A simple LMWHA diagnostic could be used as a companion diagnostic to identify responders to N-butyryl HA therapy

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A “Cytokine storm” and inflammation-mediated severe lung damage are the main underlying reasons for morbidity and mortality in SARs-COV-2 infected patients (COVID-19 patients). Biological data is building of the importance of low molecular weight HA (LMWHA) in the progression of COVID-19 in hospitalized patients. Queen’s researchers believe N-butyryl hyaluronic acid, by antagonizing LMWHA agonism of TLR4 and other LMWHA-binding proteins, represents a solution to this challenge and could save lives.

Early reports from China noted that hyaluronic acid (HA) could be a potential cause of fatalities and noted that autopsies have found lungs filled with clear liquid jelly, speculating it was HA(1). Another Chinese group then published examining expression of HA with severity of COVID-19. They discovered the HA was an indicator of fibrosis and could be used as early warning indicators of poor prognosis for critical patients with COVID-19(2). Kaber et al. investigated whether HA was a component of the excessive respiratory secretions in COVID-19(3). They examined the sputum of COVID-19 patients and compared it to sputum of healthy volunteers and cystic fibrosis patients (positive control). They found HA content is increased ~ 20-fold in both CF and COVID-19 patients compared to healthy controls. Interestingly, the hyaluronan in COVID-19 samples was comprised of low-molecular weight fragments, the form of HA linked with pro-inflammatory functions. They also found elevated HA concentrations in cadaveric lungs of COVID-19 patients. It should be note that elevated HA has been reported to be elevated in sputum and BALF of acute respiratory patients (ARDS) prior to COVID-19(4,5). Mong et al. note the long-standing association of HA and ARDS(6). They conducted autopsies on 6 COVID-19 patients and found the average lung of patients determined to have died from SARS-CoV-2 was ~2.5x normal lung weight and that hyaline membranes were consistently identified on histologist sections.

LMWHA can be synthesized enzymatically by HAS3 which produces polymers of < 300 kDa, produced by degradation of HMWHA by HYAL2 into ~20 kDa fragments, and by oxidative degradation. LMWHA is known to interact with several binding proteins including toll-like receptor-4 (TLR4), layilin, Receptor for Hyaluronan-Mediated Motility (RHAMM), LYVE-1, and others. The TLR4 pathway has been implicated in pathological inflammation during COVID-19 with the TLR4 ligand, S100A9, being overexpressed(7). TLR4 is the only TLR to transduce innate immune signals through both MyD88 and Toll-IL-1 receptor-domain-containing adaptor-inducing IFN-β to activate NF-κB. The NF-κB signaling pathway is required for induction of pro-inflammatory cytokine/chemokine generation – “cytokine storm” seen in COVID-19 patients. Molecular modelling studies looked at binding of SARS-CoV-2 spike protein (S protein) to TLRs and found TLR4 was predicted to have the strongest predicted binding energy(8). Further supporting the potential role of TLR4 in COVID-19 is the demonstration of SARS-CoV-2 spike protein ( S protein) binding to the TLR4 ligand, E.coli lipopolysaccharide (LPS)(9). S protein, when combined with low levels of LPS stimulated cytokine production via NF-κB pathway in the monocytic cell line THP-1 and human blood. The researchers then validated these findings in vivo using a NF-κB reporter mouse model and bioimaging. They found that the combination of S protein and LPS significantly increased the inflammatory response compared to either alone. LMWHA-induced pulmonary inflammatory effects are mediated in part via activation of the Pi3K/AKT pathway in neutrophils(10). TLR4 is known to induce Pi3K and AKT activation are known to increase NF-κb mediated transcriptional activity(11). This further reinforces the role of LMWHA in COVID-19 and respiratory inflammation as neutrophil activation dysregulation is associated with COVID-19 pathophysiology and disease severity(13).

Other HA-binding proteins have been evaluated in ARDS and lung injury. Hyaluronic acid binding protein 2 (HABP2) levels and activity are increased in the BAL fluid of mechanically ventilated patients with early ARDS compared with patients with cardiogenic pulmonary edema or healthy controls(13). Furthermore, form the same studies, elevated levels of HABP2 were found in alveolar macrophages, bronchial epithelial and pulmonary endothelial cells by immunohistochemistry. In experimental models of lung injury, comparing wild-type RHAMM mice to transgenic RHAMM mice(14). RHAMM knock-out mice had less weight loss, less increase in respiratory rate, reduced fibrosis, and fewer CD45+ cells in the lung compared to wild-type. In contrast, bleomycin-induced lung injury in RHAMM overexpressing transgenic mice had exaggerated inflammatory and fibrotic responses compared to wild-type mice including 4-5-fold overexpression of HA in BALF compared to wild-type.

A Queen’s University researcher has identified that N-acyl HA, in particular N-butyryl HA, can prevent the binding and activation of LMWHA via TLR4 and likely other LMWHA-binding proteins(15). In these studies N- butyryl HA prevented cytokine expression by TLR4 ligands LMWHA and LPS but had no stimulatory activity of its own. The in vivo benefits of N-butyryl HA have been testing in preclinical inflammatory models of gout, hyperuricemia, rheumatoid arthritis, and a rat excisional wound healing model(16,17). In these studies N-butyryl HA demonstrated benefit on reducing pro-inflammatory mediators such as TNFα, IL-6, and IL-1β. The concurrent reduction in p65 and p38 indicate inhibition of the NFκB and MAPK pathways. The researcher believes the activity of N-butyryl HA will be directed at LMWHA-binding sites in general and not limited to TLR4. Hence, it should have benefits beyond TLR4-selective drugs while preventing the activity of LMWHA to several different binding sites.

