U.S. patent application number 17/728769 was filed with the patent office on 2022-09-29 for method of simultaneously treating viral disease caused coronavirus, its variants and mutants using a pharmaceutical micronutrient composition.
This patent application is currently assigned to MATTHIAS W RATH. The applicant listed for this patent is MATTHIAS W RATH. Invention is credited to Anna Goc, Vadim O Ivanov, Aleksandra Niedzwiecki, MATTHIAS W Rath.
Application Number | 20220304976 17/728769 |
Document ID | / |
Family ID | 1000006290893 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220304976 |
Kind Code |
A1 |
Niedzwiecki; Aleksandra ; et
al. |
September 29, 2022 |
Method of simultaneously treating viral disease caused coronavirus,
its variants and mutants using a pharmaceutical micronutrient
composition
Abstract
A method of preventing, inhibiting and treating a mammal
suffering from viral infections using a pharmaceutical
micronutrient composition including mixture D is disclosed. The
middle east respiratory syndrome-related coronavirus and severe
acute respiratory syndrome-related coronavirus as well as their
variants and mutants affecting mammals and causing infection are
successfully treated using mixture D. Mixture D contains key
micronutrients such as an ascorbate, N-acetylcysteine, theaflavins,
resveratrol, cruciferous plant extracts, curcumin, quercetin,
naringenin, and baicalin and a combination thereof. Mixture D and
seemed to have beneficial effects to prevent and treat diseases
where viruses use the angiotensin converting enzyme 2 (ACE2)
receptor on the surface of epithelial cells, endothelial cells and
other cell types for viral entry.
Inventors: |
Niedzwiecki; Aleksandra;
(Henderson, NV) ; Rath; MATTHIAS W; (Henderson,
NV) ; Ivanov; Vadim O; (CASTRO VALLEY, CA) ;
Goc; Anna; (Sanjose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RATH; MATTHIAS W |
HENDERSON |
NV |
US |
|
|
Assignee: |
RATH; MATTHIAS W
HENDERSON
NV
|
Family ID: |
1000006290893 |
Appl. No.: |
17/728769 |
Filed: |
April 25, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17212727 |
Mar 25, 2021 |
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17728769 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/198 20130101;
A61K 45/06 20130101; A61K 31/05 20130101; A61K 31/7048 20130101;
A61K 36/31 20130101; A61K 31/352 20130101; A61K 31/375 20130101;
A61K 31/12 20130101 |
International
Class: |
A61K 31/352 20060101
A61K031/352; A61K 31/375 20060101 A61K031/375; A61K 31/198 20060101
A61K031/198; A61K 45/06 20060101 A61K045/06; A61K 36/31 20060101
A61K036/31; A61K 31/12 20060101 A61K031/12; A61K 31/7048 20060101
A61K031/7048; A61K 31/05 20060101 A61K031/05 |
Claims
1. A method of treating a mammal using a pharmaceutical
micronutrient composition, comprising: administering the
pharmaceutical micronutrient composition containing an ascorbate in
the range of 10 mg to 200,000 mg, N-acetylcysteine in the range of
2 mg to 30,000 mg, theaflavin in the range 5 mg to 3,000 mg,
resveratrol in the range of 10 mg to 5,000 mg, cruciferous plant
extracts in the range of 5 mg to 5,000 mg, curcumin in the range of
5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg,
naringenin in the range of 5 mg to 3,000 mg, polyphenol extract
from green tea in the range of 1 mg to 10,000 mg, brazilin in the
range of 1 mg to 5,000 mg and baicalin in the range of 5 mg to
3,000 mg to treat the mammal infected by a virus.
2. The method of claim 1, wherein the ascorbate is at least one of
or a combination of a L-ascorbic acid, magnesium ascorbate, calcium
ascorbate, ascorbyl palmitate, ascorbyl phosphate, sodium ascorbyl
phosphate and another pharmaceutically acceptable form of
ascorbate.
3. The method of claim 1, wherein the pharmaceutical micronutrient
composition consists of the ascorbate in the range of 10 mg to
200,000 mg, theaflavin in the range 5 mg to 3,000 mg, resveratrol
in the range of 10 mg to 5,000 mg, polyphenol extract from green
tea in the range of 1 mg to 10,000 mg, cruciferous plant extracts
in the range of 5 mg to 5,000 mg, curcumin in the range of 5 mg to
10,000 mg, quercetin in the range of 5 mg to 2,000 mg, naringenin
in the range of 5 mg to 3,000 mg, and baicalin in the range of 5 mg
to 3,000 mg.
4. The method of claim 1, wherein the pharmaceutical micronutrient
composition consists of the ascorbate in the range of 10 mg to
200,000 mg, N-acetylcysteine in the range of 2 mg to 30,000 mg,
resveratrol in the range of 10 mg to 5,000 mg, polyphenol extract
from green tea in the range of 1 mg to 10,000 mg, cruciferous plant
extracts in the range of 5 mg to 5,000 mg, curcumin in the range of
5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg,
naringenin in the range of 5 mg to 3,000 mg, and baicalin in the
range of 5 mg to 3,000 mg.
5. The method of claim 1, wherein the pharmaceutical micronutrient
composition consists of the ascorbate in the range of 10 mg to
200,000 mg, N-acetylcysteine in the range of 2 mg to 30,000 mg,
theaflavin in the range 5 mg to 3,000 mg, resveratrol in the range
of 10 mg to 5,000 mg, cruciferous plant extracts in the range of 5
mg to 5,000 mg, curcumin in the range of 5 mg to 10,000 mg,
quercetin in the range of 5 mg to 2,000 mg, naringenin in the range
of 5 mg to 3,000 mg, and baicalin in the range of 5 mg to 3,000
mg.
6. The method of claim 1, wherein the baicalein is from natural and
synthetic source, theaflavin is from a plant source, curcumin is
from a plant source, resveratrol is from a plant source, quercetin
is from a plant source, cruciferous plant extract is from a plant
source, naringenin is from a plant source, and N-acetylcysteine is
from a plant source.
7. The method of claim 1, wherein a viral infectious disease is
treated using the pharmaceutical micronutrient composition.
8. The method of claim 1, wherein the human and other species are
treated for a viral infection by administering the pharmaceutical
micronutrient composition.
9. The method of claim 8, wherein the viral infection or viral
disease is that which uses a cellular receptor for a viral entry on
a surface of an epithelial cells, endothelial cells or other cell
types.
10. The method of claim 9, wherein the viral infection or viral
disease is that which uses an angiotensin converting enzyme 2
(ACE2) receptor on the surface of an epithelial cell, endothelial
cell and other cell types, for the viral entry.
11. The method of claim 10, wherein the pharmaceutical
micronutrient composition is used to treat the human and other
species with severe acute respiratory syndrome-related to a
coronaviruses (SARS-CoV-1), SARS-CoV2 and their variants or mutants
that use angiotensin converting enzyme 2 (ACE2) receptors on the
surface of epithelial cells, endothelial cells and other cell
types, for viral entry.
12. The method of claim 11, wherein the pharmaceutical
micronutrient composition is used to treat the human and other
species with a Middle East respiratory syndrome-related coronavirus
(MERS-CoV), and its variants or mutants that use the angiotensin
converting enzyme 2 (ACE2) receptor on the surface of epithelial
cells, endothelial cells and other cell types, for viral entry.
13. A method of treating a mammal, comprising: formulating a
pharmaceutical micronutrient composition consisting of an ascorbate
in the range of 10 mg to 200,000 mg, N-acetylcysteine in the range
of 2 mg to 30,000 mg, theaflavin in the range of 5 mg to 3,000 mg,
resveratrol in the range of 10 mg to 5,000 mg, cruciferous plant
extracts in the range of 5 mg to 5,000 mg, curcumin in the range of
5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg,
naringenin in the range of 5 mg to 3,000 mg, and baicalin in the
range of 5 mg to 3,000 mg; and administering the pharmaceutical
micronutrient composition to treat a human and other mammals with a
Middle East respiratory syndrome-related coronavirus (MERS-CoV),
SARS CoV, SARS-CoV2 and their variants or mutants that use the
angiotensin converting enzyme 2 (ACE2) receptor on the surface of
epithelial cells, endothelial cells and other cell types for viral
entry.
14. The method of claim 13, wherein the pharmaceutical
micronutrient composition is formulated as an oral, non-invasive
peroral, topical (for example, transdermal), enteral, transmucosal,
targeted delivery, sustained-release delivery, delayed-release,
pulsed-release and parenteral form.
15. The pharmaceutical micronutrient composition according to claim
13, wherein the pharmaceutical micronutrient composition is
introduced with a food, drinking water, tube feeding, and as an
adjunct to other medicinal treatment.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The current application is a continuation of and claims
priority to pending U.S. application Ser. No. 17/212,727 filed on
25 Mar. 2021. The said US application is hereby incorporated by
reference in its entireties for all of its teachings.
