Pharmacology of Heparin and Related Drugs: An Update.

Heparin has been used extensively as an antithrombotic and anticoagulant for close to 100 years. This anticoagulant activity is attributed mainly to the pentasaccharide sequence, which potentiates the inhibitory action of antithrombin, a major inhibitor of the coagulation cascade. More recently it has been elucidated that heparin exhibits anti-inflammatory effect via interference of the formation of neutrophil extracellular traps and this may also contribute to heparin's antithrombotic activity. This illustrates that heparin interacts with a broad range of biomolecules, exerting both anticoagulant and nonanticoagulant actions. Since our previous review, there has been an increased interest in these nonanticoagulant effects of heparin, with the beneficial role in patients infected with SARS2-coronavirus a highly topical example. This article provides an update on our previous review with more recent developments and observations made for these novel uses of heparin and an overview of the development status of heparin-based drugs. SIGNIFICANCE STATEMENT: This state-of-the-art review covers recent developments in the use of heparin and heparin-like materials as anticoagulant, now including immunothrombosis observations, and as nonanticoagulant including a role in the treatment of SARS-coronavirus and inflammatory conditions.

Inhaled nebulised unfractionated heparin (UFH) for the treatment of hospitalised patients with COVID-19: A randomised controlled pilot study.

There is a strong scientific rationale to use nebulised unfractionated heparin (UFH) in treating patients with 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 to two hospitals 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 h 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. However, in the mITT population, nebulised UFH reduced mortality (OR 0.2, p = 0.035). Length of hospital stay was similar between groups, but at day 29, there was a greater improvement in ordinal score following treatment with UFH in the ITT and mITT populations (p = 0.076 and p = 0.012 respectively), while mechanical ventilation rates were lower with UFH in the mITT population (OR 0.31; p = 0.08). Nebulised UFH did not cause any significant adverse events. In conclusion, nebulised UFH added to SOC in hospitalised patients with COVID-19 was well tolerated and showed clinical benefit, particularly in patients who received at least 6 doses of heparin. This trial was funded by The J.R. Moulton Charity Trust and registered under REBEC RBR-8r9hy8f (UTN code: U1111-1263-3136).

Can nebulised HepArin Reduce morTality and time to Extubation in patients with COVID-19 Requiring invasive ventilation Meta-Trial (CHARTER-MT): Protocol and statistical analysis plan for an investigator-initiated international meta-trial of prospective randomised clinical studies.

There is significant interest in the potential for nebulised unfractionated heparin (UFH), as a novel therapy for patients with COVID-19 induced acute hypoxaemic respiratory failure requiring invasive ventilation. The scientific and biological rationale for nebulised heparin stems from the evidence for extensive activation of coagulation resulting in pulmonary microvascular thrombosis in COVID-19 pneumonia. Nebulised delivery of heparin to the lung may limit alveolar fibrin deposition and thereby limit progression of lung injury. Importantly, laboratory studies show that heparin can directly inactivate the SARS-CoV-2 virus, thereby prevent its entry into and infection of mammalian cells. UFH has additional anti-inflammatory and mucolytic properties that may be useful in this context. METHODS AND INTERVENTION: The Can nebulised HepArin Reduce morTality and time to Extubation in Patients with COVID-19 Requiring invasive ventilation Meta-Trial (CHARTER-MT) is a collaborative prospective individual patient data analysis of on-going randomised controlled clinical trials across several countries in five continents, examining the effects of inhaled heparin in patients with COVID-19 requiring invasive ventilation on various endpoints. Each constituent study will randomise patients with COVID-19 induced respiratory failure requiring invasive ventilation. Patients are randomised to receive nebulised heparin or standard care (open label studies) or placebo (blinded placebo-controlled studies) while under invasive ventilation. Each participating study collect a pre-defined minimum dataset. The primary outcome for the meta-trial is the number of ventilator-free days up to day 28 day, defined as days alive and free from invasive ventilation.

Inhaled nebulised unfractionated heparin for the treatment of hospitalised patients with COVID-19: A multicentre case series of 98 patients.

AIMS: To determine the safety and efficacy-potential of inhaled nebulised unfractionated heparin (UFH) in the treatment of hospitalised patients with COVID-19. METHODS: Retrospective, uncontrolled multicentre single-arm case series of hospitalised patients with laboratory-confirmed COVID-19, treated with inhaled nebulised UFH (5000 IU q8h, 10 000 IU q4h, or 25 000 IU q6h) for 6 ± 3 (mean ± standard deviation) days. Outcomes were activated partial thromboplastin time (APTT) before treatment (baseline) and highest-level during treatment (peak), and adverse events including bleeding. Exploratory efficacy outcomes were oxygenation, assessed by ratio of oxygen saturation to fraction of inspired oxygen (FiO2 ) and FiO2 , and the World Health Organisation modified ordinal clinical scale. RESULTS: There were 98 patients included. In patients on stable prophylactic or therapeutic systemic anticoagulant therapy but not receiving therapeutic UFH infusion, APTT levels increased from baseline of 34 ± 10 seconds to a peak of 38 ± 11 seconds (P < .0001). In 3 patients on therapeutic UFH infusion, APTT levels did not significantly increase from baseline of 72 ± 20 to a peak of 84 ± 28 seconds (P = .17). Two patients had serious adverse events: bleeding gastric ulcer requiring transfusion and thigh haematoma; both were on therapeutic anticoagulation. Minor bleeding occurred in 16 patients, 13 of whom were on therapeutic anticoagulation. The oxygen saturation/FiO2 ratio and the FiO2 worsened before and improved after commencement of inhaled UFH (change in slope, P < .001). CONCLUSION: Inhaled nebulised UFH in hospitalised patients with COVID-19 was safe. Although statistically significant, inhaled nebulised UFH did not produce a clinically relevant increase in APTT (peak values in the normal range). Urgent randomised evaluation of nebulised UFH in patients with COVID-19 is warranted and several studies are currently underway.

