Background and Purpose: Platelet function during inflammation is dependent on activation by endogenous nucleotides acting on purinergic receptors. The P2Y14 receptor (P2Y14R) has been reported to be expressed on platelets and is involved in leukocyte recruitment during inflammation. However, a role for P2Y14R receptors on platelet function has not yet been determined. Experimental Approach: Platelets obtained from healthy human volunteers were incubated with the P2Y14R agonist, UDP-Glucose (UDP-G), and PPTN, a selective P2Y14R antagonist. Platelet activation was quantified using Ca2+ mobilization, aggregation, and chemotaxis assays. Cooperativity with P2Y1 receptor (P2Y1R) activation was also assessed after stimulation with UDP-G in the presence of MRS2500, a selective P2Y1R antagonist. Key Results: Ca2+ mobilization occurred in platelets after incubation with UDP-G in a concentration-dependent manner, and this was suppressed in platelets treated with PPTN. Platelets did not aggregate, or bind to fibrinogen after incubation with UDP-G. However, platelet chemotaxis towards f-MLP was dependent on P2Y14R stimulation with UDP-G and this was reduced by Rho-GTPase inhibitors. Furthermore, UDP-G induced Ca2+ mobilization and chemotaxis were also inhibited when platelets were pretreated with MRS2500. Conversely, ADP induced Ca2+ mobilization, chemotaxis and aggregation were not affected by the incubation with PPTN. Conclusion and Implications: Platelets can be activated via P2Y14R stimulation to induce chemotaxis but not aggregation. Furthermore, this was dependent on concomitant activation of P2Y1R. Activation of P2Y14Rs on platelets may therefore be relevant during inflammation, but cooperation with P2Y1R activation is required.

There is a strong scientific rationale to use nebulised unfractionated heparin (UFH) in COVID-19. This pilot study investigated whether nebulised UFH was safe and had any impact on mortality, length of hospitalisation and clinical progression, in the treatment of hospitalised patients with COVID-19. This parallel group, open label, randomised trial included adult patients with confirmed SARS-CoV-2 infection admitted hospital in Brazil. One hundred patients were planned to be randomised to either “standard of care” (SOC) or SOC plus nebulized UFH. The trial was stopped after randomisation of 75 patients due to falling COVID-19 hospitalisation rates. Significance tests were 1-sided test (10% significance level). The key analysis populations were intention to treat (ITT) and modified ITT (mITT) which excluded (from both arms) subjects admitted to ITU or who died within 24 hrs of randomisation. In the ITT population (n=75), mortality was numerically lower for nebulised UFH (6 out of 38 patients; 15.8%) versus SOC (10 out of 37 patients; 27.0%), but not statistically significant; odds ratio (OR) 0.51, p=0.24. In the mITT population, nebulised UFH reduced mortality (OR 0.2, p=0.035).

The INHALE-HEP meta-trial is a prospective collaborative individual participant data meta-analysis of randomised controlled trials and early phase studies, to evaluate whether inhaled nebulised UFH in hospitalised patients with COVID-19 who do not require immediate invasive mechanical ventilation, significantly reduces intubation (or death, for patients who died before intubation) at day 28 compared to standard care alone. Objective: In keeping with best practice and with the published protocol, a pre-specified statistical analysis plan has been described and made public before completion of patient recruitment and data collection into the INHALE-HEP meta-trial. Methods: Our statistical analysis plan was designed by the INHALE-HEP executive committee and statisticians and approved by the INHALE-HEP steering committee. We reviewed the data collected as specified in the meta-trial protocol and collected in individual contributing studies. We present information pertaining to data collection, pre-specified subgroups, and study outcomes. Primary and secondary outcomes are defined, and additional subgroup analyses of pre-defined variables are described. Results: We have described our methods for presenting the trial profile and baseline characteristics, as well as our Bayesian approach to monitoring and meta-analysing individual patient data, outcomes and adverse events. All analyses will follow the intention-to-treat principle, considering all participants in the treatment group to which they were assigned, except for cases lost to follow-up or withdrawn. Conclusion: To minimise analytical bias, we have developed a statistical analysis plan and made this available to the public domain before completion of patient recruitment and data collection into the INHALE-HEP meta-trial.