FIELD OF STUDY
[0002] This application discloses method of treating viral
infection caused by coronavirus and its variants and mitigates
coronavirus infection in mammals by administering pharmaceutical
micronutrient composition.
BACKGROUND
[0003] The emergence and rapid spread of the coronavirus pandemic
has resulted in millions of deaths and is compromising human health
and economies on a global scale. Sequencing the whole genome of the
virus from patient samples from Wuhan, China (Zhu et al., 2020)
identified a new coronavirus that was named severe acute
respiratory syndrome coronavirus-2 (SARS-CoV-2) by the Coronavirus
Study Group (CSG) of the International Committee on Taxonomy of
Viruses (Gorbalenya et al., 2020). The disease caused by the virus
was named coronavirus disease 2019 (COVID-19) by the World Health
Organization (WHO).
[0004] The coronavirus is a rapidly mutating virus and, within one
year of the pandemic, several mutations of this virus have emerged
in United Kingdom, South Africa, Brazil and other countries, with
each of these mutations potentially giving rise to further
coronavirus subtypes. Clinical reports show that the British
mutation of the coronavirus can infect patients who have received
the vaccine developed against the original coronavirus SARS-CoV-2,
thereby challenging any claim of a universal efficacy of the
available vaccines against all coronavirus mutations.
[0005] Thus, it is foreseeable that the ultimate control of the
ongoing pandemic caused by the rapidly mutating coronavirus will be
compromised by the need to develop new vaccines potentially for
every new coronavirus mutation, and by the related scientific,
economic and social consequences of such a strategy.
[0006] A promising scientific avenue towards this goal is to focus
on the "docking structure" of the coronavirus on the surface of
cells, the angiotensin-converting-enzyme 2 (ACE 2) receptor.
Significantly, all known coronaviruses, including SARS CoV-2 and
its mutations, use this very same receptor as docking structure and
entry port for infections. This fact makes a detailed understanding
of the regulation of the production/expression of this receptor on
the surface of human cells--as well as related cellular
mechanisms--a prime target towards developing global health
strategies to control the pandemic characterized by a multitude of
current and future viral mutations.
[0007] The cell entry mechanisms of coronaviruses, including
SARS-CoV-2, have been extensively studied. To enter host cells,
coronaviruses first bind to a cell surface receptor for viral
attachment, subsequently enter cell endosomes, and eventually fuse
viral and lysosomal membranes (Li et al., 2016). Coronavirus entry
is mediated by a spike protein anchored on the surface of the
virus. On mature viruses, the spike protein is present as a trimer,
with three receptor-binding S1 heads sitting on top of a trimeric
membrane fusion S2 stalk.
[0008] The spike S1 protein on SARS-CoV-2 contains a
receptor-binding domain (RBD) that specifically recognizes its
cellular receptor, angiotensin-converting enzyme 2 (ACE2). As such,
the receptor-binding domain on SARS-CoV-2 spike protein part S1
head binds to a target cell using the human ACE2 (hACE2) receptor
on the cell surface and is proteolytically activated by human
proteases. Coronavirus entry into host cells is an important
determinant of viral infectivity and pathogenesis (Du et al, 2009,
Du et al. 2017).
[0009] The cellular receptor for the virus binding is
angiotensin-converting enzyme 2 or ACE2, which is an integral
membrane protein present on many cells throughout the human body,
with strong expression in the heart, vascular system,
gastrointestinal system and kidneys, as well as in type II alveolar
cells in the lungs. (Zhu et al., 2019, Li et al., 2003, Hoffman et
al., 2005). Cellular infections by the coronavirus, as well as
intracellular viral replication, is facilitated by several host
enzymatic proteins, including transmembrane protease, serine 2
(TMPRSS2), furin, cathepsins, as well as RNA-dependent RNA
polymerase (RdPp) catalyzing viral RNA multiplication.
[0010] COVID-19 infections have been associated with a high
inflammatory response in the host, termed a "cytokine storm",
thrombosis and other patho-mechanisms that can trigger a fateful
cascade of clinical events associated with advanced coronavirus
infections. In evaluating new approaches to inhibiting coronavirus
infectivity, the ability of such new approaches to ameliorate such
infection-related complications should be an additional target.
Thus, there exists an urgent need for preventive and therapeutic
strategies for inhibiting the infective mechanisms of all
coronaviruses--irrespective of mutation and/or subtype--thereby
offering new avenues towards the global control of the
pandemic.
SUMMARY
[0011] The instant pharmaceutical micronutrient composition
prevents, inhibits, treats and delays attachment, penetration,
biosynthesis, maturation and release of a coronavirus SARS-Cov-2
virus in a mammal. In one embodiment, the phytochemicals in
combination with other vitamins prevents various steps of infection
in a mammal. In one embodiment, various combinations of individual
micronutrients are called mixtures. In one embodiment, mixture D, a
pharmaceutical micronutrient composition is made up of resveratrol,
cruciferous plant extract, curcumin, quercetin, naringenin,
baicalein, theaflavin, vitamin C and N-actylcysteine.
[0012] In another embodiment, a pharmaceutical micronutrient
compound comprises an ascorbate in the range of 10 mg to 200,000
mg, N-acetylcysteine in the range of 2 mg to 30,000 mg, theaflavins
in the range 5 mg to 3,000 mg, resveratrol in the range of 10 mg to
5,000 mg, cruciferous plant extracts in the range of 5 mg to 5000
mg (or equivalent amount of its active compound, sulforaphane),
curcumin in the range of 5 mg to 10,000 mg, quercetin in the range
of 5 mg to 2,000 mg, naringenin in the range of 5 mg to 3,000 mg,
and baicalein in the range of 5 mg to 3,000 mg.
[0013] In another embodiment, additional micronutrients are added
to form a pharmaceutical micronutrient compound such as a phenolic
acid, gallic acid, tannic acid, chlorogenic acid and rosmarinic
acid; a flavonoid such as fisetin, morin, myricetin, kaempferol,
rutin, luteolin, baicalin, scutellarin, naringenin, hesperidin,
hesperetin, apigenin, genistein, phloroglucinol, schisandrin,
urolithin A, punicalagin, brazilin, hispidulin, papaverine,
silymarin, procyanidin B2, procyanidin B3, stilbenes and
pterostilbene; an alkaloid such as palmatine, berberine,
cannabidiol, castanospermine, usnic acid, malic acid, terpenes,
D-limonene and carnosic acid.
[0014] In another embodiment, a pharmaceutical micronutrient
mixture consists of an ascorbate in the range of 10 mg to 200,000
mg, N-acetylcysteine in the range of 2 mg to 30,000 mg, theaflavins
in the range 5 mg to 3,000 mg, resveratrol in the range of 10 mg to
5,000 mg, cruciferous plant extracts in the range of 5 mg to 5,000
mg (or equivalent amount of its active compound, sulforaphane),
curcumin in the range of 5 mg to 10,000 mg, quercetin in the range
of 5 mg to 2,000 mg, naringenin in the range of 5 mg to 3,000 mg,
and baicalein in the range of 5 mg to 3,000 mg. In another
embodiments, the ascorbates are at least one of or a combination of
L-ascorbic acid, magnesium ascorbate, calcium ascorbate, ascorbyl
palmitate, ascorbyl phosphate, sodium ascorbyl phosphate and/or or
another pharmaceutically acceptable form of ascorbate.
[0015] In another embodiment, the pharmaceutical micronutrient
composition further consists of at least one of the theaflavins in
the range 5 mg to 3,000 mg, resveratrol in the range of 10 mg to
5,000 mg, cruciferous plant extracts in the range of 5 mg to 5,000
mg, curcumin in the range of 5 mg to 10,000 mg, quercetin in the
range of 5 mg to 2,000 mg, and a combination thereof.
[0016] In another embodiment, several additional ingredients are
added, to form a pharmaceutically acceptable formulation for
various forms of use, such as oral, injectable, absorbable, etc.
The pharmaceutical micronutrient composition is in the form of
oral, non-invasive peroral, topical (for example, transdermal),
enteral, transmucosal, targeted delivery, sustained-release
delivery, delayed release, pulsed release and parenteral
methods.
[0017] In one embodiment, wherein the viral infection and/or viral
disease uses a cellular receptor for a viral entry on a surface of
an epithelial cells, endothelial cells and/or other cell types.
[0018] In another embodiment, the viral infection and/or viral
disease is that which uses an angiotensin converting enzyme 2
(ACE2) receptor on the surface of an epithelial cell, endothelial
cell and other cell types, for the viral entry, is treated,
prevented and mitigated using pharmaceutical micronutrient
composition.
