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

New pathogenic virus outbreaks with increasing regularity are leading us to explore novel approaches, which will reduce the reliance on a time-consuming vaccine mode to halt the strike. 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 by targeting the lipids membranes, common to all viruses and bacteria. The ongoing pandemic of the SARS-coronavirus 2 (SARS-CoV-2) has restated 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 the SARS-COV-2. Naturally derived substances, such as Cyclodextrin and sterols, reduce the infectivity of various types of viruses, including the coronavirus like SARS-COV-2, by modifying the 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 of 96 ng/ml). Compared to the inhibitor Camostat Mesylate (26000 ng/ml), it is 270 times more potent. Also, Metadichol ® is a moderate inhibitor of ACE2 @ 31 µg/ml. In the SARS-COV2 anti-viral assay using CACO2 cells, it has an EC90 of 0.16 µg/ml.


Introduction
There is today an increasing need for a broad-spectrum antimicrobial agent, which could inactivate human pathogens such as bacteria and viruses. Rapid resistance has propelled this approach by microorganisms to focused drugs. The most recent trigger is the fear of a future pandemic caused by new, poorly studied virulent strains, like the present SARS-COV-2.
Background To Sars-cov-2 The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) (COVID- 19), is a pandemic 1 , which has caused global havoc within a few months. To medically control a rapidly spreading viral pandemic utilizing speci c Antivirals and vaccines will prove expensive, time-consuming, and carries with it compromise on the safety and e cacy. An alternative approach is to test molecules that are already proven safe and tested to be effective against SARS-COV-2. Among the candidates being tested are Camostat mesylate (a 35year Japanese drug) Avigan (another Japanese Drug) and Gilead Science Inc's Remdesivir 2 To enter a host cell, the SARS COV-2 needs TMPRSS2 3 , a serine protease, and ACE 2 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 syndromerelated coronavirus (MERS-CoV). Camostat mesylate (CM), an inhibitor of TMPRSS2, inhibited SARS-CoV in a mouse model 5, 6 Hoffmann et al., 7 determined that the SARS-CoV-2 requires TMPRSS2. They showed that CM blocks the virus entry into lungs. So far there are no clinical data on the use of the CM in patients.
The other receptor used by viruses to gain entry into the host cell is ACE2. SARS-CoV-2 has a spike (S) protein on its viral envelope (exterior) that binds to the transmembrane protein angiotensin-converting enzyme 2 (ACE2), which is present in human cells. ACE2 protein is essential for viral entry. However, ACE2 also regulates blood pressure and blood volume; blocking this entirely would be detrimental. A solution that partially regulates ACE2 in concert with inhibition of TMPRSS2 would thus be an ideal solution.

Lipids and Viruses
Viral envelope lipid plays a role in both viral stability as well as its infective capabilities. For example, substances that affect the lipid envelop like Phospholipases, organics solvents, and surfactants like soaps have shown to affect the viral infectability. Causing envelope disintegration, they stop the virus transmission to a new host. Active ingredients 8 in a number of the cleaning agents, wipes, and tissues target the viral lipid envelop to render the virions non-viable. Snipes and coworkers 9 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.Â Hilmarsson et al. 10,11,12 studied the virucidal effects of medium and long-chain (8 to 18 carbon) fatty alcohols and corresponding lipids against HSV-1 and HSV-2 respiratory syncytial virus (RSV) and human virus type 2 (HPIV2) and enveloped viruses, at various concentrations, times and pH levels. After 10-minute incubation at 37 deg C and ten mM concentration, 14 of the lipids tested caused a 100 000-fold or more signi cant reduction in HSV titer. Testing between pH 7 and 4.2 showed that the pH to 4.2 caused a more rapid inactivation of HSV-1 virus titer in one minute. These long-chain alcohols may act by penetrating the envelope of the virus by hydrophobic effect, making it permeable to small molecules and thus inactivating the virus, the degree of penetration into lipid membranes due to the chain length of a lipid compared with the thickness of the membrane. 13 Metadichol is a nano lipid formulation of long chain alcohols 14 . Metadichol has been shown to inhibit viruses in vitro and in vivo 15,16,17 . Metadichol was tested for it inhibitory actions against ACE2, TMPRSS2 and anti-viral assay with SARS-COV-2.

