Azeliragon may have the potential to prevent severe COVID-19 illness in the form of a once-a-day pill


Human Lung Chip reveals the effects of breathing motions on lung immune responses and leads to repurposing of potential therapeutics for respiratory diseases, including COVID-19

The average person will take more than 600 million breaths over the course of their life. Every breath stretches the lungs’ tissues with each inhale and relaxes them with each exhale. The mere motions of breathing are known to influence vital functions of the lungs, including their development in babies, the production of air-exchange-enhancing fluid on their inner surfaces, and maintenance of healthy tissue structure. Now, new research from the Wyss Institute at Harvard University has revealed that this constant pattern of stretching and relaxing does even more – it generates immune responses against invading viruses.

Using a Human Lung Chip that replicates the structures and functions of the lung air sac, or “alveolus,” the research team discovered that applying mechanical forces that mimic breathing motions suppresses influenza virus replication by activating protective innate immune responses.

They also identified several drugs that reduced the production of inflammatory cytokines in infected Alveolus Chips, which could be useful in treating excessive inflammation in the lung. Based on these studies, one of those drugs was licensed to Cantex Pharmaceuticals for the treatment of COVID-19 and other inflammatory lung diseases. Data from the research were recently included in the company’s Investigational New Drug (IND) application to the FDA to initiate a Phase 2 clinical trial for COVID-19.

The Human Alveolus Chip contains hollow side channels that allow suction to be applied to the chips, applying cyclic strain that mimics the motions of normal human breathing (left). A permeable membrane separates human alevolus cells in the upper channel from human blood vessel cells in the lower channel, allowing them to exchange molecular signals (right). Credit: Wyss Institute at Harvard University

“This research demonstrates the importance of breathing motions for human lung function, including immune responses to infection, and shows that our Human Alveolus Chip can be used to model these responses in the deep portions of the lung, where infections are often more severe and lead to hospitalization and death,” said co-first author Haiqing Bai, Ph.D., a Wyss Technology Development Fellow at the Institute.

“This model can also be used for preclinical drug testing to ensure that candidate drugs actually reduce infection and inflammation in functional human lung tissue.” The results are published today in Nature Communications.

Creating a flu-on-a-chip

As the early phases of the COVID-19 pandemic made painfully clear, the lung is a vulnerable organ where inflammation in response to infection can generate a “cytokine storm” that can have deadly consequences. However, the lungs are also very complex, and it is difficult to replicate their unique features in the lab. This complexity has hindered science’s understanding of how the lungs function at the cell and tissue levels, in both healthy and diseased states.

The Wyss Institute’s Human Organ Chips were developed to address this problem, and have been shown to faithfully replicate the functions of many different human organs in the lab, including the lung. As part of projects funded by the NIH and DARPA since 2017, Wyss researchers have been working on replicating various diseases in Lung Airway and Alveolus Chips to study how lung tissues react to respiratory viruses that have pandemic potential and test potential treatments.

During his Ph.D. training, Bai studied diseases that affect the tiny air sacs deep inside the lungs where oxygen is rapidly exchanged for carbon dioxide. That foundation prepared him to tackle the challenge of recreating a flu infection in an Alveolus Chip so that the team could study how these deep lung spaces mount immune responses against viral invaders.

In both the upper alveolar channel (top) and lower blood vessel channel (bottom) of the Alveolus Chips, the cells formed intact tissues held together by proteins between the cells called ZO1 and VE-cadherin, which are stained red and green, respectively. Credit: Wyss Institute at Harvard University

Bai and his team first lined the two parallel microfluidic channels of an Organ Chip with different types of living human cells – alveolar lung cells in the upper channel and lung blood vessel cells in the lower channel – to recreate the interface between human air sacs and their blood-transporting capillaries.

To mimic the conditions that alveoli experience in the human lung, the channel lined by alveolar cells was filled with air while the blood vessel channel was perfused with a flowing culture medium containing nutrients that are normally delivered via the blood. The channels were separated by a porous membrane that allowed molecules to flow between them.

Previous studies at the Wyss Institute have established that applying cyclical stretching to Alveolus Chips to imitate breathing motions produces biological responses that mimic those observed in vivo. This is accomplished by applying suction to hollow side chambers adjacent to the cell-lined fluidic channels to rhythmically stretch and relax the lung tissues by 5%, which is what human lungs typically experience with every breath.

When the team infected these “breathing” Alveolus Chips with H3N2 influenza by introducing the virus into the air channel, they observed the development of several known hallmarks of influenza infection, including the breakdown of junctions between cells, a 25% increase in cell death, and the initiation of cellular repair programs. Infection also led to much higher levels of multiple inflammatory cytokines in the blood vessel channel including type III interferon (IFN-III), a natural defense against viral infection that is also activated in in vivo flu infection studies.

