The results reported open the gateway to effective and safe therapies for COVID-19. Metadichol inhibits ACE2 sufficiently to prevent SARS-CoV-2 entry into host cells. and at the same time the concentrations for inhibition of viral passage is not high enough to affect physiological functions the host.
The results also demonstrate Metadichol’s direct anti-viral effect against SARS-COV-2 virus itself, in CACO-2 cells with an EC90 of 0.15 µg/ml. Comparatively this result gives it a 2000 fold higher effectiveness than Remdesivir and 4000 fold potency over Hydroxy chloroquine phosphate 18.
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.Table 5. Metadichol assay without virus present in Vero cells
Table 5. Metadichol assay without virus present in Vero cells
Units are ug/mL unless noted
|
|
|
|
|
|
|
|
|
ug/mL Metadichol
|
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 6. Metadichol vs Various viruses as measured by Neutral Red assay
ug/mL Metadichol
|
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
|
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.
Table 7 List of Viruses Inhibited by Metadichol In Vitro
Adenovirus
|
Rift valley
|
Japanese Encephalitis
|
Marburg
|
Tacaribe
|
SARS
|
Powassan
|
Respiratory Syncytial Virus
|
Zika
|
Chikungunya
|
Ebola
|
Influenza A (H1N1)
|
Yellow fever
|
Dengue
|
West Nile Virus
|
HIV
|
Vitamin D and SARS-COV-2 infection.
An out of control inflammatory response to SARS-COV-2 is the major cause of disease severity and death in patients with COVID-19 25 and is associated with high levels of circulating cytokines, TNF, CCl2, NF-KB, CRP, Ferritin. Metadichol (see Ref 14) is an inhibitor of CCl2 (also known as MCP-1), TNF, NF-κB, and CRP which, is a surrogate marker for cytokine storm 26 and is associated with Vit D deficiency.
Vitamin D3 is generated in the skin through the action of UVB radiation, reaching 7-dehydrocholesterol 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, a DNA binding protein interacts with regulatory sequences near target genes that participate genetically and epigenetically in the transcriptional output of genes needed for functioning 27. Vitamin D reduces the risk of infections.by mechanisms that include inducing cathelicidins and defensins 28 resulting in lowered viral replication rates and reducing concentrations of pro-inflammatory cytokines. 29. Supplementation with 4000 IU/d of vitamin D decreased dengue virus infection 30. Inflammatory cytokines 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 tumor necrosis factor and interferon 31.
Vitamin D is a modulator of adaptive immunity 32 and suppresses responses mediated by the T helper cell type 1 (Th1) by primarily repressing the production of inflammatory cytokines IL-2 and interferon-gamma (INF) 33. Additionally, 1,25(OH)2D3 promotes cytokine production by the T helper type 2 (Th2) cells, which helps enhance the indirect suppression of Th1 cells by complementing this with actions mediated by a multitude of cell types 34.
1,25(OH)2D3 promotes the T regulatory cells' induction, thereby inhibiting inflammatory processes 35. It is known that COVID-19 infection is associated with the increased production of pro-inflammatory 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 significant strain on the body. CD8 T cells that mediate adaptive immunity 39 to protect the body from microbial invaders can easily reach their Hayflick 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 inflammation 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 , influenza virus type 46, and rabies virus in vitro 47.
Vitamin C deficiency 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-inflammatory 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 specificity. 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
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.