Nonantimicrobial Actions of Macrolides: Overview and Perspectives for Future Development.

Macrolides are among the most widely prescribed broad spectrum antibacterials, particularly for respiratory infections. It is now recognized that these drugs, in particular azithromycin, also exert time-dependent immunomodulatory actions that contribute to their therapeutic benefit in both infectious and other chronic inflammatory diseases. Their increased chronic use in airway inflammation and, more recently, of azithromycin in COVID-19, however, has led to a rise in bacterial resistance. An additional crucial aspect of chronic airway inflammation, such as chronic obstructive pulmonary disease, as well as other inflammatory disorders, is the loss of epithelial barrier protection against pathogens and pollutants. In recent years, azithromycin has been shown with time to enhance the barrier properties of airway epithelial cells, an action that makes an important contribution to its therapeutic efficacy. In this article, we review the background and evidence for various immunomodulatory and time-dependent actions of macrolides on inflammatory processes and on the epithelium and highlight novel nonantibacterial macrolides that are being studied for immunomodulatory and barrier-strengthening properties to circumvent the risk of bacterial resistance that occurs with macrolide antibacterials. We also briefly review the clinical effects of macrolides in respiratory and other inflammatory diseases associated with epithelial injury and propose that the beneficial epithelial effects of nonantibacterial azithromycin derivatives in chronic inflammation, even given prophylactically, are likely to gain increasing attention in the future. SIGNIFICANCE STATEMENT: Based on its immunomodulatory properties and ability to enhance the protective role of the lung epithelium against pathogens, azithromycin has proven superior to other macrolides in treating chronic respiratory inflammation. A nonantibiotic azithromycin derivative is likely to offer prophylactic benefits against inflammation and epithelial damage of differing causes while preserving the use of macrolides as antibiotics.

Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations.

BACKGROUND AND PURPOSE: Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID-19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is 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 needed. EXPERIMENTAL APPROACH: Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined. KEY RESULTS: All the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 µg·ml-1 , whereas LMWHs were less inhibitory by ~150-fold (IC50 range 3.4-7.8 mg·ml-1 ). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor. CONCLUSION AND IMPLICATIONS: This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS-CoV-2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.

INHALEd nebulised unfractionated HEParin for the treatment of hospitalised patients with COVID-19 (INHALE-HEP): Protocol and statistical analysis plan for an investigator-initiated international metatrial of randomised studies.

AIMS: 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 anticoagulant properties, which limit progression of lung injury and vascular pulmonary thrombosis. METHODS: The INHALEd nebulised unfractionated HEParin for the treatment of hospitalised patients with COVID-19 (INHALE-HEP) metatrial 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 metatrial 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 metatrial 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. Results of this study will be shared with the World Health Organisation, published in scientific journals and presented at scientific meetings.

Animal models of mechanisms of SARS-CoV-2 infection and COVID-19 pathology.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 infections has led to a 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 partly model the mechanisms involved 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 reproduce the key mechanisms of COVID-19 severe acute respiratory distress syndrome and 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. LINKED ARTICLES: This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.