In addition, the blood vessel cells of infected chips expressed higher levels of adhesion molecules, which allowed immune cells including B cells, T cells, and monocytes in the perfusion medium to attach to the blood vessel walls to help combat the infection. These results confirmed that the Alveolus Chip was mounting an immune response against H3N2 that recapitulated what happens in the lung of human patients infected with flu virus.

Focus on your breath

The team then carried out the same experiment without mechanical breathing motions. To their surprise, chips exposed to breathing motions ​​had 50% less viral mRNA in their alveolar channels and a significant reduction in inflammatory cytokine levels compared to static chips. Genetic analysis revealed that the mechanical strain had activated molecular pathways related to immune defense and multiple antiviral genes, and these activations were reversed when the cyclical stretching was stopped.

“This was our most unexpected finding – that mechanical stresses alone can generate an innate immune response in the lung,” said co-first author Longlong Si, Ph.D., a former Wyss Technology Development Fellow who is now a Professor at the Shenzhen Institute of Advanced Technology in China.

Knowing that sometimes the lungs experience greater than 5% strain, such as in chronic obstructive pulmonary disorder (COPD) or when patients are put on mechanical ventilators, the scientists increased the strain to 10% to see what would happen. The higher strain caused an increase in innate immune response genes and processes, including several inflammatory cytokines.

“Because the higher strain level resulted in greater cytokine production, it might explain why patients with lung conditions like COPD suffer from chronic inflammation, and why patients who are put on high-volume ventilators sometimes experience ventilator-induced lung injury,” Si explained.

From a chip to clinical trials 

The scientists then went a step further, comparing the RNA molecules present in cells within strained vs. static Alveolus Chips to see if they could pinpoint how the breathing motions were generating an immune response. They identified a calcium-binding protein, called S100A7, that was not detected in static chips but highly expressed in strained chips, suggesting that its production was induced by mechanical stretching. They also found that increased expression of S100A7 upregulated many other genes involved in the innate immune response, including multiple inflammatory cytokines.

These immunofluorescence micrographs (at different magnifications) show the 3D cellular structure that develops within the alveolar channel and mimics the microstructure of human alveoli. Credit: Wyss Institute at Harvard University

S100A7 is one of several related molecules known to bind to a protein on cells’ membranes called the receptor for advanced glycation end products (RAGE). RAGE is more highly expressed in the lung than in any other organ in the human body, and has been implicated as a major inflammatory mediator in several lung diseases.

The drug azeliragon is a known inhibitor of RAGE, so the scientists perfused azeliragon through the blood vessel channel of strained Alveolus Chips for 48 hours, then infected the chips with H3N2 virus. This pretreatment prevented the cytokine-storm-like response that they had observed in untreated chips.

Based on this promising result, the team then infected strained Alveolus Chips with H3N2 and administered azeliragon at its therapeutic dose two hours after infection. This approach significantly blocked the production of inflammatory cytokines – an effect that was further enhanced when they added the antiviral drug molnupiravir (which was recently approved for patients with COVID-19) to the treatment regimen.

These results caught the eye of Cantex Pharmaceuticals, which owns patent rights to azeliragon and was interested in using it to treat inflammatory diseases. Based in part on the Wyss team’s work in Alveolus Chips, Cantex licensed azeliragon for the treatment of COVID-19 and other inflammatory lung diseases in early 2022.

Given the drug’s excellent safety record in previous Phase 3 clinical trials, the company has applied for FDA approval to start a Phase 2 trial in patients with COVID-19 patients, and plans to follow with additional Phase 2 trials for other diseases including COPD and steroid-resistant asthma.

“Thanks to the great work of the scientists at the Wyss Institute, we now have compelling evidence that azeliragon may have the potential to prevent severe COVID-19 illness in the form of a once-a-day pill. We’re excited to have the opportunity to conduct clinical trials of azeliragon for this disease, seeking to bring this groundbreaking therapy to patients to prevent the life-threatening inflammation that is a major cause of hospitalization and death,” said Stephen Marcus, M.D., CEO of Cantex.

While azeliragon is a promising anti-inflammatory drug, the scientists warn that more studies are needed to determine a safe and effective treatment regimen in humans. RAGE is a vital player in initiating beneficial inflammation against pathogens in the early stages of an infection, and inhibiting it too soon could prevent a patient from mounting a sufficient immune response.

Given the Alveolus Chip’s many advantages over traditional preclinical models, the Wyss team is exploring the incorporation of additional cell types such as macrophages into the chips to increase their complexity and model more biological processes, such as adaptive immunity. They are also using their existing model to study the efficacy of new compounds, drugs, and biologics (such as mRNA therapeutics) against influenza, SARS-CoV-2, and other diseases.