[0019] The pharmaceutical micronutrient composition, in one
embodiment, is used to treat the human and other species with
severe acute respiratory syndrome-related coronaviruses
(SARS-CoV-1, SARS-CoV2 and their variants) that use angiotensin
converting enzyme 2 (ACE2) receptors on the surface of epithelial
cells, endothelial cells and other cell types, for viral entry.
[0020] The pharmaceutical micronutrient composition, in one
embodiment, is used to treat the human and other species with
Middle East respiratory syndrome-related coronavirus (MERS-CoV),
and its variants that use the angiotensin converting enzyme 2
(ACE2) receptors on the surface of epithelial cells, endothelial
cells and other cell types, for viral entry. The pharmaceutical
micronutrient composition, in one embodiment, is mixture D, which
is used in humans to treat, prevent, inhibit and stop inflammation
caused by severe acute respiratory syndrome-related coronaviruses
(SARS-CoV-1, SARS-CoV-2 and their variants), and Middle East
respiratory syndrome-related coronavirus (MERS-CoV) and its
variants.
[0021] Others features of the present embodiments will be apparent
from the accompanying drawings and from the detailed description
that follows.
BRIEF DESCRIPTION OF DRAWINGS
[0022] Example embodiments are illustrated by way of example and
not limitation in the figures of the accompanying drawings, in
which like references indicate similar elements and in which:
[0023] FIGS. 1A and 1B show several cellular and systemic
mechanisms of coronavirus infection.
[0024] FIG. 2 shows the results of binding of the receptor binding
domain (RBD) of SARS-CoV-2 to the human ACE2 receptor.
[0025] FIG. 3 shows a dose-dependent binding of SARS-CoV-2
pseudo-virions to immobilized epithelial cells overexpressing
hACE2.
[0026] FIGS. 4A, 4B and 4C show viability of cells upon treatment
with indicated polyphenols for 1 h, 3 h, and 48 h.
[0027] FIGS. 5A and 5B show SARS-CoV-2 pseudo-virions binding to
cells at different patterns of treatment.
[0028] FIGS. 6A and 6B show SARS-CoV-2 pseudo-virions' entry to
cells at different pattern of treatment.
[0029] FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7J, 7K show images
of syncytia taken after treatment with indicated polyphenols.
[0030] FIG. 8 shows quantification of syncytia after treatment with
indicated polyphenols.
[0031] FIG. 9 shows selection of the most effective formulation
based on RBD to ACE2 binding inhibition of various micronutrient
mixtures.
[0032] FIG. 10 shows the test for safety for mixture D in human
small alveolar epithelial cells.
[0033] FIG. 11 shows inhibition of RBD binding and efficacy of the
Mixture alone and its combination with Vitamin D.
[0034] FIG. 12 shows inhibition of cellular internalization of the
mutated forms of SARS-CoV-2: viral strains from the UK, Brazil, and
South Africa.
[0035] FIG. 13 shows inhibition of cellular entry of the mutated
forms of SARS-CoV-2: viral strains from the UK, Brazil, and South
Africa, upon application of different patterns of treatment.
[0036] FIG. 14 shows inhibition of ACE2 expression under normal and
pro-inflammatory conditions.
[0037] FIG. 15 shows inhibition of viral RNA-dependent RNA
polymerase (RdRp) activity by mixture D with and without vitamin
D.
[0038] FIG. 16 shows inhibition of furin activity by mixture D.
[0039] FIG. 17 shows inhibition of cellular activity of native
cathepsin L by mixture D applied individually and with vitamin
D.
[0040] FIG. 18 shows mixture D's inhibitory effect on activity of
recombinant cathepsin L and the effects of additional vitamin
D.
[0041] FIG. 19 shows anti-inflammatory effect: inhibition of IL6
secretion under normal and pro-inflammatory conditions by mixture D
alone and combined with vitamin D.
[0042] Other features of the present embodiments will be apparent
from the detailed description that follows.
DETAILED DESCRIPTION
[0043] The life cycle of the virus with the host consists of the
following five steps: attachment, penetration, biosynthesis,
maturation, and release. Once viruses bind to host receptors
(attachment), they enter host cells through endocytosis or membrane
fusion (penetration). Once viral contents are released inside the
host cells, viral RNA enters the nucleus for replication. Viral
messenger RNA (mRNA) is used to make viral proteins (biosynthesis).
New viral particles are then made (maturation) and released.
Coronaviruses consist of four structural proteins: spike (S),
membrane (M), envelope (E) and nucleocapsid (N). Spike is composed
of a transmembrane trimetric glycoprotein protruding from the viral
surface, which determines the diversity of coronaviruses and host
tropism.
[0044] Since several mechanisms are involved in the pathogenicity
of SARS-CoV-2, all of which are ultimately regulated at the level
of cellular metabolism, the most effective approach to viral
infectivity suppression is by identifying molecules that are able
to safely regulate and/or inhibit the expression of
infection-pathway-related proteins.
[0045] FIG. 1A shows the cellular mechanism of viral entry and
several entry points for the SARS-CoV-2 virus and others through
ACE2 receptors, which, having entered, require furin and cathepsin
L for replication, protein synthesis, maturation and release into
the bloodstream. FIG. 1B shows the systemic effect of the release
of interleukin 6 (IL-6) in response to inflammation caused by viral
infection. IL-6 may be a therapeutic target for inhibiting the
cytokine storm and cytokine storm-associated organ damage. We would
show that this is a good target to prevent organ damage.
[0046] The safest and most effective molecules able to exert such a
regulatory role are natural compounds, namely micronutrients. These
natural compounds are by their very nature able to affect
simultaneously, multiple biochemical processes in cellular
metabolism.
[0047] A "mammal" to be treated by the subject method may mean
either a human or non-human animal, such as mice, primates and
vertebrates. The specific diseases that would be targets for a
treatment using a pharmaceutical micronutrient composition are
infections caused by SARS-CoV-2, SARS-CoV-2 variants (such as the
UK, Nigeria, South Africa and Brazil variants, and 19 other
mutations), MERS-CoV (the beta coronavirus that causes Middle East
respiratory syndrome, or MERS), SARS-CoV (the beta coronavirus that
causes severe acute respiratory syndrome, or SARS), SARS-CoV-2, and
all their subtypes, four main sub-groupings of coronaviruses, known
as alpha, beta, gamma and delta.
[0048] Our earlier study showed that a natural micronutrient
composition containing vitamin C, certain minerals, amino acids and
plant extracts was effective in significantly decreasing cellular
ACE2 expression in human lung alveolar epithelial and vascular
endothelial cells. Also, a combination of phytobiological compounds
demonstrated efficacy in inhibiting viral binding to ACE2 cellular
receptors and affecting other mechanisms associated with viral
infectivity.
[0049] Here we claim the efficacy of certain combinations of
micronutrients in significantly inhibiting coronavirus infectivity,
including viral binding to the ACE2 receptor, viral entry into the
cell, intracellular viral replication, and other mechanisms. In
this study we tested the efficacy of a specific nutrient
compositions containing vitamin C, N-acetylcysteine, resveratrol,
theaflavins, curcumin, quercetin, naringenin, baicalin and extracts
of cruciferous plants (broccoli, cabbage, cauliflower) on key
aspects of CoV infectivity: inhibition of viral RBD binding to ACE2
receptors, cellular expression of ACE2 receptors, inhibition of key
enzymes involved in coronavirus activity, and anti-inflammatory and
anti-coagulant effects of this formulation.
[0050] The results show that this micronutrient composition was
effective in inhibiting RBD binding of spike protein of SARS-CoV-2
to the ACE2 receptor (by about 75% at 5 mcg/ml and 85% inhibition
at 10 mcg/ml). At these concentrations, this micronutrient
composition should be considered as a safe and affordable approach
in controlling the current COVID-19 pandemic.
Material and Methods
[0051] Cell cultures: Human Small Airways Epithelial Cells (HSAEpC,
purchased from ATCC) were cultured in Airways Epithelial Cells
Growth Medium (ATCC) in plastic flasks at 37.degree. C. and 5%
CO.sub.2. For the experiment HSAEpC, passage 5-7, were plated to
collagen-covered 96-well plastic plates (Corning) in 100 .mu.L
growth medium and were grown to confluent layer for 4-7 days. Human
cell lung epithelial cell line A549 (obtained from ATCC) was
cultured in DMEM supplemented with 10% fecal bovine serum.
[0052] Micronutrient composition: the micronutrient combination
used in our experiments was developed at the Dr. Rath Research
Institute (San Jose, Ca). The composition of all five mixtures
tested is presented in Table 1.