ACCORD: A Multicentre, Seamless, Phase 2 Adaptive Randomisation Platform Study to Assess the Efficacy and Safety of Multiple Candidate Agents for the Treatment of COVID-19 in Hospitalised Patients: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Stage 1: To evaluate the safety and efficacy of candidate agents as add-on therapies to standard of care (SoC) in patients hospitalised with COVID-19 in a screening stage. Stage 2: To confirm the efficacy of candidate agents selected on the basis of evidence from Stage 1 in patients hospitalised with COVID-19 in an expansion stage. TRIAL DESIGN: ACCORD is a seamless, Phase 2, adaptive, randomised controlled platform study, designed to rapidly test candidate agents in the treatment of COVID-19. Designed as a master protocol with each candidate agent being included via its own sub-protocol, initially randomising equally between each candidate and a single contemporaneous SoC arm (which can adapt into 2:1). Candidate agents currently include bemcentinib, MEDI3506, acalabrutinib, zilucoplan and nebulised heparin. For each candidate a total of 60 patients will be recruited in Stage 1. If Stage 1 provides evidence of efficacy and acceptable safety the candidate will enter Stage 2 where a total of approximately 126 patients will be recruited into each study arm sub-protocol. Enrollees and outcomes will not be shared across the Stages; the endpoint, analysis and sample size for Stage 2 may be adjusted based on evidence from Stage 1. Additional arms may be added as new potential candidate agents are identified via candidate agent specific sub-protocols. PARTICIPANTS: The study will include hospitalised adult patients (≥18 years) with confirmed SARS-CoV-2 infection, the virus that causes COVID-19, that clinically meet Grades 3 (hospitalised - mild disease, no oxygen therapy), Grades 4 (hospitalised, oxygen by mask or nasal prongs) and 5 (hospitalised, non-invasive ventilation or high flow oxygen) of the WHO Working Group on the Clinical Characteristics of COVID-19 9-point category ordinal scale. Participants will be recruited from England, Northern Ireland, Wales and Scotland. INTERVENTION AND COMPARATOR: Comparator is current standard of care (SoC) for the treatment of COVID-19. Current candidate experimental arms include bemcentinib, MEDI3506, acalabrutinib, zilucoplan and nebulised heparin with others to be added over time. Bemcentinib could potentially reduce viral infection and blocks SARS-CoV-2 spike protein; MEDI3506 is a clinic-ready anti-IL-33 monoclonal antibody with the potential to treat respiratory failure caused by COVID; acalabrutinib is a BTK inhibitor which is anti-viral and anti-inflammatory; zilucoplan is a complement C5 inhibitor which may block the severe inflammatory response in COVID-19 and; nebulised heparin has been shown to bind with the spike protein. ACCORD is linked with the UK national COVID therapeutics task force to help prioritise candidate agents. MAIN OUTCOMES: Time to sustained clinical improvement of at least 2 points (from randomisation) on the WHO 9-point category ordinal scale, live discharge from the hospital, or considered fit for discharge (a score of 0, 1, or 2 on the ordinal scale), whichever comes first, by Day 29 (this will also define the "responder" for the response rate analyses). RANDOMISATION: An electronic randomization will be performed by Cenduit using Interactive Response Technology (IRT). Randomisation will be stratified by baseline severity grade. Randomisation will proceed with an equal allocation to each arm and a contemporaneous SoC arm (e.g. 1:1 if control and 1 experimental arm; 1:1:1 if two experimental candidate arms etc) but will be reviewed as the trial progresses and may be changed to 2:1 in favour of the candidate agents. BLINDING (MASKING): The trial is open label and no blinding is currently planned in the study. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): This will be in the order of 60 patients per candidate agent for Stage 1, and 126 patients for Stage 2. However, sample size re-estimation may be considered after Stage 1. It is estimated that up to 1800 patients will participate in the overall study. TRIAL STATUS: Master protocol version ACCORD-2-001 - Master Protocol (Amendment 1) 22nd April 2020, the trial has full regulatory approval and recruitment is ongoing in the bemcentinib (first patient recruited 6/5/2020), MEDI3506 (first patient recruited 19/5/2020), acalabrutinib (first patient recruited 20/5/2020) and zilucoplan (first patient recruited 19/5/2020) candidates (and SoC). The recruitment dates of each arm will vary between candidate agents as they are added or dropped from the trial, but will have recruited and reported within a year. TRIAL REGISTRATION: EudraCT 2020-001736-95 , registered 28th April 2020. FULL PROTOCOL: The full protocol (Master Protocol with each of the candidate sub-protocols) is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Nebulised heparin as a treatment for COVID-19: scientific rationale and a call for randomised evidence.

Nebulised unfractionated heparin (UFH) has a strong scientific and biological rationale and warrants urgent investigation of its therapeutic potential, for COVID-19-induced acute respiratory distress syndrome (ARDS). COVID-19 ARDS displays the typical features of diffuse alveolar damage with extensive pulmonary coagulation activation resulting in fibrin deposition in the microvasculature and formation of hyaline membranes in the air sacs. Patients infected with SARS-CoV-2 who manifest severe disease have high levels of inflammatory cytokines in plasma and bronchoalveolar lavage fluid and significant coagulopathy. There is a strong association between the extent of the coagulopathy and poor clinical outcomes.The anti-coagulant actions of nebulised UFH limit fibrin deposition and microvascular thrombosis. Trials in patients with acute lung injury and related conditions found inhaled UFH reduced pulmonary dead space, coagulation activation, microvascular thrombosis and clinical deterioration, resulting in increased time free of ventilatory support. In addition, UFH has anti-inflammatory, mucolytic and anti-viral properties and, specifically, has been shown to inactivate the SARS-CoV-2 virus and prevent its entry into mammalian cells, thereby inhibiting pulmonary infection by SARS-CoV-2. Furthermore, clinical studies have shown that inhaled UFH safely improves outcomes in other inflammatory respiratory diseases and also acts as an effective mucolytic in sputum-producing respiratory patients. UFH is widely available and inexpensive, which may make this treatment also accessible for low- and middle-income countries.These potentially important therapeutic properties of nebulised UFH underline the need for expedited large-scale clinical trials to test its potential to reduce mortality in COVID-19 patients.