“This important paper led to the discovery of RAGE inhibitors’ promise for treating inflammatory lung diseases, which was the basis for the recent license of azeliragon to Cantex and its movement toward human clinical trials for COVID-19. I am extremely proud of this team and how quickly this scientific finding was translated into commercialization that will hopefully lead to lifesaving treatment for patients.

This is what the Wyss Institute is all about,” said senior author Donald Ingber, M.D., Ph.D., who is the Wyss Institute’s Founding Director as well as the Judah Folkman Professor of Vascular Biology at Harvard Medical School (HMS) and Boston Children’s Hospital, and Hansjörg Wyss Professor of Bioinspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

Additional authors of the study include Amanda Jiang, Chaitra Belgur, M.S., Yunhao Zhai, Ph.D., Melissa Rodas, and Aditya Patil and Girija Goyal, Ph.D. from the Wyss Institute, and former Wyss Institute members Roberto Plebani, Ph.D., Crystal Oh, Atiq Nurani, M.S., Sarah Gilpin, Ph.D., Rani Powers, Ph.D. and Rachelle Prantil-Baun, Ph.D.

This research was supported by the Wyss Institute for Biologically Inspired Engineering at Harvard University, the US Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement HR0011-20-2-0040, and the National Institutes of Health under grants UG3-HL-141797 and UH3-HL-141797.

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Coronavirus Disease 2019 (COVID-19) is a novel communicable disease caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), with remarkably heterogeneous and protean manifestations, from asymptomatic to life-threatening severe pneumonia. In some patients, especially the elderly, COVID-19 infection causes acute respiratory distress syndrome (ARDS), multiorgan failure, and death[1], [2], [3]. Various predisposing factors and comorbidities, such as aging, sex, ethnicity, obesity, diabetes, high blood pressure, malignancies, kidney and liver disorders, have been identified as COVID-19 risk factors of mortality [4].

Among these variables, age confers a substantial risk to COVID-19 mortality. A recent cohort study with a large sample volume showed that aging was substantially associated with increased death risk among people aged 80 by 20-fold compared to 50–59-year-old age group people [5]. Cardiovascular disease, metabolic disorders, malignancies, neurodegeneration, and autoimmune diseases, and age-related inflammatory conditions or inflammaging are among age-associated medical conditions that can enhance morbidity and mortality of COVID-19 infection (Fig. 1) [6], [7].

Finding aging-related mechanisms that underpin COVID-19 fatality and putting in place preemptive measures potentially may help to mitigate the overwhelming effects of the COVID-19 pandemic on health care systems. COVID-19, in essence, is a hyperinflammatory reaction, considering that the cytokine storm plays a substantial role in its pathogenesis, any pre-existing inflammatory condition, including inflammaging, may exacerbate COVID-19- associated morbidity and mortality [8], [9].

Fig 1. The schematic illustration between AGEs and COVID-19 risk factors. All of the disorders related to AGE/RAGE pathway could be as COVID-19 associated morbidity and mortality.

Nevertheless, the exact mechanism of inflammaging and its potential adverse effects on health outcomes remain unrecognized [7]. Advanced glycation end products (AGEs), a highly heterogeneous group of compounds produced by glycation of amino acids, lipids, and DNA molecules, have been shown to contribute to the age-associated increase in inflammation or ‘‘inflammaging’’[10].

Both aging and hyperglycemia accelerate the formation of AGEs, which serve as ligands to several cellular receptors, including the receptor for advanced glycation endproducts (RAGE), which is expressed mainly by the alveolar epithelial cells and macrophages, that have a central role in the lung inflammation caused by COVID-19 [11], [12], [13], [14].

Furthermore, increased levels of AGEs are associated with conditions, including diabetes, obesity, and cardiovascular disease, which increase COVID-19 related morbidity and mortality [15]. Thus, we hypothesize that AGEs may contribute to several aspects of COVID-19 pathogenesis in the elderly. Considering the importance of the RAGE pathway as a novel therapeutic target for COVID-19 management [14], [16], [17] here, for the first time, we reviewed the RAGE receptor with an emphasis on its ubiquitous ligands, AGEs, and its probable association with severe COVID-19 risk factors, including aging, hypertension, obesity, and hyperglycemia (Fig. 1), in the following, we propose potential treatments interfere with AGE-RAGE interaction.

  1. Ages formation and contribution to inflammation and oxidative stress through RAGE signaling
    AGEs are the principal product of the sequential non-enzymatic glycation of protein by sugars such as glucose and fructose [18]. The production of AGEs is primarily driven by the metabolism of glucose and fructose and, to a lesser extent by threonine and lipid peroxidation. These reactions result in the generation of highly reactive alpha dicarbonyl groups, which propagate the generation of AGEs [10]. Methylglyoxal (MGO) is potentially the most crucial propagator molecule for AGEs generation, primarily produced as a byproduct in glycolysis during the conversion of dihydroxyacetone phosphate to glyceraldehyde-3 phosphate [19]. The AGEs are created through the alteration of lipids, nucleotides, or amino acids (lysine or arginine) by propagators such as MGO [10].