TABLE-US-00001 TABLE 1 All micronutrients used in different
combinations as mixtures: Micronutrient- Mixture D Vitamin C
N-acetylcysteine Theaflavin-3,3'-digallate Resveratrol Cruciferous
plant extracts Curcumin Quercetin Naringenin Baicalin
TABLE-US-00002 TABLE 2 Mixture A, mixture B, mixture C, mixture D
and mixture E are represented in corresponding column A, B, C, D
and E. Ingredients A B C D E Green Tea Extract X X X 1 mg to 10,000
mg Resveratrol X X X X X 10 mg to 5,000 mg Cruciferous plant X X X
X X extract 5 mg to 5,000 mg Curcumin X X X X X 5 mg to 10,000 m
Quercetin X X X X X 5 mg to 2,000 mg Naringenin X X X X X 5 mg to
3,000 mg Baicalin X X X X X 5 mg to 3,000 mg Theaflavin X X X 5 mg
to 3,000 mg Vitamin C X X X X X 10 mg to 200,000 mg
N-acetylcysteine X X 2 mg to 30,000 mg Fucoidan X X
[0053] Cell-Cell fusion assay: Cell-cell fusion assay was performed
according to Ou et al. Briefly, A549 cells transduced with
eGFP-luciferase-SARS-CoV-2 spike S1 lentivirus vector (GenScript,
Piscataway, N.J.) were detached with 1 mM EDTA, treated with
indicated concentrations of selected polyphenols for 1b. at
37.degree. C. and overlaid on 80-95% confluent human A549 lung
epithelial cells overexpressing hACE2. After 4 h. incubation at
37.degree. C., images of syncytia were captured with a Zeiss Axio
Observer A1 fluorescence microscope (Carl Zeiss Meditec, Inc,
Dublin, Calif.). Positive control was 20 .mu.g/ml anti-ACE2
antibody. Results are expressed as a percentage of polyphenol-free
control (mean+/-SD, n=3).
[0054] Cell supplementation: The micronutrient mixture was
dissolved in DMSO either as 1 mg/ml or 10 mg/ml stock solutions.
For ACE2 expression experiments HSAEpC cells were supplemented with
indicated doses of the formulation in 100 .mu.L/well cell growth
medium for 3-7 days. Applied nutrient concentrations were expressed
as micrograms per ml (ug/ml).
[0055] ACE-2 expression assay (ELISA): Human Small Airways
Epithelial Cells (HSAEpC) were supplied by ATCC (American Type
Culture Collection, Manassas, Va.) and cultured in Small Airways
Epithelial Cells culture medium (ATCC). HSAEpC cells were seeded in
96-well plates covered with collagen at 6 passage and grown to
confluent layer. Cell culture medium was supplemented with
indicated amounts of mixture D and 50 mcg/ml ascorbic acid in 100
mcl per well. After 72 h. cells were supplemented with fresh medium
and the same addition for another 72 h. After 6 days' incubation,
cell layers were washed twice with phosphate-buffered saline (PBS)
and fixed with 3% formaldehyde in PBS with 0.5% Triton X100 for 1
h. at 4.degree. C. Fixed cells were washed four times with PBS and
incubated with 1% bovine serum albumin (BSA) in PBS overnight at
4.degree. C. ACE2 expression was measured with ELISA assay using
primary anti-ACE2 polyclonal antibodies (SIGMA) and secondary goat
anti-mouse IgG antibodies conjugated with horseradish peroxidase
(HRP, Rockland). Amounts of retained HRP were determined by HRP
substrate colored reaction as optical density at 450 nm using a
microplate reader. Results were calculated with Microsoft Excel
software and presented as percentage of unsupplemented controls (an
average of three repetitions+/-standard deviation).
[0056] Receptor binding and entry assays: cell lines and
pseudoviruses: Human alveolar epithelial cell line A549 was
obtained from ATCC. Human alveolar epithelial cell line A549,
stably overexpressing hACE2 receptor (hACE2/A549), was obtained
from GenScript (Piscataway, N.J.). Both cell lines were maintained
in Dulbecco's MEM containing 10% fetal bovine serum (FBS), 100 U/ml
penicillin and 100 .mu.g/ml streptomycin. Pseudovirus particles
with spike glycoprotein as the envelope protein, with eGFP and
luciferase (eGFP-luciferase-SARS-CoV-2 spike glycoprotein
pseudotyped particles) and pseudotyped .DELTA.G-luciferase
(G*.DELTA.G-luciferase) rVSV, were purchased from Kerafast (Boston,
Mass.). Bald pseudovirus particles with eGFP and luciferase
(eGFP-luciferase-SARS-CoV-2 pseudo-typed particles) were purchased
from BPS Bioscience (San Diego, Calif.). Lentiviral particles
carrying human TMPRSS2 were from Addgene (Watertown, Mass.).
[0057] Test compounds, antibodies, recombinant proteins and
inhibitors: Curcumin, tea extract standardized to 85% theaflavins,
theaflavin-3,3'-digallate, gallic acid, tannic acid, Andrographis
paniculata extract, andrographolide, licorice extract, glycyrrhizic
acid, broccoli extract, L-sulforaphane, usnic acid, malic acid,
D-limonene and ammonia chloride were purchased from Sigma (St.
Louis, Mo.). All other polyphenols and camostat mesylate were
obtained from Cayman Chemical Company (Ann Arbor, Mich.). All
antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).
TMPRSS2 recombinant protein was from Creative BioMart (Shirley,
N.Y.).
[0058] SARS-CoV-2 RBD binding to hACE2: Binding/neutralization
reaction was performed using a SARS-CoV-2 surrogate virus
neutralization test kit that can detect either antibody or
inhibitors that block the interaction between the receptor binding
domain (RBD) of the SARS-CoV-2 spike protein and the hACE2 cell
surface receptor (GenScript, Piscataway, N.J.). For screening assay
tested polyphenols at 100 .mu.g/ml were incubated with either
HRP-conjugated receptor-binding domain (RBD fragment) of SARS-CoV-2
spike S1 domain, or with hACE2 immobilized on 96-well plate for 30
min. at 37.degree. C. Next, the samples that were incubated with
RBD fragment were transferred into 96-well plate with immobilized
hACE2 receptor and incubated for additional 15 min. at 37.degree.
C., whereas hACE2 immobilized plates already incubated with
different polyphenols were washed four times with washing buffer
and treated with HRP-conjugated RBD fragments, and then incubated
for 15 min. at 37.degree. C. Subsequently, all plates were washed
four times with washing buffer and developed with
tetramethylbenzidine (TMB) substrate solution for up to 5 min.
followed by the addition of stop buffer. Optical density was
measured immediately at 450 nm with a plate reader (Molecular
Devices, San Jose, Calif.). Positive and negative controls were
provided by the manufacturer. Results are expressed as a percentage
of polyphenol-free control (mean+/-SD, n=5).
[0059] RBD binding: This assay was performed using a GenScript
SARS-CoV-2 surrogate virus neutralization test kit that can detect
either antibody or inhibitors that block the interaction between
the RBD of the viral spike protein with the ACE2 cell surface
receptor. All test samples with indicated concentrations, and
positive and negative controls (provided by the manufacturer) were
diluted with the sample dilution buffer with a volume ratio of 1:9.
In separate tubes, HRP-conjugated RBD was also diluted with the HRP
dilution buffer with a volume ratio of 1:99. Binding/neutralization
reaction was performed according to manufacturer's protocol.
Briefly, diluted positive and negative controls as well as the test
samples with indicated concentrations were mixed with the diluted
HRP-RBD solution with a volume ratio of 1:1 and incubated for 30
min. at 37.degree. C. Next, 100 .mu.L each of the positive control
mixture, negative control mixture, and the test sample mixtures
were added to the corresponding wells with immobilized ACE2
receptor and incubated for 15 min. at 37.degree. C. Subsequently,
the plates were washed four times with 260 .mu.l/well of the
1.times. wash solution, and TMB solution was added to each well
(100 .mu.l/well). Plates were incubated in the dark at room
temperature for up to 5 min. Next, 50 .mu.l/well of stop solution
was added to quench the reaction, and the absorbance was measured
immediately in plate reader at 450 nm. Experiment was performed
three times in duplicates. Data are presented as % of control.
[0060] Binding of pseudo-typed virion mutants of SARS-CoV-2 to
hACE2 receptor: The experiment was conducted according to GenScript
recommendations with small modifications. Briefly,
eGFP-luciferase-SARS-CoV-2 spike protein encapsulated
pseudo-virions were incubated at 37.degree. C. with 5 and 10
.mu.g/ml of mixture D and simultaneously added to hACE2/A549 cells.
Cells were incubated for an additional 1 h. at 37.degree. C.
Subsequently, cells were washed three times with washing buffer,
and primary antibody against SARS-CoV-2 spike protein at 1:1000
dilution, followed by HRP-conjugated secondary antibody at 1:2500
dilution, were employed in ELISA assay. The transduction efficiency
was quantified by recording of the luciferase activity, utilizing a
luciferase assay system (Promega, Madison, Wis.) and a
spectrofluorometer (Tecan Group Ltd., Switzerland). Positive and
negative controls were provided by the manufacturer. Data are
presented as % of control without mixture addition (mean+/-SD,
n=6).
