U.S. patent application number 17/006103 was filed with the patent office on 2021-12-16 for method for inhibiting coronavirus infection and replication.
The applicant listed for this patent is CHANG GUNG UNIVERSITY. Invention is credited to Huan-Jung CHIANG, Hsin-Ping CHIU, Yu-Nong GONG, Chiung-Guei HUANG, Peng-Nien HUANG, Sheng-Yu HUANG, Chuan-Tien HUNG, Yu-An KUNG, Shin-Ru SHIH.
Application Number | 20210386725 17/006103 |
Document ID | / |
Family ID | 1000005100995 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210386725 |
Kind Code |
A1 |
SHIH; Shin-Ru ; et
al. |
December 16, 2021 |
METHOD FOR INHIBITING CORONAVIRUS INFECTION AND REPLICATION
Abstract
Disclosed herein is a method for inhibiting coronavirus
infection, including administering to a subject in need thereof an
effective amount of pioglitazone or a pharmaceutically acceptable
salt thereof. Also disclosed is a method for inhibiting coronavirus
replication, including contacting a coronavirus with pioglitazone
or a pharmaceutically acceptable salt thereof.
Inventors: |
SHIH; Shin-Ru; (New Taipei
City, TW) ; KUNG; Yu-An; (Taoyuan City, TW) ;
CHIANG; Huan-Jung; (Taoyuan City, TW) ; HUNG;
Chuan-Tien; (Taichung City, TW) ; GONG; Yu-Nong;
(Taoyuan City, TW) ; CHIU; Hsin-Ping; (Taoyuan
City, TW) ; HUANG; Chiung-Guei; (Taoyuan City,
TW) ; HUANG; Peng-Nien; (Taoyuan City, TW) ;
HUANG; Sheng-Yu; (Taoyuan City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHANG GUNG UNIVERSITY |
Taoyuan City |
|
TW |
|
|
Family ID: |
1000005100995 |
Appl. No.: |
17/006103 |
Filed: |
August 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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63037889 |
Jun 11, 2020 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/16 20180101;
A61K 9/0053 20130101; A61K 31/4436 20130101 |
International
Class: |
A61K 31/4436 20060101
A61K031/4436; A61K 9/00 20060101 A61K009/00; A61P 31/16 20060101
A61P031/16 |
Claims
1. A method for inhibiting coronavirus infection, comprising
administering to a subject in need thereof an effective amount of
pioglitazone or a pharmaceutically acceptable salt thereof.
2. The method as claimed in claim 1, wherein the coronavirus
infection is caused by a coronavirus selected from the group
consisting of a member of Alphacoronavirus genus, a member of
Betacorovirus genus, a member of Gammacoronavirus genus, a member
of Deltacoronavirus genus, and combinations thereof.
3. The method as claimed in claim 2, wherein the coronavirus is
selected from the group consisting of bat coronavirus CDPHE 15, bat
coronavirus HKU10, Rhinolopus ferrumequinum alphacoronavirus
HuB-2013, human coronavirus 229E (HCoV-229E), Lucheng Rn rat
coronavirus, mink coronavirus 1, Miniopterus bat coronavirus 1,
Miniopterus bat coronavirus HKU8, Myotis ricketti alphacoronavirus
Sax-2011, Nyctalus velutinus alphacoronavirus SC-2013, Pipistrellus
kuhlii coronavirus 3398, porcine epidemic diarrhea virus,
Scotophilus bat coronavirus 512, Rhinolophus bat coronavirus HKU2,
human coronavirus NL63, NL63-related bat coronavirus strain
BtKYNL63-9b, Sorex araneus coronavirus T14, Suncus murinus
coronavirus X74, Alphacoronavirus 1, bovine coronavirus, human
coronavirus OC43 (HCoV-OC43), China rattus coronavirus HKU24, human
coronavirus HKU1, murine coronavirus, Myodes coronavirus 2JL14, bat
Hp-betacoronavirus Zhejiang 2013, Hedgehog coronavirus 1, Middle
East respiratory syndrome-related coronavirus, Pipistrellus bat
coronavirus HKU5, Tylonycteris bat coronavirus HKU4, Eidolon bat
coronavirus C704, Rousettus bat coronavirus GCCDC1, Rousettus bat
coronavirus HKU9, severe acute respiratory syndrome coronavirus
(SARS-CoV), severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2), bat SARS-like coronavirus WIV1, and combinations
thereof.
4. The method as claimed in claim 3, wherein the coronavirus is
HCoV-229E.
5. The method as claimed in claim 3, wherein the coronavirus is
HCoV-OC43.
6. The method as claimed in claim 3, wherein the coronavirus is
SARS-CoV-2.
7. The method as claimed in claim 1, wherein the pioglitazone or
the pharmaceutically acceptable salt thereof is in a dosage form
for oral administration.
8. A method for inhibiting coronavirus replication, comprising
contacting a coronavirus with pioglitazone or a pharmaceutically
acceptable salt thereof.