The RAGE is a single receptor for multiple ligands first identified and isolated from the bovine lung [20]. RAGE belongs to the heterogeneous group of pattern recognition receptors (PRRs) that can recognize a typical pattern within diverse ligands, including S100 proteins with calcium-binding properties and cytokine-like functions, High mobility group box-1 protein (HMGB1), β2-Integrin, Macrophage 1 antigen (Mac-1), or CD11b and various other ligands [21]. RAGE has three immunoglobulin-like domains, with the transmembrane and cytoplasmic domains. The two other forms of RAGE: dominant-negative RAGE (DN-RAGE) and endogenous secretory RAGE (esRAGE), are alternative splicing products. The DN-RAGE lacks the cytoplasmic domain, and both the cytoplasmic and transmembrane domain is absent in esRAGE [21].

Several signaling pathways contribute to AGEs-RAGE interactions. The actin-binding molecule, mDia1 (diaphanous1), serves as an adaptor molecule for RAGE signaling in the various cell types [22]. The activation of the RAGE signaling pathway through phosphatidylinositol-3 kinase (PI-3 K), Ki-Ras, and the mitogen-activated protein kinase (MAPKs), Erk1, and Erk2 lead to nuclear factor-kB (NF-kB) activation and range of inflammatory response, which is mediated by cytokine such as IL-6 [21], [22]. RAGE also induces a spectrum of pathological effects through the activation of oxidative stress by activation of Rac, and subsequently, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an enzyme that has a critical function in the production of the free radical superoxide[22].

  1. The association of AGEs and risk factors for COVID-19 death
    3.1. Aging
    The clinical and epidemiological reports of COVID-19 reveals that age and different comorbidities raise the risk of infection with more critical lung involvement and death[5], [23]. COVID-19 has highlighted the susceptibility of the elderly to emerging infectious diseases[6]. The study of 5700 patients in New York City revealed the importance of aging in the COVID-19-related mortality in people older than 80 years old compared to all other age groups [24]. Similarly, the study in the UK showed that more than 90% of the COVID-19-related deaths were in people over 60[5]. The accumulation of AGEs during aging is associated with an enhanced risk of developing various chronic diseases that disproportionally afflict older individuals[18]. AGEs accumulate gradually with age in adults aged 65 and older, and their levels correlate with an increased risk of mortality due to all-cause or cardiovascular disease (CVD) [25], [26]. We and others have recently reviewed the evidence for the accumulation of AGEs with age and its relevance to aging and age-related diseases, including diabetes [22], [27]. The AGEs may potentially trigger COVID-19 severity and mortality through their classic receptor RAGE, which is mainly expressed on the surface of type one and type two alveolar epithelial cells as well as alveolar macrophages, the cells that have indispensable roles in COVID-19-related acute lung injury [28], [29].

3.2. Hypertension
Hypertension is the most prevalent comorbidity reported in hospitalized COVID-19 patients, with an association with higher risks of adverse outcomes, including mortality, ICU admission, and heart failure[30]. Hypertension is considered a remarkable all-cause mortality risk in COVID-19 patients[31]. The Renin-angiotensin system (RAS) has a critical role in controlling blood pressure[32]. The angiotensin-converting enzyme-2 (ACE2), which serves as the receptor for SARS-CoV-2, exert a pivotal role in this complex system and virtually has a crucial impact on susceptibility to the severe form of COVID-19[33]. ACE2, a homologue of angiotensin-converting enzyme (ACE), was primarily described by Donoghue et al. in 2000[34]. The ACE2 is a type 1 integral membrane glycoprotein composed of 805 amino acids, and its gene is located on the short arm of the X chromosome (Xp22)[34]. It is constitutively expressed by the epithelial cells of the lungs, kidney, intestine, and blood vessels[35]. ACE2 has several critical roles, including regulation of blood pressure, cardiac function, and it serves as the principal receptor for three viruses in the coronaviridae family with clinical relevance, including HCoV-NL63, SARS-CoV, and SARS-CoV-2 [36], [37]. Several lines of evidence show close interaction between AGE-RAGE and RAS systems, which may be mediated by reactive- oxygen (ROS) produced by the AGE-RAGE pathway as well as angiotensin II created in the RAS system [38], [39]. Angiotensin II has both inflammatory and thrombotic properties, which are prominent findings in patients with COVID-19. Angiotensin II ignites the inflammatory reactions through the Angiotension 1 (AT1) receptor along with induction of RAGE expression by endothelial cells [12], [40]. It can be postulated that the down-regulation of ACE2 by SARS-CoV-2[41], [42] may lead to angiotensin II accumulation[42] which may further trigger the inflammatory reaction through the RAGE pathway. Considering the cross-talk between the AGE-RAGE pathway and the RAS system also the role of RAGE in the pathogenesis of severe form of COVID-19 [14], [43], AGE-RAGE interaction may affect COVID-19 death through its impact on the RAS system that regulates blood pressure, which needs to be documented[43].