Queen’s have granted patents in US, Japan, and pending in Canada claiming pharmaceutical compositions and uses. The researcher would like to propose testing N-butyryl HA in a COVID-19 model of SARS-CoV-2 infected K18-hACE2 mice. This model produces a robust Th1/2/17 cytokine storm in the lungs and spleens from two days post infection indicating the translational relevance of this mouse in recapitulating the critical human clinical features of COVID-19. The researchers would need to examine lung secretions/tissues for presence of LMWHA and TLR4 as well as other LMWHA binding proteins including RHAMM, CD44, lyve-1, and layilin. Currently the researchers don’t understand the behavior of N-butyryl HA in vivo. However, with the minimal structural differences between it and native HA it is likely to behave similarly. Systemically injected HA has a short half-life and is removed by the liver. To that end the researcher proposes conducting initial PK studies comparing intravenous to inhaled formulations. Inhaled formulations will likely enable the highest concentration of N-butyryl HA in the lungs and provide the best chance for efficacy. To that end the researcher would then propose to complete in vivo proof-of-concept in COVID-19 models, cystic fibrosis or CPOD/ARDS to demonstrate ability to clear lungs of LMWHA and restore normal function.

1. Shi Y, et al. COVID-19 infection: the perspectives on immune responses. Cell Death Differ. 2020.

2. Ming Ding et al. Correlation analysis of the severity and clinical prognosis of 32 cases of patients with COVID-19. Respir Med. 167: 105981, 2020.

3. Kaber, G. et al. Hyaluronan is abundant in COVID-19 respiratory secretions. medRxiv . 2020 Sep 11;, 2020 Preprint.

4. Hällgren R. et al. Accumulation of hyaluronan (hyaluronic acid) in the lung in adult respiratory distress syndrome. Am Rev Respir Dis 139:682-7, 1989.

5. Esposito AJ et al. Hyaluronic acid is associated with organ dysfunction in acute respiratory distress syndrome. Critical Care volume 21, Article number: 304, 2017.

6. Mong AJ et al. Accelerated hyaluronan concentration as the primary driver of morbidity and mortality in high-risk COVID-19 patients: with therapeutic ntroduction of an oral hyaluronan inhibitor in the prevention of “Induced Hyaluronan Storm” Syndrome. Medrxiv. August 20, 2020. Preprint

7. Mok Sohn K et al. COVID-19 Patients Upregulate Toll-like Receptor 4-mediated Inflammatory Signaling That Mimics Bacterial Sepsis. J Korean Med Sci. 2020 Sep 28;35(38):e343

8. Choudhury A, Mukherjee S. In silico studies on the comparative characterization of the interactions of SARS‐CoV‐2 spike glycoprotein with ACE‐2 receptor homologs and human TLRs. J Med Virol. 2020;92:2105–2113.

9. Petruk G et al. SARS-CoV-2 Spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity. bioRxiv. June 29,2020. Preprint

10. Zhao et al. Low-molecular-mass hyaluronan induces pulmonary inflammation by up-regulation of Mcl-1 to inhibit neutrophil apoptosis via PI3K/Akt1 pathway. Immunology, 155, 387–395, 2018.

11. Fang W. et al. Identification and activation of TLR4-mediated signalling pathways by alginate-derived guluronate oligosaccharide in RAW264.7 macrophages. Scientific Reports. 7:1663, 2017.

12. Overmeyer, KA. et al. Large-Scale Multi-omic Analysis of COVID-19 Severity. Cell Systems. 12: 1–18, 2021.

13. Wygreka M et al. Raised protein levels and altered cellular expression of factor VII activating protease (FSAP) in the lungs of patients with acute respiratory distress syndrome (ARDS). Thorax. 62(10):880-8, 2007.

14. Ciu Z., et al. The Receptor for Hyaluronan-Mediated Motility (CD168) promotes inflammation and fibrosis after acute lung injury. Matrix Biol. 78-79: 255–271, 2019.

15. Babasola O et al. Chemically modified N-acylated hyaluronan fragments modulate proinflammatory cytokine production by stimulated human macrophages. J Biol Chem. 5;289(36):24779-91, 2014.

16. Gao Y et al. A Low Molecular Weight Hyaluronic Acid Derivative Accelerates Excisional Wound Healing by Modulating Pro-Inflammation, Promoting Epithelialization and Neovascularization, and Remodeling Collagen. Int J Mol Sci. 20(15):3722, 2019.

17. Li L et al. N-Butyrylated hyaluronic acid ameliorates gout and hyperuricemia in animal models. Pharm Biol. 57(1):717-728, 2019.

Key Benefits

The development of a simple LMWHA diagnostic could be used as a companion diagnostic to identify responders to N-butyryl HA therapy.

Potential for early identification of patients for poor prognosis and treat with N-butyryl HA to prevent poor outcomes.

This treatment would uniquely preventing the negative effects of over-expressed LWMHA, reduce the associated pro-inflammatory environment and save lives.

Applications

Diagnostic identification of poor outcomes by measuring LMWHA

Treat lung diseases where LMWHA plays a role in pathology including COVID-19, cystic fibrosis, COPD and ARDS.

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