Metadichol ® a novel nano lipid that inhibits In Vitro, SARS-COV-2 and a multitude of pathological viruses

New pathogenic virus outbreaks, occurring with increasing regularity, are leading us to explore novel approaches, which will reduce the reliance on time-consuming vaccine modes to halt the outbreaks. The requirement is to nd a universal approach to disarm any new and as yet unknown viruses as they appear. A promising approach could be targeting lipid membranes, which are common to all viruses and bacteria. The ongoing pandemic of severe acute respiratory syndrome-coronavirus 2 (SARS-COV-2) has rearmed the importance of interactions between components of the host cell plasma membrane and the virus envelope as a critical mechanism of infection. Metadichol®, a nano lipid emulsion, has been examined and shown to be a strong candidate to help stop the proliferation of SARS-COV-2. Naturally derived substances, such as long-chain saturated lipid alcohols, reduce the infectivity of various types of viruses, including coronaviruses such as SARS-COV-2, by modifying lipid-dependent attachment to human host cells. SARS-COV-2 uses the receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. Metadichol®, a nano lipid formulation of long-chain alcohols, has been shown to inhibit TMPRSS2 (EC50 96 ng/ml). Compared to the inhibitor camostat mesylate (EC50 26000 ng/ml), it is 270 times more potent. Additionally, Metadichol® is also a weak inhibitor of ACE2 at 31 µg/ml. Further a live virus assay in Caco2 cells, Metadichol® inhibited SARS-CoV-2 replication with an EC90 of 0.16 µg/ml. A pilot study done by a third party Kasturaba Hospital in Mumbai India, on thirty COVID–19 patients with minor symptoms showed the absence of a virus in 75% of patients after 4 days of Metadichol treatment @ 20 mg per day. To validate this further, we have been initiated a larger study in collaboration with government agencies where we will have Metadichol treatment group and control groups, with only Standard Care. We hope to communicate these results in the near future..


Introduction
There is currently an increasing need for a broad-spectrum antimicrobial agent that could inactivate human pathogens, such as bacteria and viruses. Rapid resistance by microorganisms has propelled this approach to the development of focused drugs. The most recent trigger is the fear of a future pandemic caused by new, poorly studied virulent strains, such as the present SARS-COV-2.

Background information on SARS-COV-2
Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) (causative agent of COVID-19) is causing a pandemic1 that has produced global havoc within a few months. Medically controlling a rapidly spreading viral pandemic utilizing speci c antivirals and vaccines will prove expensive and time consuming and is accompanied by compromises on safety and e cacy. An alternative approach is to test molecules that are already proven safe for effectiveness against SARS-COV-2. Among the candidates being tested are camostat mesylate (a 35-year-old Japanese drug), Avigan (another Japanese drug) and Gilead Science Inc.'s remdesivir2.
To enter a host cell, SARS-COV-2 requires transmembrane protease serine 2 (TMPRSS2)3, a serine protease, and angiotensin-converting enzyme 2 (ACE2)4 to bind and thus facilitate its entry. Blocking both receptors can effectively stop the cell entry mechanism used by the virus.
TMPRSS2 is a protease that primes the spike protein of SARS-COV and the Middle East respiratory syndrome-related coronavirus (MERS-COV). Camostat mesylate (CM), an inhibitor of TMPRSS2, inhibited SARS-COV in a mouse model5,6. Hoffmann et al.7 determined that SARS-COV-2 requires TMPRSS2.
They showed that CM blocks virus entry into the lungs. To date, there are no clinical data on the use of CM in patients.
The other receptor used by viruses to enter the host cell is ACE2. SARS-COV-2 has a spike (S) protein on its viral envelope (exterior) that binds to the transmembrane protein ACE2, which is present on human cells. ACE2 is essential for viral entry. However, ACE2 also regulates blood pressure and blood volume; blocking ACE2 would be detrimental to health. An approach that partially regulates ACE2 in concert with inhibition of TMPRSS2 would thus be an ideal solution.

Lipids and viruses
Viral envelope lipids play a role in both viral stability and infective capabilities. For example, substances that affect the lipid envelope, such as phospholipases, organic solvents, and surfactants, such as soaps, have been shown to affect viral infectivity. Causing envelope disintegration, they stop virus transmission to a new host. Active ingredients8 in a number of cleaning agents, wipes, and tissues target the viral lipid envelope to render the virions nonviable. Snipes and coworkers9 showed that saturated alcohols could inactivate viruses with chain lengths from 10 to 14 carbons. Their studies established that inactivation of enveloped viruses by lipids varies greatly, depending on both the nature of the lipid and the type of virus.
Hilmarsson et al.10-12 studied the virucidal effects of medium-and long-chain (8 to 18 carbon) fatty alcohols and corresponding lipids against herpes simplex viruses (HSV-1 and HSV-2), respiratory syncytial virus (RSV), human parain uenza virus type 2 (HPIV2) and enveloped viruses at various concentrations, times and pH levels. After a 10-minute incubation at 37 °C with a 10 mM concentration, 14 of the lipids tested caused a 100000-fold or more signi cant reduction in HSV titre. Testing between pH 7 and 4.2 showed that a pH of 4.2 caused a more rapid inactivation of HSV-1 virus titre in one minute than higher pH values. These long-chain alcohols may act by penetrating the envelope of the virus by hydrophobic effects, making it permeable to small molecules and thus inactivating the virus; the degree of penetration into lipid membranes is based on the chain length of a lipid compared with the thickness of the membrane13.
Metadichol ® is a nano lipid formulation of long-chain alcohols14. Metadichol has been shown to inhibit viruses in vitro and in vivo15-17. Metadichol was tested for its inhibitory actions against ACE2 and TMPRSS2 and in an antiviral assay with SARS-COV-2.

