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Metadichol®, a Novel Nano Lipid Formulation that Inhibits SARS-COV-2 and a Multitude of Pathological Viruses in vitro

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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 find 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 reaffirmed 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 extremely 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.
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Metadichol®, a Novel Nano Lipid Formulation that
Inhibits SARS-COV-2 and a Multitude of Pathological
Viruses in vitro
Palayakotai Raghavan ( raghavan@nanorxinc.com )
NANORX Inc.
Research
Keywords: Coronavirus, SARS-COV-2, COVID-19, ACE2, TMPRSS2, VDR, Metadichol
DOI: https://doi.org/10.21203/rs.3.rs-34021/v7
License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read
Full License
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Abstract
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 rearmed 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 extremely 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.
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.
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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.
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..
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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, gures 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 gures 4 and
6 ) but at the same time, not signicant to affect the physiological functions of the host.
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
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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. 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 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.
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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
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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.
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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.
Metadichol is a safe, nontoxic product made from renewable sources and have been commercially available
for the last six years, with no reported side effects. This unique property allows for the use of Metadichol as
an immune modulator to prevent future occurrence of SARS-COV-2 and possibly other infections being
predicted, facilitating a rapid return to normal human social and economic activity worldwide.
Abbreviations
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Gene Description
VDR vitamin D receptor
AHR aryl hydrocarbon receptor
TERT telomerase reverse transcriptase
KL klotho
PAI1 (SERPINE1) serpin family E member 1
HIF 1 alpha hypoxia-inducible factor 1-alpha
CCL2 C-C motif chemokine ligand 2
ICAM1 intercellular adhesion molecule 1
TNF tumour necrosis factor
ACE angiotensin I-converting enzyme
ACE2 angiotensin I-converting enzyme 2
AGTR1 (ANG1) angiotensin II receptor type 1
AGTR2 (ANG2) angiotensin II receptor type 2
TMPRSS2 transmembrane serine protease 2
SIRT1 sirtuin 1
TNF tumour necrosis factor
FURIN furin, paired basic amino acid cleaving enzyme
CD 147 (BSG) Basigin (BSG), also known as extracellular matrix metalloproteinase inducer
IL6 interleukin 6
IL10 interleukin 10
CCL3 C-C motif chemokine ligand 3
IL2 interleukin 2
IL7 interleukin 7
CSF3 Colony-stimulating factor 3
IL2RA interleukin 2 receptor subunit alpha
CXCL8 C-X-C motif chemokine ligand 8
Declarations
Competing interest: Author has no competing interests
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Correspondence and requests for materials should be addressed to raghavan@nanorxinc.com
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Methods
All assays were on a fee-for-service contract basis and outsourced to bioanalytical testing companies
worldwide. SARS-COV-2 antiviral assays were performed by a bio-safety level 3 (BSL3) facility at the Anti-
viral research Institute at Utah State University in the USA. The other assays were performed at Southern
Research Infectious Disease Research Facility in Frederick, Maryland, USA and IBT Bio services in Rockville,
Maryland, USA. ACE2 and TMPRSS2 Assays were carried out by Skanda Life Sciences Pvt Ltd Bangalore ,
India.
Antiviral assay
Metadichol was serially diluted using eight half-log dilutions in test medium (MEM supplemented with 2%
FBS and 50 µg/mL gentamicin) so that the starting (high) test concentration was 100 µg/ml. Each dilution
Page 15/22
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 was prepared to achieve the lowest possible multiplicity of infection (MOI) that
would yield >80% cytopathic effect (CPE) within ve days. M128533 (protease specic for SARS-COV) was
tested in parallel as a positive control. The plates were incubated at 37±2°C and 5% CO2. On day three post-
infection, once untreated virus control wells reached maximum CPE, plates were stained with neutral red dye
for approximately 2 hours (±15 minutes). The supernatant dye was removed, and the wells were rinsed with
PBS. 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 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 (ATCC) 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
Page 16/22
calculated to determine the inhibitory effects of the test sample. CM ( Cayman Chemicals) at a two-fold
range of concentrations from 1.56 to 100 nM was used as a positive control for TMPRSS2 protease
inhibition.
ACE2 Inhibition assay
An ACE2 Inhibitor Screening Assay Kit, catalogue no. 79923 (BPS Biosciences, San Diego, USA), was used to
measure the exopeptidase activity of ACE2 and inhibition by Metadichol and the control inhibitor DX600.