9. The method as claimed in claim 8, wherein the coronavirus is
selected from the group consisting of a member of Alphacoronavirus
genus, a member of Betacorovirus genus, a member of
Gammacoronavirus genus, a member of Deltacoronavirus genus, and
combinations thereof.
10. The method as claimed in claim 9, wherein the coronavirus is
selected from the group consisting of bat coronavirus CDPHE 15, bat
coronavirus HKU10, Rhinolopus ferrumequinum alphacoronavirus
HuB-2013, human coronavirus 229E (HCoV-229E), Lucheng Rn rat
coronavirus, mink coronavirus 1, Miniopterus bat coronavirus 1,
Miniopterus bat coronavirus HKU8, Myotis ricketti alphacoronavirus
Sax-2011, Nyctalus velutinus alphacoronavirus SC-2013, Pipistrellus
kuhlii coronavirus 3398, porcine epidemic diarrhea virus,
Scotophilus bat coronavirus 512, Rhinolophus bat coronavirus HKU2,
human coronavirus NL63, NL63-related bat coronavirus strain
BtKYNL63-9b, Sorex araneus coronavirus T14, Suncus murinus
coronavirus X74, Alphacoronavirus 1, bovine coronavirus, human
coronavirus OC43 (HCoV-OC43), China Rattus coronavirus HKU24, human
coronavirus HKU1, murine coronavirus, Myodes coronavirus 2JL14, bat
Hp-betacoronavirus Zhejiang 2013, Hedgehog coronavirus 1, Middle
East respiratory syndrome-related coronavirus, Pipistrellus bat
coronavirus HKU5, Tylonycteris bat coronavirus HKU4, Eidolon bat
coronavirus C704, Rousettus bat coronavirus GCCDC1, Rousettus bat
coronavirus HKU9, severe acute respiratory syndrome coronavirus
(SARS-CoV), severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2), bat SARS-like coronavirus WIV1, and combinations
thereof.
11. The method as claimed in claim 10, wherein the coronavirus is
HCoV-229E.
12. The method as claimed in claim 10, wherein the coronavirus is
HCoV-OC43.
13. The method as claimed in claim 10, wherein the coronavirus is
SARS-CoV-2.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority of U.S. Provisional
Application No. 63/037,889, filed on Jun. 11, 2020.
FIELD
[0002] The present disclosure relates to a method for inhibiting
coronavirus infection and replication using pioglitazone.
BACKGROUND
[0003] Coronaviruses are a group of related RNA viruses belonging
to the Coronaviridae family which infect mammals and birds. These
viruses have viral envelope with a positive-sense, single-stranded
RNA genome, and are characterized by club-shaped spikes that
project from their surface, which create an image reminiscent of
solar corona in electron microscope, from which their name
derives.
[0004] To date, there are seven known coronaviruses which infect
humans, including human coronavirus OC43 (HCoV-OC43), human
coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E),
and human coronavirus NL63 (HCoV-NL63) which produce generally mild
symptoms of common cold, and Middle East respiratory
syndrome-related coronavirus (MERS-CoV), severe acute respiratory
syndrome coronavirus (SARS-CoV), and severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) which produce symptoms that are
potentially severe.
[0005] Notably, SARS-CoV-2 is identified as the viral strain that
causes the current outbreak of coronavirus disease 2019 (COVID-19),
the rapid spread of which was declared as a global pandemic known
as COVID-19 pandemic. Till date, more than 20 million confirmed
cases of COVID-19 had been reported worldwide, with a fatality rate
of approximately 3.5%. Transmission of SARS-CoV-2 between humans
primarily occurs during close contact, usually via small droplets
produced by coughing, sneezing and talking. Symptoms of COVID-19
may be relatively non-specific, including fever, cough, fatigue,
phlegm production, loss of sense of smell, shortness of breath,
muscle and joint pain, headache, and chills, among others. Among
COVID-19 patients who develop symptoms, approximately one in five
may become seriously ill and have difficulty in breathing. Further
development of COVID-19 may lead to complications, including
pneumonia, acute respiratory distress syndrome, sepsis, septic
shock, and kidney failure. Due to travel restrictions, lockdowns,
workplace hazard controls, and facility closures imposed by
governmental authorities worldwide, COVID-19 pandemic has caused
global social and economic disruption, including the largest global
recession since the Great Depression. With no known vaccine or
specific antiviral treatment currently available, the need to find
and/or develop drugs effective against coronavirus, particularly
SARS-CoV-2, has become pressing.
[0006] Drug repositioning (also known as drug repurposing) is the
investigation of existing drugs for new therapeutic purposes. This
research direction, along with development of COVID-19 vaccines and
convalescent plasma transfusion, is being actively pursued to
develop safe and effective COVID-19 treatments. In fact, several
existing antiviral medications, previously developed or used in
treatments for SARS, MERS, HIV/AIDS, and malaria, are being
investigated as COVID-19 treatment candidates. A few of these
medications, such as chloroquine and hydroxychloroquine,
dexamethasone, favipiravir, lopinavir/ritonavir, remdesivir, etc.,
have advanced into clinical trials. However, based on published
randomized controlled trials, none of these medications has yet
been shown to be clearly effective in reducing mortality of
COVID-19 patients. Therefore, there is still an urgent need to find
other classes of drugs which are effective against SARS-CoV-2.