3.3. Obesity
Obesity is a risk factor for various diseases, including metabolic, kidney, and cardiovascular disorders [44]. Recently, it has been reported that obese individuals show a poor prognosis for COVID-19, such as the need for hospitalization and ventilation, in addition to respiratory failure [45], [46], [47]. The recent study reported a significant relationship between obesity and ICU admission in COVID-19 patients[47], [48]. Additionally, obesity-related complications such as type 2 diabetes mellitus (T2DM) and hypertension are independent risk factors for a severe form of COVID-19 [48], [49]. There is a close relation between RAGE, adiposity, and innate immune system activation[50], [51]. It has been documented that inhibition of RAGE signaling may have therapeutic implications for obesity and metabolic disorders[52]. Furthermore, the reduction of methylglyoxal (MGO), one of the precursors of AGEs, has been shown to reduce obesity in mice. Dietary genistein reduces MGO and advanced glycation end-product accumulation in obese mice treated with a high-fat diet [53]. Gaens et al. confirmed the high expression of carboxymethyl lysine-AGE and RAGE receptor in adipocytes[54]. The AGE-RAGE interaction on the surface of adipocyte and macrophages in white adipocyte tissue induces the inflammatory cascade mediated by nuclear factor kappa B (NF-κB), which leads to cytokine and chemokine production[51]. It seems that the AGE-RAGE interaction in adipocytes may exacerbate the inflammatory reactions that occur in COVID-19 obese individuals, which need to be documented.

3.4. Diabetes and hyperglycemia
Data showed a remarkable association between diabetes mellitus and hyperglycemia with COVID-19 severity and increased mortality[55]. The study conducted by Codo et al. revealed that elevated glucose levels and enhanced glycolysis promotes SARS-CoV-2 replication and cytokine elaboration in monocytes [56]. Parallel to these findings, evidence showed that Glucose-lowering drugs routinely used to control blood sugar might reduce COVID-19 severity[55], [57]. Increased blood glucose promotes inflammatory reactions through the generation of AGE compounds [58]. The accumulation of AGEs is associated with an increased risk of morbidity and mortality in diabetic patients [10]. AGEs are not merely biomarkers of a hyperglycemic and pro-inflammatory condition; rather, they also contribute to the pathogenesis of diabetic complications, such as peripheral neuropathy, nephropathy, and cardiomyopathy, mainly through their interactions with their principal receptor, RAGE[25], [59], [60], [61]. AGEs are produced not only from glucose but also from intermediary molecules produced during glycolysis. Methylglyoxal (MGO) is non-enzymatically generated during glycolysis and is a critical precursor for several AGEs [10]. A recent study showed that SARS-CoV-2 induces glycolysis gene at the transcriptional levels at 24 post-infection to provide one-carbon metabolism necessary for its replication[62]. It can be postulated that striking induction of glycolysis reported in recent studies may expedite the production of AGEs during the initial phase of SARS-CoV-2 replication, especially in diabetic and hyperglycemic patients. AGEs up-regulates the expression of its receptor, RAGE, on the surface of endothelial cells, and its subsequent interactions with RAGE can induce exaggerated inflammatory and oxidative reactions through the elaboration of pro-inflammatory cytokines, including TNF-α, IL-1, and IL-6, and production of reactive oxygen and nitrogen intermediates respectively which may lead to endothelial dysfunction and hypercoagulation[58], [63]. Considering the similarity between mechanistic principles that underlie a critical form of COVID-19 and the inflammation provoked by AGE and RAGE interaction, as well as the effect of the diabetes mellitus on COVID-19 severity, it can be hypothesized that diabetes mellitus can affect COVID-19 exacerbation through the accelerating the formation of AGE compounds. On the other hand, SARS-CoV-2 promotes glycolytic reactions that may serve as a source for amplification of AGEs production[62].

  1. AGEs, cellular senescence and inflammaging
    The chronic sterile low-grade Inflammation that occurs with age is defined as inflammaging [64]. A principal feature of inflammaging is the persistent activation of the innate immune system, in which the macrophage has a pivotal role [61]. A couple of self and non-self- molecules, including microbial pathogens, microbiota, nutrients, and damaged-associated molecules (DAMPs), underpin the inflammaging process through the activation of PRR receptors such as Toll-like receptors (TLRs) and induce the production of pro-inflammatory cytokines during inflammaging [64].

