Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND COMPOSITIONS FOR TREATING CORONAVIRUS INFECTION
[001]
FIELD OF THE INVENTION
[002] The present invention concerns an antiviral composition and its use
for treating
Arenaviridae infection, Bunyaviridae infection, Flaviviridae infection,
Togaviridae infection,
Paramyxoviridae infection, Retroviridae infection, Coronaviridae infection, or
Filoviridae
infection in mammals. Some embodiments concern treatment of hemorrhagic viral
infection.
BACKGROUND OF THE INVENTION
[003] Nerium oleander, a member of the Nerium species, is an ornamental
plant widely
distributed in subtropical Asia, the southwestern United States, and the
Mediterranean. Its
medical and toxicological properties have long been recognized. It has been
proposed for use,
for example, in the treatment of hemorrhoids, ulcers, leprosy, snake bites,
cancers, tumors,
neurological disorders, warts, and cell-proliferative diseases. Zibbu et al.
(J. Chem. Pharm.
Res. (2010), 2(6), 351-358) provide a brief review on the chemistry and
pharmacological
activity of Nerium oleander.
[004] Extraction of components from plants of Nerium species has
traditionally been
carried out using boiling water, cold water, supercritical fluid, or organic
solvent.
[005] ANVIRZELTM (US 5,135,745 to Ozel) contains the concentrated form or
powdered
form of the hot-water extract of Nerium oleander. Muller et al. (Pharmazie.
(1991) Sept. 46(9),
657-663) disclose the results regarding the analysis of a water extract of
Nerium oleander. They
report that the polysaccharide present is primarily galacturonic acid. Other
saccharides include
rhamnose, arabinose and galactose. Polysaccharide content and individual sugar
composition
of polysaccharides within the hot water extract of Nerium oleander have also
been reported by
Newman et al. J. Herbal Pharmacotherapy, (2001) vol 1, pp.1-16). Compositional
analysis
Date Recue/Date Received 2021-05-13
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of ANVIRZELTM, the hot water extract, was described by Newman et al. (Anal.
Chem. (2000),
72(15), 3547-3552). U.S. Patent No. 5,869,060 to Selvaraj et al. pertains to
extracts of Nerium
species and methods of production. To prepare the extract, plant material is
placed in water
and boiled. The crude extract is then separated from the plant matter and
sterilized by filtration.
The resultant extract can then be lyophilized to produce a powder. U.S. Patent
No. 6,565,897
(U.S. Pregrant Publication No. 20020114852 and PCT International Publication
No. WO
2000/016793 to Selvaraj et al.) discloses a hot-water extraction process for
the preparation of a
substantially sterile water extract. Ishikawa et al. (J. Nutr. Sci. Vitaminol.
(2007), 53, 166-173)
discloses a hot water extract of Nerium oleander and fractionation thereof by
liquid
chromatography using mixtures of chloroform, methanol, and water. They also
report that
extracts of the leaves of N. oleander have been used to treat Type II
diabetes. US20060188585
published Aug. 24, 2006 to Panyosan discloses a hot water extract of Nerium
oleander. US
10323055 issued June 18, 2019 to Smothers discloses a method of extracting
plant material
with aloe and water to provide an extract comprising aloe and cardiac
glycoside.
US20070154573 published July 5, 2007 to Rashan et al. discloses a cold-water
extract of
Nerium oleander and its use.
[006] Erdemoglu et al. (.1 Ethnopharmacol. (2003) Nov. 89(1), 123-129)
discloses results
for the comparison of aqueous and ethanolic extracts of plants, including
Nerium oleander,
based upon their anti-nociceptive and anti-inflammatory activities. Fartyal et
al. (J. Sci. Innov.
Res. (2014), 3(4), 426-432) discloses results for the comparison of methanol,
aqueous, and
petroleum ether extracts of Nerium oleander based upon their antibacterial
activity.
[007] Organic solvent extracts of Nerium oleander are also disclosed by
Adome et al. (Afr.
Health Sci. (2003) Aug. 3(2), 77-86; ethanolic extract), el-Shazly et al. (.1
Egypt Soc. Parasitol.
(1996), Aug. 26(2), 461-473; ethanolic extract), Begum et al. (Phytochemisny
(1999) Feb.
50(3), 435-438; methanolic extract), Zia et al. (.1 Ethnolpharmacol. (1995)
Nov. 49(1), 33-39;
methanolic extract), and Vlasenko et al. (Farmatsiia. (1972) Sept.-Oct. 21(5),
46-47; alcoholic
extract). Turkmen et al. (.1 Planar Chroma. (2013), 26(3), 279-283) discloses
an aqueous
ethanol extract of Nerium oleander leaves and stems. US 3833472 issued Sept.
3, 1974 to
Yamauchi discloses extraction of Nerium odorum SOL (Nerium oleander Linn)
leaves with
Date Recue/Date Received 2021-04-13
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water, organic solvent, or aqueous organic solvent, wherein the leaves are
heated to 60 -170 C
and then extracted, and the organic solvent is methanol, ethanol, propyl ether
or chloroform.