[0007] Pioglitazone is a drug classified as a thiazolidinedione
compound which is approved by the U.S. Food and Drug Administration
(FDA) for the treatment of type 2 diabetes. The drug was developed
by Takeda Pharmaceutical Company and is being sold under trade
names Actos.RTM., Glustin@, Glizone.RTM., among others.
Pioglitazone works by modulating the transcription of genes
involved in the control of glucose and lipid metabolism in the
muscle, adipose tissues, and the liver, thereby reducing insulin
resistance in the liver and peripheral tissues, decreasing
gluconeogenesis in the liver, and reducing the amount of glucose
and glycated hemoglobin in the bloodstream. However, it remains
unknown whether pioglitazone has an antiviral effect, particularly
an inhibitory effect on coronavirus infection.
SUMMARY
[0008] Therefore, an object of the present disclosure is to provide
a method for inhibiting coronavirus infection which can alleviate
at least one of the drawbacks of the prior art.
[0009] According to the present disclosure, the method for
inhibiting coronavirus infection includes administering to a
subject in need thereof an effective amount of pioglitazone or a
pharmaceutically acceptable salt thereof.
[0010] Another object of the present disclosure is to provide a
method for inhibiting coronavirus replication which can alleviate
at least one of the drawbacks of the prior art.
[0011] According to the present disclosure, the method for
inhibiting coronavirus replication includes contacting a
coronavirus with pioglitazone or a pharmaceutically acceptable salt
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Other features and advantages of the present disclosure will
become apparent in the following detailed description of the
embodiments with reference to the accompanying drawings, of
which:
[0013] FIG. 1 shows cell viability of mock-infected and
HCoV-229E-infected LLC-MK2 cells pretreated with different
concentrations of pioglitazone at 7 days post-infection (d.p.i.),
in which the symbols "*", "**", "***" and "****" respectively
represent p<0.05, p<0.01, p<0.005 and p<0.001;
[0014] FIG. 2 shows virus titers of HCoV-229E in a control group
and infected LLC-MK2 cells pretreated with 20 .mu.M pioglitazone
(upper panel) and virus titers of HCoV-OC43 in a control group and
infected Vero E6 cells pretreated with 20 .mu.M pioglitazone (lower
panel) at 1, 2, and 3 d.p.i., in which the symbols "*", "**" and
"***" respectively represent p<0.05, p<0.01 and
p<0.005;
[0015] FIG. 3 shows RNA copy numbers of E gene (upper panel) and
RdRp gene (lower panel) of SARS-CoV-2 in a control group and
infected LLC-MK2 cells pretreated with 20 .mu.M pioglitazone at 1,
2, 3, and 4 d.p.i., in which the symbols "*" and "****"
respectively represent p<0.05 and p<0.001;
[0016] FIG. 4 shows fluorescence microscopy images of expression of
spike (S) and nucleocapside (NP) proteins of SARS-CoV-2 in a
control group, mock-infected LLC-MK2 cells and virus-infected
LLC-MK2 cells pretreated with pioglitazone at 4 d.p.i., in which
the nucleus of LLC-MK2 cells was stained with DAPI and the scale
bar of each images is 50 .mu.m; and
[0017] FIG. 5 shows distribution and size of viral plaques of
SARS-CoV-2 in a control group and infected Vero E6 cells treated
with pioglitazone at 3 d.p.i.
DETAILED DESCRIPTION
[0018] It is to be understood that, if any prior art publication is
referred to herein, such reference does not constitute an admission
that the publication forms a part of the common general knowledge
in the art, in Taiwan or any other country.
[0019] For the purpose of this specification, it will be clearly
understood that the word "comprising" means "including but not
limited to", and that the word "comprises" has a corresponding
meaning.
[0020] Unless otherwise defined, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this disclosure belongs. One skilled in
the art will recognize many methods and materials similar or
equivalent to those described herein, which could be used in the
practice of this disclosure. Indeed, this disclosure is in no way
limited to the methods and materials described.
[0021] In the development of drugs that can be used to treat
coronavirus infection, the applicants unexpectedly found that
pioglitazone can significantly reduce the viral titer and inhibit
viral replication of coronavirus in infected host cells, and hence
is expected to be effective against coronavirus infection.
[0022] Therefore, the present disclosure is directed to use of
pioglitazone or a pharmaceutically acceptable salt thereof for the
manufacture of a medicament for inhibiting coronavirus infection
and/or coronavirus replication.
[0023] The present disclosure also provides a method for inhibiting
coronavirus infection, including administering to a subject in need
thereof an effective amount of pioglitazone or the pharmaceutically
acceptable salt thereof.