Disease Name
|
Disease Categories
|
P-value
|
Corrected P-value
|
Annotated Genes Quantity
|
Annotated Genes
|
COVID-19
|
Respiratory tract disease, Viral disease
|
4.49E-50
|
3.10E-47
|
13
|
ACE2,AGT,CCL2,CCL3,CSF3,CXCL10,IL10,IL2,IL2RA,IL6,IL7,TMPRSS2,TNF
|
Pneumonia, Viral
|
Respiratory tract disease,Viral disease
|
6.28E-49
|
4.34E-46
|
13
|
ACE2,AGT,CCL2,CCL3,CSF3,CXCL10,IL10,IL2,IL2RA,IL6,IL7,TMPRSS2,TNF
|
Coronaviridae Infections
|
Viral disease
|
2.51E-47
|
1.74E-44
|
13
|
ACE2,AGT,CCL2,CCL3,CSF3,CXCL10,IL10,IL2,IL2RA,IL6,IL7,TMPRSS2,TNF
|
Coronavirus Infections
|
Viral disease
|
2.51E-47
|
1.74E-44
|
13
|
ACE2,AGT,CCL2,CCL3,CSF3,CXCL10,IL10,IL2,IL2RA,IL6,IL7,TMPRSS2,TNF
|
Nidovirales Infections
|
Viral disease
|
2.51E-47
|
1.74E-44
|
13
|
ACE2,AGT,CCL2,CCL3,CSF3,CXCL10,IL10,IL2,IL2RA,IL6,IL7,TMPRSS2,TNF
|
RNA Virus Infections
|
Viral disease
|
7.12E-30
|
4.92E-27
|
13
|
ACE2,AGT,CCL2,CCL3,CSF3,CXCL10,IL10,IL2,IL2RA,IL6,IL7,TMPRSS2,TNF
|
Virus Diseases
|
Viral disease
|
2.51E-28
|
1.73E-25
|
13
|
ACE2,AGT,CCL2,CCL3,CSF3,CXCL10,IL10,IL2,IL2RA,IL6,IL7,TMPRSS2,TNF
|
Sexually Transmitted Diseases, Viral
|
Viral disease
|
1.99E-15
|
1.38E-12
|
7
|
CCL2,CCL3,IL10,IL2,IL2RA
,IL6,TNF
|
HIV Infections
|
Immune system disease,Viral disease
|
2.26E-15
|
1.56E-12
|
7
|
CCL2,CCL3,IL10,IL2,IL2RA,
IL6,TNF
|
Lentivirus Infections
|
Viral disease
|
2.26E-15
|
1.56E-12
|
7
|
CCL2,CCL3,IL10,IL2,IL2RA,IL6,TNF
|
Retroviridae Infections
|
Viral disease
|
2.26E-15
|
1.56E-12
|
7
|
CCL2,CCL3,IL10,IL2,IL2RA,
IL6,TNF
|
HIV Wasting Syndrome
|
Immune system disease,Metabolic disease,Nutrition disorder,Viral disease
|
5.79E-07
|
4.00E-04
|
2
|
IL6,TNF
|
Coxsackievirus Infections
|
Viral disease
|
1.45E-06
|
0.001
|
2
|
IL6,TNF
|
Enterovirus Infections
|
Viral disease
|
6.36E-06
|
0.0044
|
2
|
IL6,TNF
|
Picornaviridae Infections
|
Viral disease
|
7.52E-06
|
0.00519
|
2
|
IL6,TNF
|
We can filter 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 significant p value. These five 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.
Table 10. Diseases network of the curated genes ( figure 5 genes)
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
|
The circled ones are circle in black. The highlighted ones are SIRT1, AR, and FOS. Gilinsk 66 suggested that 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 first step to infectivity is the priming of the spike proteins in SARS-COV-2 by transmembrane protease serine 2 (TMPRSS2), which also cleaves angiotensin-converting enzyme 2 (ACE2) for augmented viral entry. This is seen in the network (Fig 6) Sirt1, which plays an active role in enhancing immunity in viral infections 70
Proteases like Furin 71 and Adam-17 have been described to activate the spikes in vitro, for viral spread and pathogenesis in the infected hosts. The VDR controls Furin expression, mediated through its interaction with SRC 72. Adam-17 is regulated via CEPBP 73,74 which is involved in the regulation of genes involved in immune and inflammatory responses. Recently Ulrich and Pilalt 75 proposed that CD147 is another receptor used as a viral entry like ACE2. CD147 is a known receptor 76 for the parasite that causes Malaria in humans “plasmodium falciparum”. Interestingly, Metadichol (See Ref 6, 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, results in a reduction of ACE2 on cells, and an increase of serum AngII 77. AngII acts as a vasoconstrictor and a pro- inflammatory 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 inflammatory 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.
Clinical
A pilot study (outside the USA) on five COVID-19 patients with minor symptoms showed the absence of virus after 2-4 days of Metadichol @ 20 mg per day. To validate this further, we have been initiated a study in collaboration with the government agencies. We have expanded the trial on over 100 patients with Metadichol vs. comparable control groups, with only Standard Care. We hope to communicate these results very shortly.