[008] A supercritical fluid extract of Nerium species is known (US 8394434,
US 8187644,
US 7402325) and has demonstrated efficacy in treating neurological disorders
(US 8481086,
US 9220778, US 9358293, US 20160243143A1, US 9877979, US 10383886) and cell-
proliferative disorders (US 8367363, US 9494589, US 9846156), and some viral
infections (US
10596186, WO 2018053123A1, W02019055119A1)
[009] Triterpenes are known to possess a wide variety of therapeutic
activities. Some of
the known triterpenes include oleanolic acid, ursolic acid, betulinic acid,
bardoxolone, maslinic
acid, and others. The therapeutic activity of the triterpenes has primarily
been evaluated
individually rather than as combinations of triterpenes.
[0010] Oleanolic acid is in a class of triterpenoids typified by compounds
such as
bardoxolone which have been shown to be potent activators of the innate
cellular phase 2
detoxifying pathway, in which activation of the transcription factor Nrf2
leads to transcriptional
increases in programs of downstream antioxidant genes containing the
antioxidant
transcriptional response element (ARE). Bardoxolone itself has been
extensively investigated
in clinical trials in inflammatory conditions; however, a Phase 3 clinical
trial in chronic kidney
disease was terminated due to adverse events that may have been related to
known cellular
toxicities of certain triterpenoids including bardoxolone at elevated
concentrations.
[0011] Compositions containing triterpenes in combination with other
therapeutic
components are found as plant extracts. Fumiko et al. (Biol. Pharm. Bull
(2002), 25(11), 1485-
1487) discloses the evaluation of a methanolic extract of Rosmarimus
officinalis L. for treating
trypanosomiasis. Addington et al. (US 8481086, US 9220778, US 9358293, US
20160243143
Al) disclose a supercritical fluid extract (SCF; PBI-05204) of Nerium oleander
containing
oleandrin and triterpenes for the treatment of neurological conditions.
Addington et al.
(US 9011937, US 20150283191 Al) disclose a triterpene-containing fraction (PBI-
04711) of
the SCF extract of Nerium oleander containing oleandrin and triterpenes for
the treatment of
neurological conditions. Jager et al. (Molecules (2009), 14, 2016-2031)
disclose various plant
extracts containing mixtures of oleanolic acid, ursolic acid, betulinic acid
and other
components. Mishra et al. (PLoS One 2016 25;11(7):e0159430. Epub 2016 Jul 25)
disclose an
Date Recue/Date Received 2021-04-13
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extract of Betula utilis bark containing a mixture of oleanolic acid, ursolic
acid, betulinic acid
and other components. Wang et al. (Molecules (2016), 21, 139) disclose an
extract of Alstonia
scholaris containing a mixture of oleanolic acid, ursolic acid, betulinic acid
and other
components. L. e Silva et al. (Molecules (2012), 17, 12197) disclose an
extract of Eriope
blanchetti containing a mixture of oleanolic acid, ursolic acid, betulinic
acid and other
components. Rui et al. (Int. J. Mol. Sci. (2012), 13, 7648-7662) disclose an
extract of
Eucaplyptus globulus containing a mixture of oleanolic acid, ursolic acid,
betulinic acid and
other components. Ayatollahi et al. (Iran. J. Pharm. Res. (2011), 10(2), 287-
294) disclose an
extract of Euphorbia microsciadia containing a mixture of oleanolic acid,
ursolic acid, betulinic
acid and other components. Wu et al. (Molecules (2011), 16, 1-15) disclose an
extract of
Ligustrum species containing a mixture of oleanolic acid, ursolic acid,
betulinic acid and other
components. Lee et al. (Biol. Pharm. Bull (2010), 33(2), 330) disclose an
extract of Forsythia
viridissima containing a mixture of oleanolic acid, ursolic acid, betulinic
acid and other
components.
[0012] Oleanolic acid (0 or OA), ursolic acid (U or UA) and betulinic acid
(B or BA) are
the three major triterpene components found in PBI-05204 (PBI-23; a
supercritical fluid extract
of Nerium oleander) and PBI-04711 (a triterpene-containing fraction 0-4 of PBI-
05204). We
(two of the instant inventors) previously reported (Van Kanegan et al., in
Nature Scientific
Reports (May 2016), 6:25626. doi: 10.1038/srep25626) on the contribution of
the triterpenes
toward efficacy by comparing their neuroprotective activity in a brain slice
oxygen glucose
deprivation (OGD) model assay at similar concentrations. We found that PBI-
05204 (PBI) and
PBI-04711 (Fraction 0-4) provide neuroprotective activity.
[0013] Extracts of Nerium species are known to contain many different
classes of
compounds: cardiac glycosides, glycones, steroids, triterpenes,
polysaccharides and others.