[0024] The present disclosure also provides a method for inhibiting
coronavirus replication, including contacting a coronavirus with
pioglitazone or the pharmaceutically acceptable salt thereof.
[0025] As used herein, the term "administration" or "administering"
means introducing, providing or delivering a pre-determined active
ingredient to a subject by any suitable routes to perform its
intended function.
[0026] As used herein, the term "subject" refers to any animal of
interest, such as humans, monkeys, cows, sheep, horses, pigs,
goats, dogs, cats, mice, and rats. In certain embodiments, the
subject is a human.
[0027] As used herein, the term "pharmaceutically acceptable salt"
refers to any salt, which, upon administration to the subject is
capable of providing (directly or indirectly) a compound as
described herein (i.e., pioglitazone) without undue toxicity,
irritation, allergic response and the like. In particular,
"pharmaceutically acceptable salt" may encompass those approved by
a regulatory agency of the federal or a state government or listed
in the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The
preparation of salts can be carried out by methods known in the
art.
[0028] For instance, the pharmaceutically acceptable salts of
pioglitazone may be acid addition salts, base addition salts or
metallic salts, and they can be synthesized from the parent
compound which contains a basic or acidic moiety by conventional
chemical methods. Generally, such salts are, for example, prepared
by reacting the free acid or base forms of these compounds with a
stoichiometric amount of the appropriate base or acid in water or
in an organic solvent or in a mixture thereof. Examples of the acid
addition salts may include mineral acid addition salts such as, for
example, hydrochloride, hydrobromide, hydroiodide, sulfate,
nitrate, and phosphate; and organic acid addition salts such as,
for example, acetate, maleate, fumarate, citrate, oxalate,
succinate, tartrate, malate, mandelate, methanesulphonate,
p-toluenesulphonate, 2-naphtalenesulphonate, and
1,2-ethanedisulphonate. Examples of the alkali addition salts may
include inorganic salts such as, for example, ammonium; and organic
alkali salts such as, for example, ethylenediamine, ethanolamine,
N,N-dialkylenethanolamine, triethanolamine, choline, glucamine, and
basic aminoacids salts. Examples of the metallic salts may include,
for example, sodium, potassium, calcium, magnesium, aluminium, and
lithium salts.
[0029] According to this disclosure, the coronavirus infection is
caused by a coronavirus selected from the group consisting of a
member of Alphacoronavirus genus, a member of Betacorovirus genus,
a member of Gammacoronavirus genus, a member of Deltacoronavirus
genus, and combinations thereof.
[0030] Examples of the member of Alphacoronavirus genus may
include, but are not limited to, Colavirus, Decacovirus,
Duvinacovirus, Luchacovirus, Minacovirus, Minunacovirus,
Myotacovirus, Nyctacovirus, Pedacovirus, Rhinacovirus,
Setracovirus, Soracovirus, Sunacovirus, Tegacovirus, and
combinations thereof. For example, the member of Alphacoronavirus
genus may be bat coronavirus CDPHE 15, bat coronavirus HKU10,
Rhinolopus ferrumequinum alphacoronavirus HuB-2013, human
coronavirus 229E (HCoV-229E), Lucheng Rn rat coronavirus, mink
coronavirus 1, Miniopterus bat coronavirus 1, Miniopterus bat
coronavirus HKU8, Myotis ricketti alphacoronavirus Sax-2011,
Nyctalus velutinus alphacoronavirus SC-2013, Pipistrellus kuhlii
coronavirus 3398, porcine epidemic diarrhea virus, Scotophilus bat
coronavirus 512, Rhinolophus bat coronavirus HKU2, human
coronavirus NL63, NL63-related bat coronavirus strain BtKYNL63-9b,
Sorex araneus coronavirus T14, Suncus murinus coronavirus X74, or
Alphacoronavirus 1.
[0031] Examples of the member of Betacoronavirus genus may include,
but are not limited to, Embecovirus, Hibecovirus, Merbecovirus,
Nobecovirus, Sarbecovirus, and combinations thereof. For example,
the member of Betacoronavirus genus may be bovine coronavirus,
human coronavirus OC43 (HCoV-OC43), China rattus coronavirus HKU24,
human coronavirus HKU1, murine coronavirus, Myodes coronavirus
2JL14, bat Hp-betacoronavirus Zhejiang 2013, Hedgehog coronavirus
1, Middle East respiratory syndrome-related coronavirus,
Pipistrellus bat coronavirus HKU5, Tylonycteris bat coronavirus
HKU4, Eidolon bat coronavirus C704, Rousettus bat coronavirus
GCCDC1, Rousettus bat coronavirus HKU9, severe acute respiratory
syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), or bat SARS-like coronavirus WIV1.
[0032] Examples of the member of Gammacoronavirus genus may
include, but are not limited to, Brangacovirus, Cegacovirus,
Igacovirus, and combinations thereof. For example, the member of
Gammacoronavirus genus may be goose coronavirus CB17, Beluga whale
coronavirus SW1, avian coronavirus, avian coronavirus 9203, or duck
coronavirus 2714.
