The results in Table 1 and 2 demonstrate Metadichol's direct antiviral effect against the SARS-COV-2 virus in Caco-2 cells, with an EC90 of 0.15 µg/ml. Comparatively, this result indicates that Metadichol has a 2000-fold higher effectiveness than Remdesivir and 4000-fold potency over hydroxychloroquine phosphate18..
A previously published work15 of antiviral data against other viruses is shown in Tables 3 and 4. Raw data show the cytotoxicity of Metadichol without a virus present in Vero cells was measured by neutral red assay. When >75% "toxicity" occurred in the absence of virus, no viral CPE value was reported.
These results suggest that it is toxic to cells at concentrations above 5 ug/ml in most cases. However, Metadichol is not toxic, as the LD50 is 5000 mg/kg19-21. It is likely that Metadichol at higher concentrations behaves in a soap-mimicking manner by disrupting the lipid membrane, and at lower concentrations, it neutralizes the virus by a different mechanism. Metadichol is not toxic to cell lines, but rather, it behaves as a "detergent" in neutralizing SARS-COV-2 and other pathogenic viruses shown in Table 5. Additionally, Metadichol® targets selectively cancer cells in this case Caco-2 cells. In a previous study22of Klotho gene expression in the cancer cell lines Mia-Paca, Colo 205, and Panc1, Metadichol was seen to be toxic to cell lines above 1 µg/ml. It is also toxic at 10 µg/ml in leukaemia cancer cells243
Metadichol also inhibits TMPRSS2 ( Table 6 figure 1,2) and is 270-fold more potent than CM24 Metadichol moderately inhibits ACE2 ( Table 7, figures 3 and 4 ) and, in combination with TMPRSS2 inhibition, likely leads to a pronounced synergistic effect in overcoming viral entry. The reported results open the gateway to effective and safe therapies for COVID-19. Metadichol is a mild inhibitor of ACE2 ( table 7 and figures 4 and 6 ) but at the same time, not significant to affect the physiological functions of the host.
Vitamin D and SARS-COV-2 infection
An uncontrolled inflammatory response to SARS-COV-2 is the major cause of disease severity and death in patients with COVID-1925 and is associated with high levels of circulating cytokines, tumor necrosis factor (TNF), CCl2, C-reactive protein (CRP), and Ferritin. Metadichol14 is an inhibitor of CCl2 (also known as MCP-1), TNF, NF-kB, and CRP, which is a surrogate marker for cytokine storms26 and is associated with vitamin D deficiency.
Vitamin D3 is generated in the skin through the action of UVB radiation, with 7-dehydrocholesterol generated in the skin, followed by a thermal reaction. Vitamin D3 is converted to 25(OH)D in the liver and then to 1,25(OH)2D (calcitriol) in the kidneys. Calcitriol binds to the nuclear vitamin D receptor (VDR); a DNA-binding protein interacts with regulatory sequences near target genes that participate genetically and epigenetically in the transcriptional output of genes needed for function27. Vitamin D reduces the risk of infections by mechanisms that include inducing cathelicidins and defensins28, resulting in lowered viral replication rates and reducing concentrations of pro-inflammatory cytokines29. Supplementation with 4000 IU/d vitamin D decreased dengue virus infection30. Inflammatory cytokine levels increase in viral and bacterial infections, as seen in COVID-19 patients. Vitamin D can reduce the production of pro-inflammatory Th1 cytokines, such as TNF and interferon (IFN)31.
Vitamin D is a modulator of adaptive immunity32 and suppresses responses mediated by T helper type 1 (Th1) cells primarily by repressing the production of the inflammatory cytokines interleukin (IL)-2 and IFN-gamma33. Additionally, 1,25(OH)2D3 promotes cytokine production by T helper type 2 (Th2) cells, which helps enhance the indirect suppression of Th1 cells by complementing this suppression with actions mediated by a multitude of cell types34.
1,25(OH)2D3 promotes T regulatory cell induction, thereby inhibiting inflammatory processes35. It is known that COVID-19 is associated with the increased production of pro-inflammatory cytokines, elevated CRP levels, increased risk of pneumonia, sepsis, acute respiratory distress syndrome (ARDS), and heart failure36. Case fatality rates (CFRs) in China were 6%-10% for those with cardiovascular disease, chronic respiratory tract disease, diabetes, and hypertension37. Metadichol is a inverse agonist/protean agonist 14 of VDR ie it binds at the same site as calcitriol but has different properties. It is the only known inverse agonist to VDR known in medical literature.
Telomerase and viral infections
Metadichol at one picogram increases h-TERT (telomerase) expression by 16-fold38. Viral infection places a significant strain on the body. CD8 T cells that mediate adaptive immunity39 to protect the body from microbial invaders can easily reach their Hayflick limit by depleting their telomeres40. This possibility is more likely if telomeres are already short. Infections put enormous strain on immune cells to replicate. Naive T and B cells are particularly important when our bodies encounter new pathogens, such as SARS-COV-2. The quantity of these cells is crucial for useful immune function.
AHR and viral infections
One of the major issues with infected COVID-19 patients has been respiratory failure. It has been suggested that the aryl hydrocarbon receptor (AHR) is activated during coronavirus infections, impacting antiviral immunity and lung cells associated with repair41. Signaling via AHR may dampen the immune response against coronavirus42. It has been reported that although some signalling is needed for coronavirus replication, excessive activation of this pathway may be deleterious for the virus. AHR limits activation and interferes with multiple antiviral immune mechanisms, including IFN-I production and intrinsic immunity. Yamada et al.43 suggested that AHR (the constitutive aryl hydrocarbon receptor) signaling constrains type I IFN-mediated antiviral innate defence and suggested a need to block constitutive AHR activity; only an inverse agonist can dampen this activity. We have shown that Metadichol® binds to AHR as an inverse/protean agonist44 and thus can reduce complications attributed to uncontrolled inflammation and cytokine storms.