[0061] Cathepsin L activity assay: Experiment was performed in cell
lysates using a Cathepsin L Activity Assay Kit (Abcam, Cambridge,
Mass.) according to the manufacturer's protocol. Briefly,
5.times.10.sup.6 A549 cells treated with mixture D at 5 and 10
.mu.g/ml concentrations for 24 h. were washed with cold
1.times.PBS, and lysed 100 .mu.l with CL buffer for 8 min. After 3
minutes of centrifugion at 4.degree. C., supernatants were
collected and enzymatic reaction was set up by mixing 50 .mu.l of
treated sample, 50 .mu.l of control sample, 50 .mu.l of background
control sample, 50 .mu.l of positive and negative controls. Next,
50 .mu.l CL buffer and 1 .mu.l mM DTT were added, followed by
addition of 2 .mu.l of 10 mM CL substrate Ac-FR-AFC, except for the
background control. Samples were incubated at 37.degree. C. for 1
h. and fluorescence was recorded at extension/emission=400/505 nm
with a fluorescence spectrometer (Tecan Group Ltd., Switzerland).
Data are presented as % of control without PB addition (mean+/-SD,
n=6).
[0062] Effect of mixture D on the activity of isolated cathepsin L
was tested using Cathepsin L Activity Screening Assay Kit (BPS
Bioscience, San Diego, Calif.) according to the manufacturer's
protocol. Briefly, mixture D at 5.0 and 10 .mu.g/ml concentrations
was added to cathepsin L (0.2 mU/.mu.l) for 15 mins at 22.degree.
C., prior to fluorogenic substrate (Ac-FR-AFC) (10 .mu.M) addition
and incubation for 60 mins at RT. Positive control contained only
cathepsin L, and negative control containing cathepsin L and
cathepsin L inhibitor E64d (25 .mu.M). The fluorescence was
recorded at extension/emission=360/480 nm with a fluorescence
spectrometer (Tecan Group Ltd., Switzerland). Data are presented as
a percentage of control without PB addition (mean+/-SD, n=6).
[0063] Furin activity assay: Effects of mixture D on furin
enzymatic activity were evaluated using a SensoLyte Rh110 Furin
Activity Assay Kit (AnaSpec, Fremont, Calif.) in accordance with
the manufacturer's protocol. Briefly, mixture D at 10 and 20
.mu.g/ml concentrations was mixed with furin recombinant protein
for 15 min., followed by the addition of fluorogenic Rh110 furin
substrate. The samples were incubated for 1 h. at 22.degree. C. and
the fluorescence was recorded at extension/emission=490/520 nm with
a fluorescence spectrometer (Perceptive Biosystems Cytofluor 4000).
Results were calculated with Microsoft Excel software and presented
as a percentage of unsupplemented controls (an average of three
repetitions+/-standard deviation).
[0064] In vitro RdRp activity: In vitro RdRp activity was examined
using a SARS-CoV-2 RNA Polymerase Assay Kit (ProFoldin, Hudson,
Mass.) according to the manufacturer's protocol. Briefly, 0.5 .mu.l
of 50.times. recombinant RdRp was incubated with 2.5 .mu.l of
50.times. buffer and 21 .mu.l of Mixture D at 5 and 10 .mu.g/ml
concentrations for 15 min at RT, followed by the addition of master
mix containing 0.5 .mu.l of 50.times.NTPs and 0.5 .mu.l of
50.times. template (as a single-stranded polyribonucleotide). The
reaction (25 .mu.l) was incubated for 2 h at 34.degree. C. and then
stopped by addition of 65 .mu.l of 10.times. fluorescence dye, and
the fluorescence signal was recorded within 10 min at
extension/emission=488/535 nm using a fluorescence spectrometer
(Tecan, Group Ltd., Switzerland). Results are expressed as a
percentage of control without PB addition (mean+/-SD, n=6).
[0065] Interleukin 6 (IL-6) assay: Human Small Airways Epithelial
Cells (HSAEpC) were supplied by ATCC and cultured in Small Airways
Epithelial Cells culture medium (ATCC). SAEC cells were seeded in
six-well plates covered with collagen at 6 passage and grown to
confluent layer. Cell culture medium was supplemented with
indicated amounts of Mixture D mixture, 50 mcg/ml ascorbic acid and
Vitamin D3 in 3 ml per well. After 72 hours incubation conditioned
media were collected and IL-6 content was measured using R&D
Systems Human IL6 ELISA assay in accordance with the manufacture's
protocol. Results were calculated with Microsoft Excel software and
presented as a percentage of unsupplemented controls (an average of
three repetitions+/-standard deviation).
Results
[0066] Our study helps to unravel previously unidentified but
important antiviral mechanisms of natural compounds and expands our
understanding of SARS-CoV-2 biology. Clinical evaluation of their
efficacy in SARS-CoV-2 pathophysiology would be particularly
interesting during later steps of the infection process. This
should include their effects on host responses following SARS-CoV-2
infection and whether or not their antiviral potential could
support or complement current pharmacological treatments.
[0067] Efficacy of polyphenols and plant extracts in preventing
binding of the RBD sequence of SARS-CoV-2 and hACE2 receptor. We
investigated the ability of several classes of polyphenols to
inhibit the binding of the RBD sequence of the SARS-CoV-2 spike
protein to the hACE2 receptor taking a two-stage approach. In the
first step we screened 51 different polyphenols and plant extracts
for their ability to inhibit binding of an HRP-conjugated RBD
fragment of SARS-CoV-2 spike protein to the immobilized hACE2
receptor and its direct binding to the hACE2 receptor itself.
[0068] As presented in Table 3 and Table 4, three polyphenols,
brazilin, theaflavin-3,3'-di-gallate, and curcumin, showed the
highest efficacy (100%) in inhibiting RBD binding to hACE2 when
used at 100 .mu.g/ml concentrations. At the same time these and
other tested polyphenols did not significantly bind to the ACE2
receptor itself.
[0069] Here, we provide in vitro experimental evidence that among
51 polyphenols selected in this study, brazilin,
theaflavin-3,3'-digallate and curcumin exhibited the highest
affinity in binding to the RBD-spike protein of SARS-CoV-2. While
curcumin, at considerably low concentrations, showed moderate
binding to hACE2 receptor, neither brazilin, nor
theaflavin-3,3'-digallate displayed binding affinity to this
receptor.
[0070] We further investigated this effect by using hA549 cells
expressing spike protein. By applying spike-protein-enveloped
pseudo-virions and a different pattern of exposure to polyphenols,
we observed that all three polyphenol compounds can inhibit viral
attachment to the cell surface ACE2 receptors after both short-term
(1 h. and 3 h.) and long-term (48 h.) exposure or incubation
pattern. When the SARS-CoV-2 virions were pre-incubated with these
compounds for 1 h., added simultaneously, or when the compounds
were added 1 h. post-infection, the virions' ability to bind to
cell surface ACE2 receptors and transduce cells was decreased by
all test compounds in dose-dependent fashion. Interestingly, the
same inhibitory effect of polyphenols, although at their higher but
still non-toxic concentrations, was observed when SARS-CoV-2
pseudo-virions where forcibly attached to the cells by spinfection.
In addition, we noticed that brazilin, theaflavin-3,3'-digallate,
and curcumin can reduce cell-cell fusion between spike-expressing
cells and hACE2 overexpressing cellular monolayer. These results
collectively indicate that all these three compounds have
inhibitory properties directed especially towards
RBD-SARS-CoV-2.
TABLE-US-00003 TABLE 3 Effects of various classes of polyphenols in
preventing RBD of SARS-CoV-2 binding and ACE2 receptor binding.
Tested polyphenols and alkaloids Binding with RBD Binging with ACE2
(0.1 mg/ml) (% of control .+-. SD) (% of control .+-. SD) Phenolic
acids Gallic acid 18.3 .+-. 4.5 6.5 .+-. 1.3 Tannic acid 79.4 .+-.