[0033] Examples of the member of Deltacoronavirus genus may
include, but are not limited to, Andecovirus, Buldecovirus,
Herdecovirus, and combinations thereof. For example, the member of
Deltacoronavirus genus may be Wigeon coronavirus HKU20, Bulbul
coronavirus HKU11, Common moorhen coronavirus HKU21, Coronavirus
HKU15, Munia coronavirus HKU13, White-eye coronavirus HKU16, or
Night heron coronavirus HKU19.
[0034] In an exemplary embodiment, the coronavirus is HCoV-229E. In
another exemplary embodiment, the coronavirus is HCoV-OC43. In yet
another exemplary embodiment, the coronavirus is SARS-CoV-2.
[0035] According to this disclosure, pioglitazone or the
pharmaceutically acceptable salt thereof may be prepared into a
pharmaceutical composition in a dosage form suitable for, e.g.,
oral administration, using technology well known to those skilled
in the art. Examples of the dosage form for oral administration may
include, but are not limited to, sterile powder, tablets, troches,
lozenges, capsules, dispersible powder, granule, solutions,
suspensions, emulsions, syrup, elixirs, slurry, and the like.
[0036] According to this disclosure, the pharmaceutical composition
may further include a pharmaceutically acceptable carrier that is
widely employed in the art of drug-manufacturing. Examples of the
pharmaceutically acceptable carrier may include, but are not
limited to, solvents, buffers, emulsifiers, suspending agents,
decomposers, disintegrating agents, dispersing agents, binding
agents, excipients, stabilizing agents, chelating agents, diluents,
gelling agents, preservatives, wetting agents, lubricants,
absorption delaying agents, liposomes, and the like. The choice and
amount of the pharmaceutically acceptable carrier are within the
expertise of those skilled in the art.
[0037] The dosage and the frequency of administration of
pioglitazone or the pharmaceutically acceptable salt thereof may
vary depending on the following factors: the severity of the
illness/viral infection to be treated and the weight, age, physical
condition and response of the subject to be treated.
[0038] The present disclosure will be further described by way of
the following examples. However, it should be understood that the
following examples are intended solely for the purpose of
illustration and should not be construed as limiting the present
disclosure in practice.
Examples
General Experimental Materials:
[0039] 1. Pioglitazone was purchased from Sigma Aldrich. 2. Primers
used in the following examples were synthesized by Taqkey Science
Co., Ltd., Taiwan. 3. Cell cultures
[0040] Rhesus monkey kidney epithelial (LLC-MK2) cells (ATCC CCL-7)
and African green monkey kidney (Vero-E6) cells (ATCC CRL-1586)
used in the following experiments were purchased from ATCC
(American Type Culture Collection, Manassas, Va., USA). Human
hepatocellular carcinoma (Huh-7) cells (Ser. No. 01/042,712) was
purchased from Sigma Aldrich. The cells of the respective type were
incubated in a cell culture dish, where LLC-MK2 cells and Vero-E6
cells were incubated in minimum essential medium (MEM; Gibco, Grand
Island, N.Y.) and Huh-7 cells were incubated in Dulbecco's Modified
Eagle's Medium (DMEM; Gibco), each supplemented with 10% fetal
bovine serum (FBS; Gibco), followed by cultivation in an incubator
with culture conditions set at 37.degree. C. or 33.degree. C. and
5% CO.sub.2. Medium change was performed every 3 days. Cell passage
was performed when the cultured cells reached 90% of
confluence.
4. Virus strains
[0041] Human coronavirus 229E (HCoV-229E) (ATCC VR-740) and human
coronavirus OC43 (HCoV-OC43) (ATCC VR-1558) used in the following
experiments were purchased from ATCC. Severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) was isolated from
nasopharyngeal swab of a patient diagnosed with coronavirus disease
2019 (COVID-19) who was followed and treated at Chang-Gung Memorial
Hospital, Linkou, Taiwan (CGMH-CGU-01).
General Experimental Procedures:
1. Virus Infection
[0042] When the cultured cells reached 80% to 90% of confluence,
infection by viruses, such as HCoV-229E, HCoV-OC43 or SARS-CoV-2,
at a given multiplicity of infection (m.o.i.), was performed in
serum-free MEM. The viruses were allowed to adsorb at 37.degree. C.
(HCoV-229E and SARS-CoV-2) or 33.degree. C. (HCoV-OC43) for 1 hour,
after which the infected cells were washed with phosphate-buffered
saline (PBS) and incubated at 37.degree. C. or 33.degree. C. in MEM
containing 2% FBS.
2. Statistical Analysis
[0043] The experimental data are expressed as mean.+-.standard
error of the mean (SEM), and were analyzed by Student's two-tailed
unpaired t-test using GraphPad Prism 6 software (GraphPad Software,
Inc., California, USA), where p-values<0.05 were considered to
be statistically significant.