Experimental Methods
All assays were on a fee for service contract basis and outsourced to Bioanalytical testing companies worldwide. Antiviral assay was done by a Bio Safety level 3 (BSL3) facility in USA.

Anti-Viral assay
Metadichol was serially diluted using eight half-log dilutions in test medium (MEM supplemented with 2% FBS and 50µg/mL gentamicin) so that the starting (high) test concentration was 100 µg/ml. Each dilution was added to 5 wells of a 96-well plate with 80-100% con uent CACO-2 cells.
Three wells of each dilution were inoculated with virus, with two wells uninoculated (as toxicity controls), six wells were inoculated and untreated (as virus controls), and six wells were uninoculated and untreated (as cell controls). SARS-CoV-2 virus was prepared to achieve the lowest possible multiplicity of infection (MOI) that would yield >80% cytopathic effect (CPE) within 5 days. M128533 (Protease inhibitor speci c for SARS virus.) was tested in parallel as a positive control. Plates were incubated at 37±2°C, 5% CO2. On day 3 post-infection, once untreated virus control wells reached maximum CPE, plates were stained with neutral red dye for approximately 2 hours (±15 minutes). Supernatant dye was removed, and wells rinsed with PBS, and the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol for >30 minutes and the optical density was read on a spectrophotometer at 540 nm.
Optical densities were converted to percent 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 EC90. Results in Table 1  For virus yield reduction (VYR) assay, the supernatant uid from each compound concentrations was collected on day 3 post infection, before neutral red staining (3 wells pooled) and tested for virus titer using a standard endpoint dilution CCID50 assay in Vero 76 cells and titer calculations using the Reed-Muench As shown in Table 2, the virus reduction assay did not follow a typical dose response, with virus reduction seen at concentration of 0.3 µg/ml and 3.2 µg/ml, but no reduction seen at a concentration of 1 µg/ml. Assuming that breakthrough of virus at 1 µg/ml was an outlier. The calculated SI ratio was 20 (Table 1), indicating EC 90 of 0.15 µg/ml.

TMPRSS2 Inhibition assay
Procedure TMPRSS puri ed from LNCaP cells ( Cayman Chemicals) was used as an enzyme source. The reaction mixture contains the puri ed TMPRSS2 protease in TBS buffer 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 mins and at 37°C. To the reaction mixture, 1µl of 10mM uorogenic trypsin substrate Cbz-Gly-Gly-Arg-AMC was added and the kinetic uorescence reading was recorded after 2 mins incubation at 37°C at 383ex and 455em at 5-10 mins using Spectramax i3X, Molecular devices. Change in uorescence (delta RFU) was calculated to determine the inhibitory effects of the test sample. Camostat mesylate at a two-fold range of concentrations from 1.56 to 100nM was used as a positive control for TMPRSS2 protease.