Inhaled nebulised unfractionated heparin (UFH) has a strong scientific and biological rationale that warrants urgent investigation of its therapeutic potential in patients with COVID-19. UFH has antiviral effects and prevents the SARS-CoV-2 virus’ entry into mammalian cells. In addition, UFH has significant anti-inflammatory and anti-coagulant properties, which limit progression of lung injury and vascular pulmonary thrombosis. Methods and intervention The INHALEd nebulised unfractionated HEParin for the treatment of hospitalised patients with COVID-19 (INHALE-HEP) meta-trial is a prospective individual patient data analysis of on-going randomised controlled trials and early phase studies. Individual studies are being conducted in multiple countries. Participating studies randomise adult patients admitted to the hospital with confirmed SARS-CoV-2 infection, who do not require immediate mechanical ventilation, to inhaled nebulised UFH or standard care. All studies collect a minimum core dataset. The primary outcome for the meta-trial is intubation (or death, for patients who died before intubation) at day 28. The secondary outcomes are oxygenation, clinical worsening and mortality, assessed in time-to-event analyses. Individual studies may have additional outcomes. Analysis We use a Bayesian approach to monitoring, followed by analysing individual patient data, outcomes and adverse events. All analyses will follow the intention-to-treat principle, considering all participants in the treatment group to which they were assigned, except for cases lost to follow-up or withdrawn. Trial registration, ethics and dissemination The meta-trial is registered at ClinicalTrials.gov ID NCT04635241. Each contributing study is individually registered and has received approval of the relevant ethics committee or institutional review board.

Introduction Inhaled nebulised unfractionated heparin (UFH) has a strong scientific and biological rationale and warrants urgent investigation of its therapeutic potential for COVID-19. UFH has antiviral effects and prevents the SARS-CoV-2 virus’ entry into mammalian cells. In addition, UFH has significant anti-inflammatory and anti-coagulant properties, which limit progression of lung injury and vascular pulmonary thrombosis. Methods and intervention This meta-trial is a prospective collaborative individual patient data meta-analysis of randomised controlled trials and early phase studies. Individual studies are conducted in multiple countries. Adult patients admitted to the hospital with confirmed SARS-CoV-2 infection, who do not require immediate mechanical ventilation, are randomised to inhaled nebulised UFH or standard care. All studies collect a minimum core dataset. The primary outcome is intubation (or death, for patients who died before intubation) at day 28, assessed in a time-to-event analysis. The secondary outcomes are oxygenation, clinical worsening and mortality, assessed in time-to-event analyses. Individual studies may have specific outcome measures in addition to the core set. Ethics and dissemination: The meta-trial is registered at ClinicalTrials.gov, ID NCT04635241. Results of this study will be shared with the WHO, published in scientific journals and presented at scientific meetings.

Background and Purpose: Currently there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently being used to treat anticoagulant anomalies in COVID-19 patients. In addition, in the UK, nebulised unfractionated heparin (UFH) is currently being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild-type SARS-CoV-2, in vitro, is thus urgently needed. Experimental Approach: A range of heparin preparations both UFH (n=4) and low molecular weight heparins (LMWH) (n=3) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Victoria/01/2020) using a plaque reduction neutralisation assay and Vero E6 cells. ND50 values for each heparin were calculated using a mid-point probit analysis. Key Results: UFH had potent antiviral effects, with ND50 values of 12.5 and 23 μg/ml for two porcine mucosal UFH tested. Bovine mucosal UFH had similar antiviral effects although it was ~50% less active (ND50, 50-75 μg/ml). In contrast, LMWHs such as Clexane and Fragmin were markedly less active by ~100-fold (ND50 values of 2.6-6.8 mg/ml). Conclusions and Implications: This comparison of a panel of clinically relevant heparins, including the UFH preparation under trial in the UK, demonstrated that distinct products exhibit different degrees of antiviral activity against live SARS-CoV-2. Porcine mucosal UFH has the strongest antiviral activity followed by bovine mucosal UFH, whereas LMWHs had the lowest amount of antiviral activity (by 100-fold). Overall the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 infections has led to substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS-CoV-2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID-19. At the time of writing, in vivo studies of SARS-CoV-2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partially model the mechanisms implicated in human severe COVID-19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID-19. These models need to recapitulate key mechanisms of COVID-19 severe acute respiratory distress syndrome and reproduce the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS-CoV-2 infection and COVID-19-related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID-19 pathogenesis and for assessing potential treatments.