Experimental and epidemiological studies show that AGEs have a particular role in inflammaging through several mechanisms, including protein cross-linking, oxidative stress, senescence, and up-regulation of inflammatory processes [10], [65]. As mentioned, above through binding several ligands, RAGE acts like a classic PRR and induces various subtle inflammatory reactions during the aging phenomenon [7], [21]. Furthermore, AGEs can also contribute to inflammaging by increasing cellular senescence. Cellular senescence, a state of stable growth arrest is accompanied by activation of inflammatory processes. Senescent cells increase with age in most mammalian tissues and can accelerate age-related diseases in part by secreting a myriad of factors, including pro-inflammatory molecules, collectively known as the senescence-associated secretory phenotype (SASP)[66], [67]. A recent review discusses how increased cellular senescence may contribute to the cytokine storm observed in COVID-19 infections[68], [69].

  1. Ages in acute respiratory distress syndrome (ARDS) and sepsis
    Acute respiratory distress syndrome (ARDS) is the potentially life-threatening inflammatory involvement of alveoli, which has a critical role in the COVID-19 associated death[70]. The alveolar macrophages and alveolar epithelial cells (AECs), including type1 and type 2 cells, have a pivotal role in the evolution of this respiratory syndrome [71], [72]. The pattern recognition receptors, including TLRs, inflammasome, and RAGE, play pivotal roles in inflammatory reactions associated with ARDS and lung injury through their interactions with several pathogens associated molecular pattern (PAMP) and damaged-associated molecules (DAMPs) [73], [74] (Fig. 2). Recently, it was shown that the expression of EN-RAGE (S100A12), a ligand for RAGE and a biomarker of pulmonary injury, strikingly increases in peripheral monocyte of COVID-19 patients and may promote pulmonary damage in these patients through its interaction with RAGE on the surface of alveolar epithelial cells [43]. This study accentuates the importance of RAGE and its ligand in the pathogenesis of COVID-19-associated lung injury. AGEs are one of the classical and primary ligands recognized for RAGEs [12], [21]. The production and accumulation of AGEs in the body, due to physiological or pathological processes like aging and hyperglycemia, may promote COVID-19 related lung pathology in the elderly [10]. The existing data reveals that a diet with a high content of AGEs worsens acute lung injury in animal models [75].
Fig 2. Contribution of AGEs in cytokine storm related to COVID-19 pathogenies. Different receptors and cells in alveolar sac are involved in cytokine storm syndrome. ACE2 as COVID-19 receptor is expressed on various cells including type one and type two alveolar epithelial cells as well as alveolar macrophages. On the other hand, RAGE receptor on the type 2 alveolar epithelial cells surface reacts with its ligands especially AGEs resulting to NF-κB activation and Cytokine storm.

Inflammasomes are multi-subunit and complex molecules, which induce the production of an active form of inflammatory cytokines, including IL-1 and IL-18, following activation by various molecules such as bacterial and viral pathogens [76]. Recent studies showed the importance of inflammasome in the development of the severe and critical form of COVID-19 [77], [78], [79]. Also, there is a close interaction between inflammasomes and RAGE in inflammatory diseases [80], [81], [82]. Considering these findings, one can postulate that the RAGE–AGE and inflammasome pathways may exert a significant role in the severe form of pneumonitis and determining the extent of lung damage in many pathological events, including severe pneumonitis in COVID-19 [82].

A previous study showed that patients with COVID-19 meet the diagnostic criteria for viral sepsis and septic shock [6]. Furthermore. RAGE signaling has a remarkable effect on the development of sepsis, and inhibition of this molecule with antibody (anti-RAGE) may mitigate the sepsis complications [83]. Thus the RAGE-AGE pathway is a potential contributor in COVID-19 associated septic, which needs to be further examined.

  1. Anti-AGEs interventions that hypothetically could be used to minimize COVID-19 complications
    Considering the association of AGEs with immune function, aging, and COVID-19 comorbidity diseases, AGEs can be a promising therapeutic target for treating patients with pre-existing conditions and severe COVID-19 symptoms. Studies that targeted AGEs for treating age-associated diseases have shown significant improvement in overall health in both animal models and human clinical trials [84]. Hence, inhibition of AGEs can be an alternate method for reducing the risk of COVID-19 and related mortalities.