Results And Discussion
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 gure 1,2) and is 270-fold more potent than CM24 Metadichol moderately inhibits ACE2 ( Table 7,

Vitamin D and SARS-COV-2 infection
An uncontrolled in ammatory 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 de ciency.
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-in ammatory cytokines29. Supplementation with 4000 IU/d vitamin D decreased dengue virus infection30. In ammatory cytokine levels increase in viral and bacterial infections, as seen in COVID-19 patients. Vitamin D can reduce the production of pro-in ammatory 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 in ammatory 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 in ammatory processes35. It is known that COVID-19 is associated with the increased production of pro-in ammatory 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 signi cant strain on the body. CD8 T cells that mediate adaptive immunity39 to protect the body from microbial invaders can easily reach their Hay ick 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. Signalling 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) signalling 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 in ammation 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, in uenza virus type46, and rabies virus in vitro47.
Vitamin C de ciency 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-in ammatory 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 di cult to achieve single target speci city. 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 lter 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 signi cant p-value. These ve 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 rst 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 in ammatory 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-in ammatory cytokine (Figure 9) via AT1R78. The Angiostensin II-AT1R axis leads to a pro-in ammatory state79, leading to infections through activation of NF-KB and to increased IL-6 levels in multiple in ammatory 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 in ammation. There are increased levels of the pro-in ammatory 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. In ltration 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 in ammatory 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.
Clinical A pilot study done by a third party Kasturaba Hospital in Mumbai India, on thirty COVID-19 patients with minor symptoms showed the absence of a virus in 75% of patients after 4 days of Metadichol treatment @ 20 mg per day. To validate this further, we have been initiated a larger study in collaboration with government agencies where we will have Metadichol treatment group and control groups, with only Standard Care. We hope to communicate these results in the near future..

Summary And Conclusions
Metadichol inhibits SARS-COV-2 entry into host cells by inhibiting TMPRSS2 and partial inhibition of ACE2 and boosts the antiviral response by enhancing innate and adaptive immunity through the vitamin D pathway and antiviral activity by endogenously increasing vitamin C levels. In addition, telomerase activity can also play a key role in maintaining the levels of naive T and B cells needed to ght infections. Metadichol modulates cytokine storms, as it is an inhibitor of TNF, ICAM1 and CCL2, which, as shown, play a key role with other cytokines. Co morbidities associated82,83 with COVID-19, such as hypertension and diabetes84,85, are also controlled by Metadichol, which could certainly improve the long-term prognosis for the affected patient population. These actions on multiple genes and via multiple pathways bring about homeostasis and prevent SARS-COV-2 infections. Metadichol's86 actions on multiple genes and proteins lead to over 2000 unique interactions with other genes and result in a network that helps bring about homeostasis.
Optical densities were converted to percent of that of cell controls, and the concentration of compound that would cause 50% cell death (CC50) in the absence of virus was calculated by regression analysis.
The selective index (SI) is the CC50 divided by the EC90. The results are shown in Table 7.
For a virus yield reduction (VYR) assay, the supernatant uid from each compound concentration was collected on day three post-infection before neutral red staining (3 wells pooled) and tested for virus titre using a standard endpoint dilution CCID50 assay in Vero 76 cells and titre calculations via the Reed-Muench (1948) equation. The concentration of compound required to reduce virus yield by one log10 was calculated by regression analysis (EC90).
As shown in Table 2 the virus reduction assay did not follow a typical dose-response relationship, with virus reduction seen at concentrations of 0.3 µg/ml and 3.2 µg/ml but no reduction seen at a concentration of 1 µg/ml. It was assumed that breakthrough of the virus at 1 µg/ml was an outlier. The calculated SI was 20 (Table 1), indicating an EC90 of 0.15 µg/ml.
The results for other viruses shown in Table 3 and 4 and 5 were carried out in a similar procedure by various labs using Vero cells.

TMPRSS2 inhibition assay
Procedure TMPRSS puri ed from LNCaP cells (Cayman Chemicals) was used as an enzyme source. The reaction mixture contained puri ed TMPRSS2 protease in TBS with or without a range of various concentrations from 1.56 to 100 ng/ml of test sample or inhibitor. The reaction mixture was incubated for 10 minutes at 37°C. To the reaction mixture, 1 µl of 10 mM the uorogenic trypsin substrate Cbz-Gly-Gly-Arg-AMC was added, and kinetic uorescence readings were recorded after 2 minutes of incubation at 37°C at Ex383 nm and Em455 nm at 5-10 minutes using a SpectraMax i3X (Molecular Devices). The change in uorescence (delta RFU) was calculated to determine the inhibitory effects of the test sample. CM at a two-fold range of concentrations from 1.56 to 100 nM was used as a positive control for TMPRSS2 protease inhibition.