ACE2 Inhibition assay
The ACE2 Inhibitor Screening Assay Kit, Catalog no 79923 (BPS biosciences, San Diego USA) was to measure the exopeptidase activity of ACE2 and inhibition by Metadichol and control inhibitor DX600. The inhibitory activity was measured based on the uorescence emitted by the cleavage of the chromogenic substrate. Procedure: Enzyme (ACE2) stocks were prepared and from the supplied kit. 20µl of enzyme solution (0.5ng/µl) was added to all the wells designated for the assay. DX600, a potent ACE2 inhibitor Metadichol also inhibits TMPRSS2, as is seen to be 270-fold more potent than Camostat Mesylate 19 . Metadichol inhibits moderately ACE2 and, in combination with TMPRSS2 inhibition, likely leading to a pronounced synergistic effect in overcoming viral entry. The anti-viral assay shown in Table 8, suggest that it is toxic to cells at concentrations above 1 µg/ml. but Metadichol is not toxic as the LD 50 is 5000 mg/kilo 20,21,22 . 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. A previously published work (see ref 15) on anti-viral assay this same "toxicity" was seen and this is shown in Tables 5 and 6.
Raw data from Cytotoxicity of Metadichol without virus present in Vero cells as measured by Neutral red assay. When >75% "toxicity" occurred in the absence of virus, no viral CPE value was reported.  It is not toxicity of Metadichol on cell lines but rather it behaves as a "detergent " in neutralizing the SARS-COV-2 and other pathogenic viruses as shown in table 7.
Also, Metadichol® targets cancer cells in CACO-2 cells. In a previous study 23 of Klotho gene expression of cancer cell lines Mia-Paca, Colo 205 and Panc1, where it was also seen to be toxic to cell lines above 1 µg/ml. It is also toxic to Leukemia CEM-SS cell lines above 5 µg/ml 24 .

1,25(OH)2D3 promotes the T regulatory cells' induction, thereby inhibiting in ammatory processes 35. It is known
that COVID-19 infection is associated with the increased production of pro-in ammatory cytokines, C-reactive protein, increased risk of pneumonia, sepsis, acute respiratory distress syndrome, and heart failure 36. Case fatality rates (CFR's) in China were 6%-10% for those with cardiovascular disease, chronic respiratory tract disease, diabetes, and hypertension 37.

Telomerase and Viral infections
Metadichol increases h-TERT ( telomerase) at one picogram by 16 fold 38. Viral infection puts a signi cant strain on the body. CD8 T cells that mediate adaptive immunity 39 to protect the body from microbial invaders can easily reach their Hay ick limit by depleting their telomeres 40. This is more so if telomeres are already short, then this is more likely to happen. Infections put enormous strain on immune cells to replicate. Naive T and B cells are particularly important when our bodies encounter new pathogens like the like COVID-19 coronavirus. The quantity of these cells is crucial for useful immune function.

Aryl Hydrocarbon receptor and Viral Infections
One of the major issues with infected COVID-19 patients has been a 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 repair 41. NF-KB signaling via AHR may dampen the immune response against coronavirus 42. It has been reported that although some NF-KB signaling is needed for coronavirus replication, excessive activation of this pathway may be deleterious for the virus. AHR limits NF-kB activation and interferes with multiple antiviral immune mechanisms, including IFN-I production and intrinsic immunity. Yamada et al.,43 suggested AHR (Constitutive aryl hydrocarbon receptor ) signaling constrains type I interferon-mediated antiviral innate defense and suggested a need to block AHR constitutive activity and only an inverse agonist can dampen this. We have shown previously that Metadichol® binds to AHR as an inverse/protean agonist 44. Metadichol is an inverse/protean agonist (see Ref 14) of vitamin D receptor and thus can reduce complications attributed to out of control in ammation and cytokine storm.