Metformin is the cornerstone drug for the management of hyperglycemia in T2DM patients. Interestingly a recent study documented the positive effects of metformin, independent of its glucose reducing capabilities, in COVID-19 patients [85]. Data shows that metformin reduces the toxic effects of AGEs and reduces diabetes-associated COVID-19 severity. Association of Cardio Vascular Diseases (CVD) with COVID −19 is clinically proven, and pre-existing CVD conditions worsen the outcome of this viral disease [86]. Algaebrium chloride (ALT-711), a drug used for treating CVD, has shown promising results in breaking AGEs cross-linking and thus improving the heart condition in dogs [87], monkeys [88], diabetic mice models [89], and humans [90]. ALT-711 treatment improves cardiovascular functions by reducing ventricle stiffness. This drug was also proven to reduce the accumulation of carboxymethyl lysine (one of the AGEs) and increase the solubility of collagen [89]. Another drug C16 is proved for its ability to limit the accumulation of AGEs in blood vessels and prevent the AGEs cross-linking in the diabetic rat model [91]. Furthermore, aminoguanidine has shown a promising effect to improve vascular elasticity, permeability and reduce complications associated with cardiac hypertrophy in diabetic mice models. Notably, aminoguanidine treatment reduced the level of AGEs in diabetes and atherosclerosis mice models [89]. Reducing AGEs has proven to be an effective treatment for improving age-associated diabetes and cardiovascular diseases [92]. Taken together, it is compelling to hypothesize that reducing AGEs through the administration of the above drugs candidates could help to maintain cardiac function during SARS-CoV-2 infections.

Increased oxidative stress leads to the accumulation of AGEs and inflammatory disease, which has been observed in patients with Rheumatoid arthritis. Treatment with Pyridoxamine and Benfotiamine has proven to reduce the accumulation of AGEs in arthritis patients and improve the inflammatory condition [93]. In the case of SARS-CoV-2 infection excessive ROS is generated due to impaired redox balance in high-risk patients. Treatment with antioxidants is expected to regain the redox balance [94]. Given the role of AGEs to induce ROS, it will be interesting to see if Pyridoxamine and Benfotiamine have a positive impact in reducing the redox imbalance in COVID-19 patients.

Also, there is an alarming concern of acute renal failure in COVID-19 patients due to excessive cytokine release, which requires intensive care in critically ill patients. Clinicians are finding it problematic to manage acute renal failure and reduce mortality. The reason behind the renal failure in COVID-19 patients remains unclear for now [95]. Similar to these observations accumulation of AGEs are the causative factor in diabetic nephropathy that results in glomerular hypertrophy and reduced urine excretion [96]. In a treatment approach, short fragments of nucleic acids (DNA or RNA) called aptamers are reported to bind with AGEs and decrease the accumulation of AGEs in the kidney of diabetic mice. The binding of aptamers to AGEs facilitates effective removal of AGEs from the system and protects the kindney from the AGEs induced oxidative stress [96]. Considering the risk of renal failure in COVID-19 patients, these AGEs reducing aptamers could be considered for treating patients with renal damage.

  1. Reducing AGE precursors to combat COVID-19
    An alternate way to reduce AGEs is to target precursors of AGEs, the reactive carbonyls, generated as a byproduct of normal glycolysis. These reactive carbonyl compounds irreversibly modify nucleic acids and proteins through a non-enzymatic reaction. Reducing the reactivity of these dicarbonyls has proved to reduce the accumulation of AGEs. Methylglyoxal (MGO) is considered a major dicarbonyl that can react with biological macromolecules to generate different AGEs. Carnisone is a naturally occurring dipeptide that is proven to effectively inhibit the activity of methylglyoxal in vitro [97]. Carnisone is effective not only against AGEs generated through glycolysis but also against aldehydes generated through lipid peroxidation [98]. This drug has been proven to negate several health complications including hyperglycemia. Similar to carnisone other peptides like homocarnisone and anserine are found to reduce the glycation process and thereby limit the generation of AGEs [99]. As these peptides are naturally occurring and proven to be safe, exploring the effectiveness of these peptides for COVID-19 treatment might serve as an alternate way to reduce the generation of AGEs and their secondary complication in patients.

LR90 is another potent inhibitor of MGO-induced cytotoxicity and serves as a potent antioxidant and anti-inflammatory drug. LR90 exhibits cytoprotective activity against MGO-induced apoptosis by preventing the release of cytochrome C and inhibiting the activation of caspase-3 and caspase-9 [100]. A recent study reported that SARS-CoV-2 infection-induced apoptosis through its accessory protein ORF3a in a similar pathway of activating the release of mitochondrial cytochrome C and activation of caspase-9 [101]. The close correlation between the pathways involved in MGO cytotoxicity and SARS-Cov-2 infection mediated cytotoxicity is striking. Hence, LR90 could be a potential drug candidate to reduce cytotoxicity in COVID-19 patients through its effects on AGEs. Analogous to LR90, antioxidants like aminoguanidine and N-acetyl cysteine are proved to better combat the cytotoxic effects of MGO in endothelial cells [102].

