich, Mercuri et al., 2016), the peak amount of UFH delivered to
the lung should be ~400 - 2500 μg/ml, though these
values could be lower if diluted by increased fluid volumes as a result
of pulmonary oedema. Even allowing for this, these values greatly exceed
the ND50 of 12.5 μg/ml reported here for the same batch
of Workhardt UFH as used in the current UK clinical trial. Thus,
nebulisation of UFH should provide strong antiviral effects, in vivo.
Importantly, inhaled UFH does not cross the bronchial mucosa;
intravenous or subcutaneous routes are required for systemic delivery of
heparin as an anticoagulant.
Previous work has demonstrated that LMWHs also bind to the SARS-CoV-2
(Mycroft-West, Su et al., 2020b). However, in the present study we
observed that LMWHs were markedly less potent in live SARS-CoV-2 virus
assays (ND50 values of 2.6 - 6.8 mg/ml) than UFH. Using
the results reported here LMWH would appear unlikely to reach sufficient
concentration to achieve significant antiviral activity for either
systemic or nebulisation delivery. The typical therapeutic range for
LMWH for anticoagulant therapy is ~5 - 8 μg/ml (0.6 - 1
IU/ml). Consistent with our data, the relationship (fold difference,
295-fold) between UFH and enoxaparin seen here was similar to that
observed by Tandon et al, 2020 using a pseudotyped lentivirus inhibition
assay where 180-fold difference was seen (Tandon, Sharp et al., 2020).
In addition, LMWHs were also observed to be less potent than UFH for
inhibition of cell binding by spike protein (Partridge, Green et al.,
2020). However, an important caveat is that the potency of UFH and LMWHs
remain to be determined in a suitable range of human cells relevant to
those affected in individuals infected with SARS-Cov-2, perhaps
especially from respiratory tract tissues.
The results of the present study also suggest a dependency on molecular
weight for different UFH and LMWH preparations. A positive correlation
between molecular weight and antiviral activity was noted for the
various porcine LMWHs (4,200 - 6,600 Da) and UFH preparations tested
which supports the hypothesis that UFH is more active due to its higher
molecular weight (12,500 - 16,300 Da). Consistent with these live virus
data, molecular weight dependency for binding of heparin and heparan
sulfate saccharides to spike protein has been observed (Liu, Chopra et
al., 2020; Mycroft-West, Su et al., 2020a).
In the present study bovine mucosal UFH (ND50, 75 μg/ml)
had a slightly lower antiviral potency (~three-fold)
compared to porcine UFH. The potent antiviral activity seen for both
porcine and bovine heparins, suggests that this property is not species
dependent. Bovine UFH may provide an additional source of heparin to use
during the coronavirus pandemic. Currently therapeutic UFH available in
Europe and US is of porcine mucosal origin; however, owing to supply
issues there is now interest, specifically in the US, employing bovine
mucosal UFH as an additional source to improve the robustness of supply
chains (Hogwood, Mulloy et al., 2017; Keire, Mulloy et al., 2015).
The antiviral ND50 data for the different UFH and LMWHs
display no obvious correlation with anticoagulant activities (IU/mg),
indicating that different structure-activity relationships exist for
antiviral activity. Importantly, this suggests that further
investigation of non-anticoagulant heparins (Cassinelli, Torri et al.,
2020; Lindahl & Li, 2020) and heparin mimetics (Guimond, Mycroft-West
et al., 2020; Lindahl & Li, 2020) is warranted. Mimetics have
significant potential to target similar antiviral mechanisms and could
be delivered systemically at higher doses to improve efficacy without
potential side effects such as bleeding. Moreover, mimetics would also
provide a fully synthetic route to bypass limitations of heparin supply.
Here we provide evidence for the first time that various types of
commercially available and clinically used UFH preparations exhibit
potent antiviral efficacy against live wild-type SARS-CoV-2 in
vitro. This activity was seen across different brands of UFH and was
also observed with both porcine and bovine heparins. These data indicate
that current clinical use of systemic UFH in the treatment of COVID-19
patients in an ICU setting may provide useful antiviral benefits.
Moreover, we predict that the delivery of UFH to the lung (via
nebulisation) should provide a strong direct antiviral therapy in
addition to other documented beneficial effects of heparins.