2.3 7.2 .+-. 2.3 Curcumin 100 .+-. 0.2 4.6 .+-. 2.4 Chlorogenic
acid 25.5 .+-. 2.5 4.7 .+-. 1.6 Rosmarinic acid 22.5 .+-. 3.8 7.9
.+-. 1.8 Flavonoids Fisetin 22.4 .+-. 1.9 6.0 .+-. 2.4 Quercetin
22.4 .+-. 6.5 7.8 .+-. 3.3 Morin 30.5 .+-. 5.8 5.6 .+-. 3.1
Myricetin 45.5 .+-. 5.4 5.6 .+-. 2.1 Kaempferol 15.6 .+-. 2.9 6.2
.+-. 2.5 Rutin 20.6 .+-. 6.3 4.8 .+-. 2.0 Luteolin 10.4 .+-. 4.7
4.8 .+-. 1.6 Baicalein 22.5 .+-. 5.1 7.4 .+-. 1.4 Baicalin 10.3
.+-. 2.9 4.9 .+-. 1.9 Scutellarin 8.1 .+-. 3.7 7.5 .+-. 1.7
Naringin 23.6 .+-. 6.4 3.7 .+-. 1.1 Naringenin .sup. 20 .+-. 5.1
8.3 .+-. 1.6 Hesperidin 90.3 .+-. 3.8 8.3 .+-. 2.3 Hesperetin 42.5
.+-. 4.6 4.9 .+-. 2.7 Apigenin 17.1 .+-. 4.1 8.3 .+-. 1.9 Genistein
22.1 .+-. 2.8 9.4 .+-. 2.7 Phloroglucinol 69.5 .+-. 3.6 5.9 .+-.
3.4 Schisandrin 22.4 .+-. 3.3 5.1 .+-. 2.7 Urolithin A 31.1 .+-.
4.6 8.8 .+-. 1.6 Punicalagin 32.3 .+-. 5.9 5.4 .+-. 2.3 Brazilin
100 .+-. 0.1 4.6 .+-. 2.2 Hispidulin 20.1 .+-. 6.0 7.4 .+-. 2.1
Papaverine 1.6 .+-. 0.2 6.5 .+-. 3.7 Silymarin 30.0 .+-. 2.6 8.8
.+-. 3.8 Procyanidin B2 31.1 .+-. 3.6 5.8 .+-. 2.7 Procyanidin B3
32.3 .+-. 3.7 7.8 .+-. 2.7 Stilbenes Trans-resveratrol 22.3 .+-.
2.9 5.5 .+-. 2.4 Pterostilbene 23.1 .+-. 2.8 9.4 .+-. 2.5 Alkaloids
Palmatine 40.4 .+-. 6.1 8.5 .+-. 2.7 Berberine 17.3 .+-. 2.7 9.4
.+-. 2.4 Cannabidiol 1.4 .+-. 0.3 5.8 .+-. 2.0 Castanospermine 8.2
.+-. 2.3 5.5 .+-. 3.1 Usnic acid 22.0 .+-. 3.4 5.7 .+-. 1.7 Malic
acid 1.2 .+-. 3.7 5.8 .+-. 1.4 Terpenes D-limonene 27.2 .+-. 6.4
6.4 .+-. 1.5 Carnosic acid 27.1 .+-. 5.1 6.9 .+-. 4.1
TABLE-US-00004 TABLE 4 Binding ability of selected plant extracts
and their major components, to RBD of SARS-CoV-2 and to ACE2
receptor. Tested plant extracts Binding to RBD Binging to ACE2 (0.1
mg/ml) (% of control .+-. DS) (% of control .+-. DS) Tea extract
(85% catechin 88.3 .+-. 3.7 5.4 .+-. 1.2 standardized)
(+)-gallocatechin 69.5 .+-. 2.8 5.7 .+-. 1.6 (-)-catechin gallate
37.4 .+-. 4.7 8.6 .+-. 1.5 (-)-gallocatechin gallate 75.4 .+-. 5.6
7.5 .+-. 1.7 (-)-gallocatechin 73.5 .+-. 6.7 3.9 .+-. 2.3
(+)-epigallocatechin gallate 87.5 .+-. 6.8 5.9 .+-. 2.0 Tea extract
(85% theaflavins 100 .+-. 0.3 5.6 .+-. 2.1 standardized) Theafalvin
27.3 .+-. 1.4 7.9 .+-. 1.9 Theaflavine-3'3-digallate 100 .+-. 0.1
5.6 .+-. 2.3 Broccoli extract 28.6 .+-. 2.6 9.7 .+-. 1.8
L-sulforaphane 30.2 .+-. 3.6 6.7 .+-. 1.5 Andrographis paniculata
18.4 .+-. 1.8 5.8 .+-. 3.6 extract Andrographolide 22.1 .+-. 2.5
5.6 .+-. 2.4 Licorice extract 18.3 .+-. 3.6 5.7 .+-. 1.4
Glycyrrhizic acid 22.2 .+-. 2.3 10.1 .+-. 2.8
[0071] As shown in FIG. 2, the inhibitory effect of these most
effective polyphenols, curcumin. theaflavin-3'3-digallate and
brazilin, on RBD-hACE2 binding, was dose dependent and ranged from
20% to 95% at the concentrations from 2.5-10 .mu.g/ml,
respectively.
[0072] In a second step, we incubated A549 cells expressing
SARS-CoV-2 spike protein with these three test polyphenols for 1 h.
and then exposed them to soluble hACE2 receptor. In this
experiment, we also observed dose-dependent interference in spike
protein-hACE2 binding ranging from 15% to 95% at 2.5-10 .mu.g/ml,
respectively, which corresponded to previously obtained results
(FIG. 3).
[0073] Cell viability tests revealed that short-term incubation
(i.e., 1 h. and 3 h.) with these polyphenols at concentrations up
to 25 pig/ml showed no cytotoxicity, as shown in FIG. 4A, FIG. 4B
and FIG. 4C. As presented on FIG. 5A, brazilin,
theaflavin-3.3-digallate, and curcumin similarly inhibited binding
of SARS-CoV-2 spike protein pseudo-typed virions to hACE2/A549 in
dose-dependent fashion, regardless of exposure time and the
application pattern. Statistically significant inhibition of
pseudo-virions binding by all test polyphenols was observed already
at 5.0 .mu.g/ml and 10 .mu.g/ml when tested before 1 h. (FIG. 5A)
and simultaneously (FIG. 5B).
[0074] Another series of experiments also revealed that brazilin,
theaflavin-3.3'-digallate and curcumin, applied at non-toxic
concentrations (i.e., 5.0-25 .mu.g/ml), have a similar
dose-dependent inhibitory effect on binding of SARS-CoV-2 spike
protein pseudo-typed virions A549 to hACE2/A549. Inhibition of
virions transduction ranged from 20% to 80% without spinfection,
and from 20% to 40% when spinfection was applied (FIG. 6A). Without
spinfection, statistically significant inhibition by test
polyphenols was observed starting from 5.0 .mu.g/ml concentration,
both when SARS-CoV-2 spike pseudo-virions were incubated with
selected polyphenols 1 h. before hACE2/A549 cells exposure, and
when they were added simultaneously with test polyphenols (FIG.
3A). When test polyphenols were added 1 h. after SARS-CoV-2 spike
pseudo-virions were exposed to hACE2/A549 cells, significant
inhibitory effect of polyphenols was observed starting from 10
.mu.g/ml concentration.
[0075] Test polyphenols showed different efficacy on cell
transduction by the pseudo-virions. When the viral transduction of
hACE2/A549 cells was forced by the application of spinfection,
curcumin showed significant inhibitory effect at lower
concentrations compared with brazilin and theaflavin-3'3-digallate.
As such, exposure of SARS-CoV-2 virions to curcumin for 1 h. before
and simultaneously with adding to hACE2/A549 cells resulted in
inhibition of transduction starting from its 5.0 .mu.g/ml
concentration. Higher (10 .mu.g/ml) concentrations of brazilin and
theaflavin-3,3'-digallate were required to achieve statistically
significant inhibitory effects using the same patterns of exposure.
All test polyphenols added 1 h. after SARS-CoV-2 virions were
applied to the cells, resulted in significant inhibition of
transduction at 10 .mu.g/ml concentration of each compound (FIG.
6B).
[0076] The effect of test polyphenols on fusion of A549 cells
expressing SARS-CoV-2 spike protein pseudo-typed virions with lung
epithelial cells expressing hACE2 is presented in FIG. 4. A549
pseudo-virion expressing cells preincubated with test polyphenols
and then layered for 4 h. on hCE2/A549 cells showed a significantly
decreased attachment. Pre-incubation with brazilin at 25 .mu.g/ml
decreased cell attachment by 40%, with theaflavin-3'3-digallate by
40% to 70% at 10-25 .mu.g/ml, and with curcumin by 70% to 95% at
the same concentrations (10-25 .mu.g/ml). These results were
consistent with the previously obtained sets of data.
[0077] FIGS. 7A, 7B, 7C, 7D. 7E, 7F, 7G, 7H, 7I, 7J, 7K and FIG. 8
show the effect of test polyphenols on fusion to the human ACE2
receptor overexpressing A549 cells. A. Cell-cell fusion of A549
cells expressing eGFP spike protein with A549 cells stably
expressing human ACE2 receptor. A549 cells expressing eGFP spike
protein were pre-treated with indicated polyphenols at different
concentrations for 1 h. at 37.degree. C. and co-cultured for an
additional 4 h. at 37.degree. C. with A549 cells stably expressing
human ACE2 receptor. The scale bar indicates 250 .mu.m. B.