Vitamin C and its role in viral infections
In infectious diseases, there is also a need to boost Innate and adaptive immunity. Micronutrients with the most robust evidence for immune support are vitamins C and D. Vitamin C is essential for a healthy and well functional host defense mechanism. The pharmacological application of vitamin C enhances immune function 45. Vitamin C has antiviral properties leading to inhibition of replication of herpes simplex virus type 1, poliovirus type 1 , in uenza virus type 46, and rabies virus in vitro 47.
Vitamin C de ciency reduces cellular 48-52 and humoral immune responses, and treatment of healthy subjects promoted and enhanced natural killer cell activities 53 underlining the immunological importance of vitamin C 54,55 and supports its role as a crucial player in various aspects of immune cell functions, such as immune cell proliferation and differentiation, besides its anti-in ammatory properties. Moreover, the newly characterized hydroxylase enzymes, which regulate the activity of the hypoxia-inducible factors (HIF), gene transcription, and cell signaling of immune cells, need vitamin C as a cofactor for optimal activity 56,57,58.
Metadichol increases Vitamin C levels endogenously by recycling Vitamin C and reaches levels not reached by oral intake. The levels reached to bring about changes in improving diverse biomarkers. 59,60,61.
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 hard to achieve single target speci city. Thus, a need to transition from targeting a single gene to multiple targeting of genes is likely to be more active, leading to blocking multiple paths of disease progression 62,63. Table 8 COVID19 and 13 Curated genes  CCL2  IL6  IL7   TNF  TMPRSS2  ACE2   IL10  CCL3  AGT   IL2  IL8  IL2RA CSF3 An analysis of the 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.org 64 that has curated genes relevant to COVID-19. Table 9 shows diseases impacted by the network of Curated genes.  10E-47  13  ACE2,AGT,CCL2,CCL3,   CSF3,CXCL10,IL10,IL2   ,IL2RA,IL6,IL7,TMPRS   S2, We can lter the 13 genes to a set 4 genes: TNF, CCL2, ACE2 and TMPRSS2 are modulated by Metadichol and AGT that is part of RAS (Renin-Angiotensin System) network that ACE2 is 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 can be generated as shown below ( Figure   6) using www.innatedb.org 65 This integrates known interactions and pathways from major public databases. Vitamin D, as a potential mitigation agent in preventing SARS-COV-2 entry.
Metadichol binds to VDR, which controls the expression of FOS 67 . AR also controls the expression of FOS as well as TMPRSS2. Based on these curated data expressions, Figure 7 was generated using web based software PACO 68 shows the relationship between genes in the network. VDR controls FOS expression, FOS controls AGT, AGT controls expression of AGTR1 and ACE and AR controls expression of TMPRSS2. Goren et al. 69 suggested that SARS-CoV-2 infection is likely to be androgen-mediated. The rst step to infectivity is the priming of the spike proteins in SARS-COV-2 by transmembrane protease serine 2 (TMPRSS2), which also cleaves angiotensinconverting enzyme 2 (ACE2) for augmented viral entry. This is seen in the network (Fig 6)  The key to entry into cells by SARS-COV-2 is ACE2 which, when endocytosed with SARS-CoV, results in a reduction of ACE2 on cells, and an increase of serum AngII 77. AngII acts as a vasoconstrictor and a pro-in ammatory cytokine ( Figure 8) via AT1R 78 . The AngII-AT1R axis also activates NF-κB 79. SARS-CoV-2 infection in the lungs can activate NF-κB, which can activate the IL-6 increase, leading to multiple in ammatory and autoimmune diseases 80.
The dysregulation of angiotensin downstream of ACE2 leads to cytokine release that is seen in COVID-19 patients, resulting in increases TNF that leads to IL6, CCl2, NF-KB, and CRP levels. The cytokine storm results in ARDS (Acute respiratory distress syndrome). Metadichol, by its binding to VDR, leads to a network of genes that are involved in mitigating entry and mitigating SARS-COV-2 infection via the Renin-Angiotensin pathway in Figure 8.

Summary And Conclusions
Metadichol, as we have shown, blocks entry of ACE2, TMPRSS2, and CD147 through inhibiting malarial parasite and also Furin, whose expression is controlled by VDR. Metadichol is a unique nano lipid emulsion that inhibits many viruses. Metadichol's 81 action on multiple genes and proteins lead to over 2000 unique interactions with other genes and resulting in a network that affects many biomarkers and diseases, thereby helping bring about Homeostasis.
Metadichol is a safe, non-toxic product, made from renewable sources and commercially available for the last six years, with no reported side effects. This property allows for use of Metadichol in situation that exists today and also to prevent future occurrence of SARS-COV-2 infections being predicted and use to it will allow a rapid return to normal human activity world wide.  Five genes and their network relationships Renin Angiotensin related genes and VDR related gene associations Figure 8 Metadichol-VDR and modulation of RAS pathway