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.