(M1430-04-22) Discovery and Development of a New Therapeutic Product for the Treatment Long Covid

Purpose: Covid symptoms persisting longer than 28 weeks, called ‘Long Covid’, are debilitating and significantly affect the quality of life of these patients. Long COVID is an illness that occurs in at least 10% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. There are several hypothesized mechanisms for long COVID pathogenesis, including immune dysregulation, microbiota disruption, autoimmunity, clotting and endothelial abnormality, and dysfunctional neurological signalling. It is hypothesized that the complex immune pathology is mediated through a convoluted profile of inflammation, coagulation and tissue damage. To target these complex events, a new flavonoid, proanthocyanidin (PAC), with multitarget modulation has been identified with high potential for rapid translation into an ideal therapeutic for prevention, and treatment of long COVID.

Methods: The potential of PAC to inhibit micro-clot formation was investigated using mouse platelets. Platelets isolated from mouse blood were incubated with the potential therapeutics at different concentrations for 15 min at 37^oC. The platelets were activated by treating with 5 µM adenosine diphosphate (ADP) for 15 min at 37^oC while shaking at 700 rpm, and platelet aggregation was measured by flow cytometry. Neuroprotective activity of PAC was tested in cerebrovascular endothelial cells (CVE), human astrocytes (HA) and mouse hippocampal neural cell line (HT-22).  A pilot in vivo study was conducted on severely COVID (WA-1 strain; 10⁵TCID/intranasal) infected AC70 mice by administering PAC at a dosage of 40 µg/mouse, i.p., to study its efficacy in reducing thrombocytopenia in lungs. This study shows that virtually all patients with severe COVID-19 develop anomalous antibodies that target an endogenous protein, platelet factor 4 (PF4), and that are the hallmark of two life-threatening disorders characterized by thrombosis and thrombocytopenia: heparin-induced thrombocytopenia and vaccine-induced thrombosis with thrombocytopenia. Anti-PF4 antibodies are the hallmark of two life-threatening disorders: heparin-induced thrombocytopenia and vaccine-induced thrombosis with thrombocytopenia. Because of its poor water solubility and permeability characteristics preclude its use as oral formulation. To address the translational development challenges with PAC, compritol-based lipid nanoparticles (LNP) loaded with PAC were developed with the hot-melt homogenization method and characterized.

Results: PAC has inhibited micro-clot formation with IC50 of < 3uM using mouse platelets. Anti-inflammatory, cholesterol-lowering efficacy at 10 uM. PAC alleviates hemin-induced injury in cerebrovascular endothelial cells (CVE), human astrocytes (HA) and mouse hippocampal neural cell line (HT-22). Higher anti-PF4 antibody levels were found in patients with the most severe disease and the most conspicuous platelet reductions. These findings suggest that anti-PF4 antibodies may play a role in the severe multiorgan disease manifestations of COVID-19. Particle size of the PAC loaded LNP was 136.8±0.15 nm with a zeta potential of -6.2 mv and encapsulation efficiency of >90%.

Conclusion: PAC was found to be a potent platelet inhibitor and it reduced LDL-cholesterol levels. PAC demonstrated remarkable antiplatelet activity compared to the control (p < 0.001). Promising neuro-protective activity was demonstrated in HT-22 cell line. In vivo efficacy of oral inhalation of LNP of PAC are in progress.


PAC alleviates hemin-induced injury in cerebrovascular endothelial cells (CVE), human astrocytes (HA) and mouse hippocampal neural cell line (HT-22; B. Dose-dependent Efficacy of PAC in inhibiting platelet aggregation using platelets isolated from Balb/c mice (n=3); C. A nanoemulsion formulation of PAC was prepared and lyophilized to get a powder with a particle of 140 nm.


A pilot study demonstrating the efficacy of PAC (40 µg/mouse, i.p.) in reducing thrombocytopenia in lungs in severely COVID (WA-1 strain; 105TCID/intranasal) infected AC70 mice.