The inhibitory activity was measured based on the uorescence emitted by the cleavage of a chromogenic
substrate.
Procedure
Enzyme (ACE2) stocks were prepared from the supplied kit. Twenty micro litres of enzyme solution (0.5 ng/
µl) was added to all the wells designated for the assay. DX600, a potent ACE2 inhibitor, was used as a
positive control for ACE2 inhibition at various concentrations ranging from 0.0156 µg/ml to 1 µg/ml. The
test sample at concentrations ranging from 0.125 µg/ml to 40 µg/ml was used. To each well containing the
enzyme solution, 5 µl of inhibitor solution was added to the respective designated wells. The reaction
mixture was incubated at room temperature for 5 minutes. After incubation, 25 µl of ACE2 substrate was
added to the mixture and incubated for 1 hour at room temperature. The RFU due to cleavage of the
substrate were read at Ex555 nm and Em585 nm using a SpectraMax i3x (Molecular Devices). The IC50
values were calculated based on the readings obtained.
Tables
Table 1. In vitro antiviral assay results
CC50 EC90 SI90
Metadichol (µg/ml) 4 0.15 20
M128533 (µg/ml) >10 0.2 >33
CC50: 50% cytotoxic concentration of compound without virus added;
EC50: 50% effective antiviral concentration;
EC90: calculated concentration to reduce virus yield by 1 log (90%);
SI: CC50/EC50.
Page 17/22
Table 2. Cytotoxicity and virus yield data for each concentration of Metadichol tested
Metadichol Concentration (µg/ml) Cytotoxicity (%) Virus Titre (CCID50 per 0.1 ml)
100 100% <0.7
32 100% <0.7
10 83% <0.7
3.2 54% 0.7
1 17% 4.3
0.3 26% 1.5
0.1 26% 5.7
0.03 26% 5.3
Table 3. Raw data for cytotoxicity of Metadichol without virus present, as measured by neutral red assay
Units are µg/ml
unless noted     
Metadichol
(µg/ml) Adenovirus Tacaribe Rift
valley SARS Japanese
encephalitis West
Nile
virus
Yellow fever
Powassan
virus
500 95% 98% 96% 96% 100% 100% 100% 100%
160 92% 98% 96% 95% 100% 100% 100% 100%
50 90% 97% 97% 95% 100% 100% 100% 100%
16 85% 95% 81% 92% 88% 77% 98% 100%
5 0% 23% 26% 35% 33% 28% 35% 44%
1.6 0% 2% 10% 15% 12% 14% 19% 6%
0.5 0% 3% 9% 0% 2% 3% 2% 0%
0.16 0% 17% 3% 0% 0% 0% 4% 0%
CC50 9.90 7.30 8.40 6.70 7.20 8.50 5.00 5.1

Table 4 Antiviral assay of Metadichol against various viruses, as measured by neutral red assay
Page 18/22
Metadichol
(µg/ml) Adenovirus Tacaribe Rift
valley
fever
SARS Japanese
encephalitis West
Nile Yellow
fever Powassan
5 100% 31% 100% 0% 56% 84% 70% 53%
1.6 100% 69% 100% 52% 87% 100% 73% 100%
0.5 100% 97% 100% 100% 100% 100% 95% 100%
0.16 100% 100% 100% 100% 100% 100% 96% 100%
EC50 >9.9 2.8 >8.4 1.7 >7.2 >8.5 >5 >5.1
Table 5. List of viruses inhibited by Metadichol in vitro
Adenovirus Rift valley
Japanese encephalitis Marburg
Tacaribe SARS
Powassan Respiratory syncytial virus
Zika Chikungunya
Ebola Inuenza A (H1N1)
Yellow fever Dengue
West Nile virus HIV
Table 6. TMPRSS2 assay data
Page 19/22
Sample Concentration RFU % Inhibition IC50
Control
0 43233358 0.00
Metadichol (ng/ml)
1.56 41305150 4.46 96.65 ng/ml
3.12 39329385 9.03
6.25 36713767 15.08
12.5 33778222 21.87
25 30695684 29.00
50 26087008 39.66
100 16009312 62.97
 
Camostat mesylate (µg/ml)
0.78 37984828 12.14 26.46 ug/ml
1.56 35235186 18.50
3.125 31685728 26.71
6.25 29234396 32.38
12.5 23276839 46.16
25 18931887 56.21