Example 1. Evaluation of the Effect of Pioglitazone on Cell
Viability of HCoV-229E-Infected Cells
Experimental Procedures:
[0044] LLC-MK2 cells were seeded in a 96-well plate at 2000 cells
per well. After 24 hours, the cells were pretreated with
pioglitazone, at concentrations of 0, 5, 10, 20, 40, 60, 80, 100,
and 120 .mu.M, respectively, for 1 hour. Subsequently, the
pretreated cells were infected with HCoV-229E at m.o.i. of 0.01 for
7 days according to the procedures as described in the preceding
section, entitled "1. Virus infection," of the General Experimental
Procedures. Mock-infected cells served as a control group.
HCoV-229E-infected cells and mock-infected cells were subjected to
cell viability determination using CellTiter96 Aqueous One Solution
Cell Proliferation Assay (Promega).
Results:
[0045] FIG. 1 illustrates cell viability of HCoV-229E-infected and
mock-infected LLC-MK2 cells that were pretreated with increasing
concentrations of pioglitazone. As shown in FIG. 1, pioglitazone is
capable of increasing cell viability of HCoV-229E-infected LLC-MK2
cells as compared to the control group, indicating that infection
of coronavirus, such as HCoV-229E, can be inhibited by
pioglitazone.
Example 2. Evaluation of the Effect of Pioglitazone on Viral Yield
in HCoV-229E-Infected and HCoV-OC43-Infected Cells
Experimental Procedures:
[0046] LLC-MK2 cells and Vero E6 cells were pretreated with 20
.mu.M of rosiglitazone for 1 hour, and then were respectively
infected with HCoV-229E and HCoV-OC43 at m.o.i. of 0.01 according
to the procedures as described in the preceding section, entitled
"1. Virus infection," of the General Experimental Procedures.
[0047] The HCoV-229E in the infected LLC-MK2 cells and the
HCoV-OC43 in the infected Vero E6 cells were harvested at 1, 2, and
3 days post-infection (d.p.i.) for determination of virus
titers.
[0048] Each of HCoV-229E and HCoV-OC43 was subjected to a ten-fold
dilution using serum-free DMEM or MEM, so as to obtain a diluted
viral solution having a dilution factor of 10 for use in the
following viral plaque assay.
[0049] Huh-7 cells and Vero E6 cells were seeded at 8.times.10, and
5.times.10.sup.5 cells per well into respective wells of 6-well
plates containing 10% FBS DMEM or MEM, and were cultured in an
incubator (37.degree. C. and 5% CO.sub.2) for 24 hours. Thereafter,
the Huh-7 cells and Vero E6 cells were infected with the diluted
solutions (500 .mu.L) of HCoV-229E and HCoV-OC43, respectively,
according to the procedures as described in the preceding section,
entitled "1. Virus infection," of the General Experimental
Procedures. After that, the HCoV-229E-infected Huh-7 cells and the
HCoV-OC43-infected Vero E6 cells were washed with PBS, and then 3
mL of an agarose overlay medium (0.3% agarose in DMEM or MEM
containing 2% FBS) was added to each well. After the agarose
overlay medium had solidified, the plates were placed in an
incubator set at 37.degree. C. for the HCoV-229E-infected Huh-7
cells or at 33.degree. C. for the HCoV-OC43-infected Vero E6
cells.
[0050] The cells in each well were fixed with 2 mL of a 10%
formaldehyde solution at room temperature for 2 hours. Next, the
agarose overlay in each well was removed, and then the fixed cells
in each well were stained with 0.5% crystal violet (Manufacturer:
Sigma Aldrich) for 2 min. After rinsing the stained cells with
water, the viral plaques in each well were counted. The viral titer
(plaque forming units (P.F.U.)/mL) was determined by the following
formula (1):
A=B/(C.times.0.5) (1)
wherein: A=viral titer [0051] B=the viral plaques counted [0052]
C=the dilution factor of the virus
[0053] In comparison, Huh-7 cells and Vero E6 cells pretreated with
dimethyl sulfoxide (DMSO) (serving as control groups) were
subjected to the same analysis.
Results:
[0054] FIG. 2 shows virus titers (expressed as log P.F.U./mL) of
the HCoV-229E and HCoV-OC43 respectively in the infected Huh-7
cells pretreated with pioglitazone (upper panel) and the infected
Vero E6 cells pretreated with pioglitazone (lower panel) at 1, 2,
and 3 d.p.i. As shown in FIG. 2, as compared to the control groups,
viral yields of the HCoV-229E and HCoV-OC43 in the infected cells
pretreated with pioglitazone are significantly lower, indicating
that pioglitazone is effective in reducing viral replication in
host cells infected with coronavirus.
Example 3. Evaluation of the Effects of Pioglitazone on Viral Gene
Expression and Viral Yields in SARS-CoV-2-Infected Cells
A. Quantitative Determination of Viral Gene Expression
[0055] LLC-MK2 cells were pretreated with 20 .mu.M of pioglitazone
for 1 hour, and then were infected with SARS-CoV-2 at m.o.i. of
0.01 according to the procedures as described in the preceding
section, entitled "1. Virus infection," of the General Experimental
Procedures.