Quantitative analysis of formed syncytia. Experiments were done in
triplicate and repeated three times. Data are presented as
percentage of control f SD; A p<0.01, * p<0.001.
Control--0.025% DMSO, positive control--20 .mu.g/ml anti-ACE2
antibody.
[0078] FIG. 9 shows Mixture D (resveratrol, cruciferous plant
extract, curcumin, quercetin, naringenin, baicalin, theaflavin,
vitamin C and N-acetylcysteine) gives the best inhibition of
binding.
[0079] FIG. 10 shows the safety of the mixture D on human alveolar
cells. The pharmaceutical micronutrient composition mixture D was
applied at 5 and 10 mcg/ml doses individually and in combinations
with vitamin D and was safe to be used on human small alveolar
epithelial cells.
[0080] FIG. 11 shows inhibition of RBD binding of the mixture D
alone and its combination with vitamin D. Mixture D was effective
in inhibiting RBD binding to ACE2 receptors by 75% at 5 mcg/ml and
by 85% at 10 mcg/ml compared to control. The mixture D in
combination with vitamin D did not further enhance this inhibitory
effect. We can safely say that mixture D alone has high efficacy
and inhibits RBD binding.
[0081] FIG. 12 shows results of inhibition of cellular
internalization of the mutated forms of SARS-CoV-2: viral strains
from the UK, Brazil and South Africa. Mixture D (10 mcg/ml) added
simultaneously with mutated virions to cells overexpressing ACE2
was equally effective in inhibiting cellular entry of these mutated
forms of SARS-CoV-2: by 48% for UK mutation, by 47% for Brazilian
mutation, by 48% for South African mutation. These effects were
concentration dependent. Exposure of viral particles to the mixture
D for 1 h, before combining them with cells also inhibited cellular
entry of these viral mutants by up to 40%. These results not only
show efficacy for inhibiting cellular entry by viral strains but
also show that the direct exposure of viral particles to this
pharmaceutical micronutrient compound helps to prevent the viral
entry.
[0082] FIG. 13 shows inhibition of cellular entry by mutated forms
of SARS-CoV-2, viral strains from the UK, Brazil and South Africa,
owing to the inhibitory effect of the mixture D when applied
simultaneously with the virions and cells.
[0083] FIG. 14 shows inhibition of ACE2 expression under normal and
pro-inflammatory conditions. Exposure of human small alveolar
epithelial cells to the mixture D for 6 days resulted in inhibition
of ACE2 expression by 73% at 12 mcg/ml. This inhibitory effect of
the mixture D on ACE2 expression persisted and was even enhanced
under pro-inflammatory conditions (inhibition between 83-86%).
[0084] FIG. 15 shows inhibition of viral RdRp activity and effects
of vitamin D. It shows mixture D alone can inhibit RdRp activity by
53% when used at 10 mcg/ml, and by 30% at 5 mcg/ml compared to
control. Combinations of the mixture D with vitamin D did not
further enhance RdRp inhibition.
[0085] FIG. 16 shows inhibition of furin activity in the cells,
owing to mixture D activity. Mixture D applied individually at 10
mcg/ml could decrease furin activity by 33%, and at 20 mcg/ml by
52%. FIG. 17 shows the test results of inhibition of cellular
activity of cathepsin L by mixture D and the effects of vitamin D
and Mixture D. Mixture D applied to the cells individually and in
combination with vitamin D shows 20% inhibition of cathepsin L
activity. Mixture D in combination with vitamin D does not further
enhance this inhibitory effect.
[0086] FIG. 18 shows anti-inflammatory effect: inhibition of IL-6
secretion under normal and pro-inflammatory conditions by the
mixture D alone and combined with vitamin D. Mixture D (10 mcg/ml)
applied to small alveolar endothelial cells for 3 days decreased
IL-6 secretion by 50%. Exposure of HSAEpC to lipopolysaccharide
(LPS, 5 mcg/ml) increased IL-6 secretion by 43%. Under this
pro-inflammatory condition, the mixture D could inhibit IL-6
secretion by 55%. This inhibitory effect was increased to 83% by a
combination of the mixture D (10 mcg/ml) with 10 mcg/ml of vitamin
D.
[0087] Drug formulations suitable for these administration routes
can be produced by adding one or more pharmacologically acceptable
carrier to the agent and then treating the micronutrient
composition through a routine process known to those skilled in the
art. The mode of administration includes, but is not limited to,
non-invasive peroral, topical (for example, transdermal), enteral,
transmucosal, targeted delivery, sustained-release delivery,
delayed release, pulsed release and parenteral methods. Peroral
administration may be administered both in liquid and dry state. In
one embodiment, pharmaceutical micronutrient composition would be
more specifically mixture D.
[0088] Formulations suitable for oral administration may be in the
form of capsules, cachets, pills, tablets, lozenges (using a
flavored bases, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin or sucrose and acacia), each
containing a predetermined amount of a subject composition as an
active ingredient. Subject compositions may also be administered as
a bolus, electuary or paste.
[0089] When an oral solid drug product is prepared, pharmaceutical
micronutrient composition is mixed with an excipient (and, if
necessary, one or more additives such as a binder, a disintegrant,
a lubricant, a coloring agent, a sweetening agent, and a flavoring
agent), and the resultant mixture is processed through a routine
method, to thereby produce an oral solid drug product such as
tablets, coated tablets, granules, powder or capsules. Additives
may be those generally employed in the art. Examples of excipients
include lactate, sucrose, sodium chloride, glucose, starch, calcium
carbonate, kaolin, microcrystalline cellulose and silicic acid.
Binders include water, ethanol, propanol, simple syrup, glucose
solution, starch solution, liquefied gelatin,
carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl
starch, methyl cellulose, ethyl cellulose, shellac, calcium
phosphate and polyvinyl pyrrolidone. Disintegrants include dried
starch, sodium arginate, powdered agar, sodium hydroxy carbonate,
calcium carbonate, sodium lauryl sulfate, monoglyceryl stearate and
lactose. Lubricants include purified talc, stearic acid salts,
borax and polyethylene glycol. Sweetening agents include sucrose,
orange peel, citric acid and tartaric acid.
[0090] When a liquid drug product for oral administration is
prepared, pharmaceutical micronutrient composition is mixed with an
additive such as a sweetening agent, a buffer, a stabilizer, or a
flavoring agent, and the resultant mixture is processed through a
routine method, to produce an orally administered liquid drug
product such as an internal solution medicine, syrup or elixir.
Examples of the sweetening agent include vanillin; examples of the
buffer include sodium citrate; and examples of the stabilizer
include tragacanth, acacia, and gelatin.
[0091] For the purposes of transdermal (e.g., topical)
administration, dilute sterile, aqueous or partially aqueous
solutions (usually in about 0.1% to 5% concentration), otherwise
similar to the above parenteral solutions, may be prepared with
pharmaceutical micronutrient composition.
[0092] Formulations containing pharmaceutical micronutrient
composition for rectal or vaginal administration may be presented
as a suppository, which may be prepared by mixing a subject
composition with one or more suitable non-irritating carriers,
comprising, for example, cocoa butter, polyethylene glycol, a
suppository wax or a salicylate, which is solid at room
temperature, but liquid at body temperature and, therefore, will
melt in the appropriate body cavity and release the encapsulated
compound(s) and composition(s). Formulations that are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0093] A targeted-release portion for capsules containing
pharmaceutical micronutrient composition can be added to the
extended-release system by means of either applying an
immediate-release layer on top of the extended release core; using
coating or compression processes, or in a multiple-unit system such
as a capsule containing extended- and immediate-release beads.
[0094] When used with respect to a pharmaceutical micronutrient
composition, the term "sustained release" is art recognized. For
example, a therapeutic composition that releases a substance over
time may exhibit sustained-release characteristics, in contrast to
a bolus type administration in which the entire amount of the
substance is made biologically available at one time. In particular
embodiments, upon contact with body fluids, including blood, spinal
fluid, mucus secretions, lymph or the like, one or more of the
pharmaceutically acceptable excipients may undergo gradual or
delayed degradation (e.g., through hydrolysis), with concomitant
release of any material incorporated therein, e.g., a therapeutic
and/or biologically active salt and/or composition, for a sustained
or extended period (as compared with the release from a bolus).
This release may result in prolonged delivery of therapeutically
effective amounts of any of the therapeutic agents disclosed
herein.
[0095] Current efforts in the area of drug delivery include the
development of targeted delivery, in which the drug is only active
in the target area of the body (for example, mucous membranes such
as in the nasal cavity), and sustained-release formulations, in
which the pharmaceutical micronutrient composition is released over
a period of time in a controlled manner from a formulation. Types
of sustained release formulations include liposomes, drug-loaded
biodegradable microspheres and pharmaceutical micronutrient
composition polymer conjugates.