Vitamin C and viral infections
In infectious diseases, there is also a need to boost innate and adaptive immunity. The micronutrients with the most robust evidence for immune support are vitamins C and D. Vitamin C is essential for a healthy and functional host defence. The pharmacological application of vitamin C enhances immune function45. Vitamin C has antiviral properties leading to inhibition of the replication of HSV-1, poliovirus type 1, influenza virus type46, and rabies virus in vitro47.
Vitamin C deficiency reduces cellular48-52and humoral immune responses, and treatment of healthy subjects promoted and enhanced natural killer (NK) cell activities53, underlining the immunological importance of vitamin C54,55 and supporting its role as a crucial player in various aspects of immune cell functions, such as immune cell proliferation and differentiation, in addition to its anti-inflammatory properties. Moreover, the newly characterized hydroxylase enzymes, which regulate the activity of hypoxia-inducible factor gene transcription and cell signalling of immune cells, need vitamin C as a cofactor for optimal activity56-58. Metadichol administration increases vitamin C levels endogenously by recycling vitamin C and produces levels not reached by oral intake, and those reached bring about changes in improving diverse biomarkers59-61.
Gene cluster network analysis
The present drug discovery paradigm is based on the idea of one gene-one target, one disease. It has become clear that it is difficult to achieve single target specificity. Thus, the need to transition from targeting a single gene to targeting multiple genes is likely to become more attractive, leading to blocking multiple paths of disease progression62,63. Gene network analysis can provide a minimum set of genes that can form the basis for targeting diseases. This clustering network of genes can modulate gene pathways and biological networks. We used www.ctdbase.org64, which has curated genes show in Table 8 relevant to COVID-19. Table 9 lists genes and diseases states that they are involved in.
We can filter the 13 genes to a set of 5 genes: TNF, CCL2, ACE2, TMPRSS2 are modulated by Metadichol and AGT, which is part of the renin-angiotensin system (RAS) network that ACE2 is a part of (Figure 5). A similar analysis of these network genes shows that they are closely networked in diseases, with a highly significant p-value. These five genes are closely related, and the network generated, is shown in Figure 6, using www.innatedb.org65. This analysis integrates known interactions and pathways from major public databases.
The highlighted ones are SIRT1, AR (androgen receptor) , and FOS. Glinsky66 suggested vitamin D as a potential mitigation agent in preventing SARS-COV-2 entry. Metadichol binds to VDR, which controls the expression of FOS67. AR also controls the expression of FOS, as well as that of TMPRSS2. Figure 7, generated below using PACO68, shows the gene network and regulation relationships. VDR controls FOS expression, FOS controls AGT, AGT controls the expression of AGTR1 and ACE, and AR controls the expression of TMPRSS2.
Wambier and Goren69 suggested that SARS-COV-2 infection is likely to be androgen mediated. The first step to infection is the priming of the SARS-COV-2 spike proteins by TMPRSS2, which also cleaves ACE2 for augmented viral entry. This pathway is seen in the network (Figure 8). SIRT1 plays an active role in enhancing immunity in viral infections70
Proteases such as Furin71 and Adam-17 have been described to activate the spike protein in vitro for viral spread and pathogenesis in infected hosts. VDR controls Furin expression, mediated through its interaction with SRC72. Adam-17 is regulated via CEPBP73,74, which is involved in the regulation of genes involved in immune and inflammatory responses. Recently, Ulrich and Pillat75 proposed that CD147, similar to ACE2, is another receptor used for viral entry. CD147 is a known receptor76 for the parasite that causes malaria in humans, Plasmodium falciparum. Metadichol (see Ref6, US patent 9,006,292) inhibits the malarial parasite.
The key to entry into cells by SARS-COV-2 is ACE2, which, when endocytosed with SARS-COV-2, results in a reduction in ACE2 on cells and an increase in serum Angiostensin II77. Angiostensin II acts as a vasoconstrictor and a pro-inflammatory cytokine (Figure 9) via AT1R78. The Angiostensin II-AT1R axis leads to a pro-inflammatory state79, leading to infections through activation of NF-KB and to increased IL-6 levels in multiple inflammatory and autoimmune diseases80.
The dysregulation of angiotensin 2 downstream of ACE2 leads to the cytokine release that is seen in COVID-19 patients, resulting in increased TNF levels that lead to elevated IL-6, CCl2, and CRP levels. Cytokine storms81 result in ARDS.
Controlling cytokine storms
A cytokine storm develops after an initial immune response by the induction of cytokines. The response to SARS-COV-2 leads to inflammation. There are increased levels of the pro-inflammatory cytokines IL-6, IL-18, TNF, and IL-1-beta by macrophages and of IFN-gamma by NK cells.
Figure 9 was generated, using of PACO (www.pathwcommons.org), shows the cytokine relationship network. The cytokines can activate T cells, which lead to tissue damage and infection in the lungs. Infiltration of T cells can also result from the upregulation of adhesion molecules, such as ICAM1, by lung endothelial cells. Metadichol is an inhibitor (see Ref14, US patent 8,722,093) of TNF alpha in vivo, and ICAM1 and CCl2 depress the hyper inflammatory cytokine response caused by SARS-COV-2 and, at the same time, enhance innate and adaptive immunity through the VDR pathways and increased vitamin C levels. Metadichol, by its binding to VDR, leads to a network of gene control of the cytokine storms illustrated in Figure 6, bringing about homeostasis.