[0056] At 1, 2, 3 and 4 d.p.i., viral RNA was extracted using
LabTurbo Viral Mini Kit with LabTurbo 48 Compact System (Taigen
Bioscience Corporation), and was used as a template for
synthesizing cDNA by reverse transcription polymerase chain
reaction (RT-PCR) using MMLV Reverse Transcription kit (Protech
Technology). Thereafter, the thus obtained cDNA, serving as a DNA
template, was subjected to quantitative PCR (qPCR), which was
performed on a LightCycler@480 System (Roche Life Science) using
the PCR reaction mixture and the reaction conditions shown in Table
1, so as to determine the RNA copy number of viral target genes,
including Envelope (E) and RNA-dependent RNA polymerase (RdRp)
genes of SARS-CoV-2. The SARS-CoV-2-E gene-specific primers and
probe and the SARS-CoV-2-RdRp gene-specific primers and probe
listed in Table 2 were designed following recommendations by the
Taiwan Center for Disease Control (CDC).
[0057] In comparison, LLC-MK2 cells pretreated with DMSO (serving
as control group) were subjected to the same analysis.
TABLE-US-00001 TABLE 1 Volume Contents (&muL) cDNA 1 E
gene-specific Forward primer (10 .mu.M) 0.5 pramers and probe
Reverse primer (10 .mu.M) 0.5 5'FAM probe (10 .mu.M) 0.5 RdRp
gene-specific Forward primer (10 .mu.M) 0.5 primers and probe
Reverse primer (10 .mu.M) 0.5 5'FAM probe (5 .mu.M) 0.5 2 .times.
qPCRBIO Probe Blue Mix Lo-ROX 5 (PCR Biosystems) DEPC-treated
d.sub.2H.sub.2O 2.5 Operation conditions: Denaturation at
95.degree. C. for 5 min, followed by 50 cycles of the following
reactions: denaturation at 95.degree. C. for 5 sec, and primer
annealing and extension at 72.degree. C. for 10 sec.
TABLE-US-00002 TABLE 2 Viral Nucleotide sequence SEQ ID gene
Primer/Probe (5'.fwdarw.3') NO. E gene Forward primer
acaggtacgttaatagtta 1 atagcgt Reverse primer atattgcagcagtacgcac 2
aca 5' FAM probe FAM-acactagccatcctt 3 actgcgcttcg-BBQ RdRp Forward
primer gtgaratggtcatgtgtgg 4 gene cgg Reverse primer
caratgttaaasacactat 5 tagcata 5' FAM probe FAM-caggtggaacctcat 6
caggagatgc-BBQ Note: FAM and BBQ respectively represent fluorescein
and BlackBerry .RTM. Quencher.
Results:
[0058] FIG. 3 illustrates RNA quantification (expressed as log RNA
copy number/mL) of E gene (upper panel) and RdRp gene (lower panel)
of SARS-CoV-2 in the infected LLC-MK2 cells pretreated with
pioglitazone. As shown in FIG. 3, as compared to the control group,
pioglitazone leads to significant reduction in RNA expression of
each of E and RdRp genes of SARS-CoV-2 in the infected LLC-MK2
cells, indicating that pioglitazone can reduce the viral gene
expression and thereby inhibit the viral replication in the host
cells.
B. Immunofluorescence Microscopy:
[0059] LLC-MK2 cells were pretreated with 20 .mu.M of pioglitazone
for 1 hour, and then were infected with SARS-CoV-2 at m.o.i. of
0.01 according to the procedures as described in the preceding
section, entitled "1. Virus infection," of the General Experimental
Procedures. In comparison, SARS-CoV-2-infected LLC-MK2 cells
pretreated with 0.08% DMSO served as an infection control group,
and mock-infected LLC-MK2 cells pretreated with pioglitazone served
as a normal control group.
[0060] At 4 d.p.i., the cells were washed with PBS and fixed with
4% formaldehyde for 10 minutes at room temperature. Then, the cells
were permeabilized and subjected to immunostaining for detection of
SARS-CoV-2 spike (S2 subunit) and nucleocapsid proteins using the
primary and secondary antibodies shown in Table 3 below, and the
nucleus of the cells was stained with DAPI (Manufacturer:
Invitrogen; Catalog no.: D1306). Thereafter, the stained cells were
examined using a fluorescence microscope (Manufacturer: Olympus
Corporation; Model No.: IX71).
TABLE-US-00003 TABLE 3 Proteins Primary antibody Secondary antibody
SARS-CoV-2 Mouse anti-SARS-CoV-2 Alexa Fluor 488 spike spike
monoclonal goat anti-mouse IgG antibody (Cat. No. A-11001, (Cat.