[0096] Delayed-release dosage formulations are created by coating a
solid dosage form with a film of a polymer, which is insoluble in
the acid environment of the stomach, but soluble in the neutral
environment of the small intestine. The delayed-release dosage
units can be prepared, for example, by coating a pharmaceutical
micronutrient composition with a selected coating material. The
pharmaceutical micronutrient composition may be a tablet for
incorporation into a capsule, a tablet for use as an inner core in
a "coated core" dosage form, or a plurality of drug-containing
beads, particles or granules, for incorporation into either a
tablet or a capsule. Preferred coating materials include
bioerodible, gradually hydrolysable, gradually water-soluble,
and/or enzymatically degradable polymers, and may be conventional
"enteric" polymers. Enteric polymers, as will be appreciated by
those skilled in the art, become soluble in the higher pH
environment of the lower gastrointestinal tract, or slowly erode as
the dosage form passes through the gastrointestinal tract, while
enzymatically degradable polymers are degraded by bacterial enzymes
present in the lower gastrointestinal tract, particularly in the
colon. Alternatively, a delayed-release tablet may be formulated by
dispersing a drug within a matrix of a suitable material such as a
hydrophilic polymer or a fatty compound. Suitable hydrophilic
polymers include, but are not limited to, polymers or copolymers of
cellulose, cellulose ester, acrylic acid, methacrylic acid, methyl
acrylate, ethyl acrylate and vinyl or enzymatically degradable
polymers or copolymers as described above. These hydrophilic
polymers are particularly useful for providing a delayed-release
matrix. Fatty compounds for use as a matrix material include, but
are not limited to, waxes (e.g., carnauba wax) and glycerol
tristearate. Once the active ingredient is mixed with the matrix
material, the mixture can be compressed into tablets.
[0097] A pulsed-release dosage is one that mimics a multiple dosing
profile without repeated dosing, and typically allows at least a
twofold reduction in dosing frequency as compared with the drug
presented as a conventional dosage form (e.g., as a solution or
prompt drug-releasing, conventional solid dosage form). A
pulsed-release profile is characterized by a time period of no
release (lag time) or reduced release, followed by rapid drug
release. These can be formulated for critically ill patients using
the instant pharmaceutical micronutrient composition.
[0098] The phrases "parenteral administration" and "administered
parenterally" as used herein refer to modes of administration other
than enteral and topical, such as injections, and include without
limitation intravenous, intramuscular, intrapleural, intravascular,
intrapericardial, intra-arterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intra-articular,
subcapsular, subarachnoid, intraspinal and intrastemal injection
and infusion.
[0099] Certain pharmaceutical compositions disclosed herein,
suitable for parenteral administration, comprise one or more
subject compositions in combination with one or more
pharmaceutically acceptable sterile, isotonic, aqueous, or
non-aqueous solutions, dispersions, suspensions or emulsions, or
sterile powders, which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, and which may contain
antioxidants, buffers, bacteriostats, solutes that render the
formulation isotonic within the blood of the intended recipient, or
suspending or thickening agents.
[0100] When an injection product is prepared, pharmaceutical
micronutrient composition is mixed with an additive such as a pH
regulator, a buffer, a stabilizer, an isotonicity agent or a local
anesthetic, and the resultant mixture is processed through a
routine method, to thereby produce an injection for subcutaneous
injection, intramuscular injection, or intravenous injection.
Examples of the pH regulator or buffer include sodium citrate,
sodium acetate and sodium phosphate; examples of the stabilizer
include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic
acid; examples of the local anesthetic include procaine
hydrochloride and lidocaine hydrochloride; and examples of the
isotonicity agent include sodium chloride and glucose.
[0101] Adjuvants are used to enhance the immune response. Various
types of adjuvants are available. Haptens and Freund's adjuvant may
also be used to produce water-in-oil emulsions of immunogens.
[0102] The phrase "pharmaceutically acceptable" is art recognized.
In certain embodiments, the term includes compositions, polymers
and other materials and/or dosage forms that are within the scope
of sound medical judgment, suitable for use in contact with the
tissues of mammals, both human beings and animals, without
excessive toxicity, irritation, allergic response or other problem
or complication, commensurate with a reasonable benefit-risk
ratio.
[0103] The phrase "pharmaceutically acceptable carrier" is art
recognized, and includes, for example, pharmaceutically acceptable
materials, compositions or vehicles, such as a liquid or solid
filler, diluent, solvent or encapsulating material involved in
carrying or transporting any subject composition from one organ or
portion of the body, to another organ or portion of the body. Each
carrier must be "acceptable" in the sense of being compatible with
the other ingredients of a subject composition, and not injurious
to the patient. In certain embodiments, a pharmaceutically
acceptable carrier is non-pyrogenic. Some examples of materials
that may serve as pharmaceutically acceptable carriers include: (1)
sugars, such as lactose, glucose and sucrose; (2) starches, such as
corn starch and potato starch; (3) cellulose and its derivatives,
such as sodium carboxymethyl cellulose, ethyl cellulose and
cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin;
(7) talc; (8) cocoa butter and suppository waxes; (9) oils, such as
peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil,
corn oil and soybean oil; (10) glycols, such as propylene glycol;
(11) polyols, such as glycerin, sorbitol, mannitol and polyethylene
glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13)
agar; (14) buffering agents, such as magnesium hydroxide and
aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;
(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;
(20) phosphate buffer solutions; and (21) other non-toxic
compatible substances employed in pharmaceutical formulations.
[0104] In certain embodiments, the pharmaceutical micronutrient
compositions described herein are formulated in a manner such that
said compositions will be delivered to a mammal in a
therapeutically effective amount, as part of a prophylactic,
preventive or therapeutic treatment to overcome the infection
caused by corona viruses (irrespective of the type).
[0105] In certain embodiments, the dosage of the pharmaceutical
micronutrient compositions, which may be referred to as therapeutic
composition provided herein, may be determined by reference to the
plasma concentrations of the therapeutic composition or other
encapsulated materials. For example, the blood samples may be
tested for their immune response to their corresponding viral load
or lack thereof.
[0106] The therapeutic pharmaceutical micronutrient composition
provided by this application may be administered to a subject in
need of treatment by a variety of conventional routes of
administration, including orally, topically, parenterally, e.g.,
intravenously, subcutaneously or intramedullary. Further, the
therapeutic compositions may be administered intranasally, as a
rectal suppository, or using a "flash" formulation, i.e., allowing
the medication to dissolve in the mouth without the need to use
water. Furthermore, the compositions may be administered to a
subject in need of treatment by controlled-release dosage forms,
site-specific drug delivery, transdermal drug delivery,
patch-mediated drug delivery (active/passive), by stereotactic
injection, or in nanoparticles.
[0107] Expressed in terms of concentration, an active ingredient
can be present in the therapeutic compositions of the present
invention for localized use via the cutis, intranasally,
pharyngolaryngeally, bronchially, intravaginally, rectally or
ocularly.
[0108] For use as aerosols, the active ingredients can be packaged
in a pressurized aerosol container together with a gaseous or
liquefied propellant, for example dichlorodifluoromethane, carbon
dioxide, nitrogen, propane and the like, with the usual adjuvants
such as cosolvents and wetting agents, as may be necessary or
desirable. The most common routes of administration also include
the preferred transmucosal (nasal, buccal/sublingual, vaginal,
ocular and rectal) and inhalation routes.
[0109] In addition, in certain embodiments, the subject
pharmaceutical micronutrient composition of the present application
may be lyophilized or subjected to another appropriate drying
technique such as spray drying. The subject compositions may be
administered once, or may be divided into a number of smaller doses
to be administered at varying intervals of time, depending in part
on the release rate of the compositions and the desired dosage.
[0110] Formulations useful in the methods provided herein include
those suitable for oral, nasal, topical (including buccal and
sublingual), rectal, vaginal, aerosol and/or parenteral
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods well known in
the art of pharmacy. The amount of a subject pharmaceutical
micronutrient composition that may be combined with a carrier
material to produce a single dose may vary depending upon the
subject being treated and the particular mode of
administration.
[0111] The therapeutically acceptable amount described herein may
be administered in inhalant or aerosol formulations. The inhalant
or aerosol formulations may comprise one or more agents, such as
adjuvants, diagnostic agents, imaging agents, or therapeutic agents
useful in inhalation therapy. The final aerosol formulation may,
for example, contain 0.005-90% w/w, for instance 0.005-50%,
0.005-5% w/w, or 0.01-1.0% w/w, of medicament relative to the total
weight of the formulation.
[0112] Examples of suitable aqueous and non-aqueous carriers that
may be employed in the pharmaceutical micronutrient composition
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol and the like), and suitable mixtures
thereof, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Proper fluidity may be maintained, for
example by the use of coating materials such as lecithin, by the
maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
* * * * *