  1. Inhibiting RAGE axis to limit COVID-19 related inflammation
    Upon activation by ligands, RAGE initiates series of pathways that end up in the generation of ROS and inflammation response. RAGE-mediated activation of pro-inflammatory signals results in tissue damage and regulation of NADPH oxidase that contributes to oxidative stress and neutrophil dysfunction [10]. Given the expression of RAGE in lungs and the critical role of ACE-2 receptors in alveolar cells for SARS-CoV-2 binding, there is a higher possibility for AGE-RAGEs to be involved in COVID-19 progression. Several small molecules have been tested for their efficacy to inhibit RAGE [103]. TTP488, also known as Azeliragon, inhibits the binding of several RAGE ligands, including HMGB1, S100B, and Aβ [104]. TTP488 treatment reduces inflammatory signaling in neurodegenerative models. At a low dose of 5 µM, this drug was found to be not only safe but also improve cognitive function in human subjects [105]. HMGB1 and S100 protein families are the damage-associated signal that are highly expressed in diabetes and microbial infections. A recent clinical study in COVID-19 patients revealed that HMGB1 and S100 A8/A9 levels were significantly high in severe infections. This was found to be associated with poor clinical outcomes, including infection-related tissue damage and cytokine storm [106]. Another small molecule FPS-ZM1, initially identified to inhibit the RAGE-Aβ interaction, was also found to reduce inflammatory signaling in mouse models. FPS-ZM1 effective in rescuing cardiac dysfunction, hypertrophy, and inflammation [107]. Pyrazole-5-carboxamides and 6-Phenoxy-2-phenylbenzoxazoles are other serirs of RAGE inhibitors, exhibited RAGE inhibitory activity in previous studies[108], [109]. Given the efficiency of these drugs to limit RAGE activation by its ligands and their role in reducing inflammatory responses, it will worth studying their effect to reduce COVID-19 associated disease outcomes.
  2. Reducing dietary AGEs to limit COVID-19 associated inflammation
    Dietary or exogenous AGEs are the primary source of AGE molecules in our body[110]. Exogenous AGEs are mainly produced during heat-processing of foods by non-enzymatic Maillard reaction between free carbonyl groups[111]. AGEs are effectively absorbed in the gastrointestinal system, and there is a direct association between plasma AGEs concentrations and dietary AGEs intake and its elimination by kidneys[112], [113]. Data from clinical trials accentuate the critical role of the high dietary intake of AGEs molecules in triggering and promoting inflammatory and oxidative reactions[110], [114]. There are several approaches for reducing the dietary AGEs including, lowering the cooking temperature, decreasing cooking time, and using higher humidity and moisture during food preparation, caloric restriction is another way for diminishing exogenous AGEs [115], [116], [117], [118]. Fats and meats tend to contain more dietary AGE per gram of weight, given that reducing butter, cream cheese, margarine, beef, and hamburger in a routine regimen can help lessen exogenous AGEs. On the other hand, foods such as low-fat milk, vegetables, yogurt, natural juice, honey, chicken, and lamb have low exogenous AGE content[118]. Reducing dietary AGEs content in routine regimen during the pandemic period by simple modifications may hypothetically be beneficial for blunting the hyperinflammatory mechanisms underlying the pathogenesis of a severe form of COVID-19.

Although the direct role of AGEs and RAGE in COVID-19 disease progression and severity is yet to be clarified there seems to be more correlation between AGEs and COVID-19 pathogenicity. Given the uncertainty of potent vaccine or drug candidates against COVID-19 it is worth testing the biomolecules that can potentially reduce the burden of AGEs as complementary treatment (Table 1), to minimize the effect of COVID-19 infections.

Table 1. Potential therapeutics to interven the AGE-RAGE signaling in COVID-19 patients and improve clinical outcomes.

S. NoCompound/DrugMechanism of Action
2Algaebrium chlorideImproves cardiac function, reduce collagen crosslinking
3CarnisoneInhibits Methylglyoxal
4HomocarnisobeReduce glycation process
5AnserineReduce glycation process
6LR90Inhibits MGO induced cytotoxicity
7Amino guanidineInhibits MGO induced cytotoxicity
8N-acetyl cystine (NAC)Inhibits MGO induced cytotoxicity
9Azeliragon (TTP488)RAGE innhibitor
10FPS-ZM1RAGE innhibitor
11Pyrazole-5-carboxamidesRAGE inhibitors
126-Phenoxy-2-phenylbenzoxazolesRAGE inhibitors
13PyridoxineRAGE signaling Inhibitor
14FlavonoidsEnhances the Glyoxalase Pathway
15DNA RNA aptamersReduce AGEs induced stress and removal of AGEs
16B alanineReduce glycation process
17HistidineReduce glycation process
183-[2-(4-Bromo-phenyl)-1-methyl-2-oxo-ethyl]-4,5,6,7-tetrahydro-benzothiazol-3-ium bromide (C16)Reduce AGEs accumulation

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