No. GTX632604, Invitrogen, GeneTex, Irvine, CA) Waltham, MA)
SARS-CoV-2 Rabbit anti-SARS-CoV-2 Alexa Fluor 594 nucleocapsid
nucleocapsid goat anti-rabbit IgG polyclonal antibody (Cat. No.
A-11012, (Cat. No. GTX135361, Invitrogen, GeneTex, Irvine, CA)
Waltham, MA)
Results:
[0061] FIG. 4 shows fluorescence microscopy images of expression of
the spike and nucleocapside proteins of SARS-CoV-2 in the LLC-MK2
cells pretreated with pioglitazone. It can be seen from FIG. 4
that, as compared to the mock-infected LLC-MK2 cells, the spike and
nucleocapside proteins are clearly expressed in the infection
control group, indicating that SARS-CoV-2 viruses have been
replicated in the infected cells. However, such viral replication
is significantly reduced in the cells pretreated with pioglitazone,
suggesting that pioglitazone exerts an inhibitory effect against
SARS-CoV-2 infection.
C. Viral Plaque Reduction Assay:
[0062] Vero E6 cells were seeded at 5.times.10' cells per well into
6-well plates containing 10% FBS MEM, and were cultured in an
incubator (37.degree. C. and 5% CO.sub.2) for 24 hours. Next, the
Vero E6 cells were pretreated with 20 .mu.M of pioglitazone for 1
hour, and then were infected with SARS-CoV-2 at 100 P.F.U.,
according to the procedures as described in the preceding section,
entitled "1. Virus infection," of the General Experimental
Procedures. After that, the SARS-CoV-2-infected Vero E6 cells were
washed with PBS, and then 3 mL of an agarose overlay medium (0.3%
agarose in MEM containing 2% FBS) containing 20 .mu.M of
pioglitazone was added to each well. After the
pioglitazone-containing agarose overlay medium had solidified, the
plates were placed in a 37.degree. C. incubator. Subsequently, the
SARS-CoV-2 viruses in the infected Vero E6 cells were harvested at
3 d.p.i. for determination of virus titers. The cells in each well
were fixed with 2 mL of a 10% formaldehyde solution at room
temperature for 2 hours. Next, the agarose overlay in each well was
removed, and then the fixed cells in each well were stained with
0.5% crystal violet (Manufacturer: Sigma Aldrich) for 2 min. After
rinsing the stained cells with water, distribution of the viral
plaques in each well was analyzed by visual observation. In
comparison, SARS-CoV-2-infected Vero E6 cells cultured in a
pioglitazone-free agarose overlay medium (0.3% agarose in MEM
containing 2% FBS), which served as a control group, were subjected
to the same analysis.
Results:
[0063] FIG. 5 shows the viral plaques of the SARS-CoV-2 in the Vero
E6 cells treated with pioglitazone. As shown in FIG. 5, the size
and number of the viral plaques of the SARS-CoV-2 were reduced in
the pioglitazone-treated Vero E6 cells as compared to the control
group.
[0064] Taken together, the above results demonstrate that
pioglitazone can effectively inhibit replication of coronavirus,
such as SARS-CoV-2.
[0065] In the description above, for the purposes of explanation,
numerous specific details have been set forth in order to provide a
thorough understanding of the embodiments. It will be apparent,
however, to one skilled in the art, that one or more other
embodiments may be practiced without some of these specific
details. It should also be appreciated that reference throughout
this specification to "one embodiment," "an embodiment," an
embodiment with an indication of an ordinal number and so forth
means that a particular feature, structure, or characteristic may
be included in the practice of the disclosure. It should be further
appreciated that in the description, various features are sometimes
grouped together in a single embodiment, figure, or description
thereof for the purpose of streamlining the disclosure and aiding
in the understanding of various inventive aspects, and that one or
more features or specific details from one embodiment may be
practiced together with one or more features or specific details
from another embodiment, where appropriate, in the practice of the
disclosure.
[0066] While the disclosure has been described in connection with
what are considered the exemplary embodiments, it is understood
that this disclosure is not limited to the disclosed embodiments
but is intended to cover various arrangements included within the
spirit and scope of the broadest interpretation so as to encompass
all such modifications and equivalent arrangements.
Sequence CWU 1
1
6126DNAArtificial SequenceForward primer for E gene of SARS-CoV-2
1acaggtacgt taatagttaa tagcgt 26222DNAArtificial SequenceReverse
primer for E gene of SARS-CoV-2 2atattgcagc agtacgcaca ca
22326DNAArtificial Sequence5'-end FAM probe for E gene of
SARS-CoV-2 3acactagcca tccttactgc gcttcg 26422DNAArtificial
SequenceForward primer for RdRp gene of SARS-CoV-2 4gtgaratggt
catgtgtggc gg 22526DNAArtificial SequenceReverse primer for RdRp
gene of SARS-CoV-2 5caratgttaa asacactatt agcata 26625DNAArtificial
Sequence5'-end FAM probe for RdRp gene of SARS-CoV-2 6caggtggaac
ctcatcagga gatgc 25
* * * * *