50 8797988 79.65
Table 7 ACE2 assay data
Page 20/22
Sample Concentration (µg/ml) RFU % Inhibition IC50 (µg/ml)
Control 0 308315546 0.00
Metadichol 0.125 290309918 5.84 30.15
0.25 260064163 15.65
0.5 249149792 19.19
1 240301136 22.06
10 212275253 31.15
20 187702504 39.12
40 139821100 54.65
 
DX600 0.0156 252855648 17.99 0.1027
0.031 231028864 25.07
0.0625 193810784 37.14
0.125 145881248 52.68
0.25 127485752 58.65
0.5 111498760 63.84
Table 8. COVID-19 and 13 curated genes
CCL2 IL6 IL7
TNF TMPRSS2 ACE2
IL10 CCL3 AGT
IL2 IL8 IL2RA
CSF3 
Table 9. Disease network of the 13 curated genes
Page 21/22
Disease name Disease categories Corrected
P-value Annotated
gene
quantity
Annotated genes
COVID-19 Respiratory tract disease,
viral disease 3.10E-47 13 ACE2, AGT, CCL2, CCL3,
CSF3, CXCL10, IL10, IL2,
IL2RA, IL6, IL7, TMPRSS2,
TNF
Pneumonia,
viral Respiratory tract disease,
viral disease 4.34E-46 13 ACE2, AGT, CCL2, CCL3,
CSF3, CXCL10, IL10, IL2,
IL2RA, IL6, IL7, TMPRSS2,
TNF
Coronaviridae
infections Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL3,
CSF3, CXCL10, IL10, IL2,
IL2RA, IL6, IL7, TMPRSS2,
TNF
Coronavirus
infections Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL3,
CSF3, CXCL10, IL10, IL2,
IL2RA, IL6, IL7, TMPRSS2,
TNF
Nidovirales
infections Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL3,
CSF3, CXCL10, IL10, IL2,
IL2RA, IL6, IL7, TMPRSS2,
TNF
RNA virus
infections Viral disease 4.92E-27 13 ACE2, AGT, CCL2, CCL3,
CSF3, CXCL10, IL10, IL2,
IL2RA, IL6, IL7, TMPRSS2,
TNF
Virus diseases Viral disease 1.73E-25 13 ACE2, AGT, CCL2, CCL3,
CSF3, CXCL10, IL10, IL2,
IL2RA, IL6, IL7, TMPRSS2,
TNF
Sexually
transmitted
diseases, viral
Viral disease 1.38E-12 7 CCL2, CCL3, IL10, IL2,
IL2RA, IL6, TNF
HIV infections Immune system disease, viral
disease 1.56E-12 7 CCL2, CCL3, IL10, IL2,
IL2RA, IL6, TNF
Lentivirus
infections Viral disease 1.56E-12 7 CCL2, CCL3, IL10, IL2,
IL2RA, IL6, TNF
Retroviridae
infections Viral disease 1.56E-12 7 CCL2, CCL3, IL10, IL2,
IL2RA, IL6, TNF
HIV wasting
syndrome Immune system disease,
metabolic disease, nutrition
disorder, viral disease
4.00E-04 2 IL6, TNF
Coxsackievirus
infections Viral disease 0.001 2 IL6, TNF
Enterovirus Viral disease 0.0044 2 IL6, TNF
Page 22/22
infections
Picornaviridae
infections Viral disease 0.00519 2 IL6, TNF
Table 10. Disease network of genes implicated in SARS-COV-2 infection
Disease name P-value Corrected P-
value Genes Annotated genes
COVID-19 1E-18 5.44E-16 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
Pneumonia, viral 1.56E-
18 8.46E-16 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
Coronaviridae infections 3.4E-18 1.85E-15 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
Coronavirus infections 3.4E-18 1.85E-15 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
Nidovirales infections 3.4E-18 1.85E-15 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
Pneumonia 9.42E-
15 5.11E-12 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
Respiratory tract
infections 3.13E-
13 1.7E-10 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
RNA virus infections 2.46E-
12 1.34E-09 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
Virus diseases 9.48E-
12 5.15E-09 5 ACE2, AGT, CCL2, TMPRSS2,
TNF
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