Specific compounds include oleandrin; neritaloside; odoroside; oleanolic acid;
ursolic acid;
betulinic acid; oleandrigenin; oleaside A; betulin (urs-12-ene-313,28-diol);
28-norurs-12-en-313-
ol; urs-12-en-3f3-ol; 313,313-hydroxy-12-oleanen-28-oic acid; 313,20a-
dihydroxyurs-21-en-28-
oic acid; 313,27-dihydroxy-12-ursen-28-oic acid; 313,1313-dihydroxyurs-11-en-
28-oic acid;
313,12oc-dihydroxyoleanan-28,1313-olide; 313,27-dihydroxy-12-oleanan-28-oic
acid; and other
components.
Date Recue/Date Received 2021-04-13
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100141 Viral hemorrhagic fever (VHF) can be caused by five distinct virus
families:
Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, and Paramyxoviridae.
The Filoviruses,
e.g. Ebolavirus (EBOV) and Marburgvirus (MARV), are among the most pathogenic
viruses
known to man and the causative agents of viral hemorrhagic fever outbreaks
with fatality rates
of up to 90%. Each virion contains one molecule of single-stranded, negative-
sense RNA.
Beyond supportive care or symptomatic treatment, there are no commercial
therapeutically
effective drugs and no prophylactic drugs available to treat EBOV (Ebolavirus)
and MARV
(Marburgvirus) infections, i.e. filovirus infections. Five species of
Ebolavirus have been
identified: Tal Forest (formerly Ivory Coast), Sudan, Zaire, Reston and
Bundibugyo.
[0015] Negative-sense single-stranded enveloped RNA virus ((-)-(ss)-
enyRNAV) includes
viruses in the Arenaviridae family, Bunyaviridae family (Bunyavirales order),
Filoviridae
family, Orthomyxoviridae family, Paramyxoviridae family, and Rhabdoviridae
family. The
negative viral RNA is complementary to the mRNA and must be converted to a
positive RNA
by RNA polymerase before translation; therefore, the purified RNA of a
negative sense virus
is not infectious by itself, as it needs to be converted to a positive sense
RNA for replication.
Exemplary viruses and infections from the Arenaviridae family include Lassa
virus, aseptic
meningitis, Guanarito virus, Junin virus, Lujo virus, Machupo virus, Sabia
virus and
Whitewater Arroyo virus. Exemplary viruses and infections from the
Bunyaviridae family
include Hantavirus, Crimean-Congo hemorrhagic fever orthonairovirus. Exemplary
viruses and
infections from the Paramyxoviridae family include Mumps virus, Nipah virus,
Hendra virus,
respiratory syncytial virus (RSV), human parainfluenza virus (HPIV), and NDV.
Exemplary
viruses and infections from the Orthomyxoviridae family include influenza
virus (A through
C), Isavirus, Thogotovirus, Quaranjavirus, H1N1, H2N2, H3N2, H1N2, Spanish
flu, Asian flu,
Hong Kong Flu, Russian flu. Exemplary viruses and infections from the
Rhabdoviridae family
include rabies virus, vesiculovirus, Lyssavirus, Cytorhabdovirus.
[0016] The Flaviviruses are positive-sense, single-stranded, enveloped RNA
viruses
((+)-(ss)-enyRNAV). They are found in arthropods, primarily ticks and
mosquitoes, and cause
widespread morbidity and mortality throughout the world. Some of the mosquito-
transmitted
viruses include Yellow Fever, Dengue Fever, Japanese Encephalitis, West Nile
Viruses, and
Zikavirus. Some of the tick-transmitted viral infections include Tick-borne
Encephalitis,
Date Recue/Date Received 2021-04-13
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Kyasanur Forest Disease, Alkhurma Disease, Omsk Hemorrhagic Fever. Although
not a
hemorrhagic infection, Powassan virus is a Flavivirus. (+)-(ss)-envRNAV
include
Coronaviridae family (human and animal pathogen), Flaviviridae family (human
and animal
pathogen), Togaviridae family (human and animal pathogen), and Arterviridae
family (animal
pathogen).
[0017]
Coronavirus (CoV) is the common name for Coronaviridae. In humans, CoV causes
respiratory infections, which are typically mild but can be lethal in rare
forms such as SARS
(severe acute respiratory syndrome)-CoV, MERS (Middle East Respiratory
Syndrome)-CoV,
and COVID-19. CoV has a nucleocapsid of helical symmetry and the genome size
ranges from
about 26 to about 32 kilobases. Other exemplary human CoV include CoV 229E,
CoV NL63,
CoV 0C43, CoV HKU1, and CoV HKU20. The envelope of CoV carries three
glycoproteins:
S- spike protein: receptor binding, cell fusion, major antigen; E-Envelope
protein: small,
envelope-associated protein; and M- Membrane protein: transmembrane - budding
& envelope
formation. In a few types of CoV, there is a fourth glycoprotein: HE-
heamagglutinin-esterase.
The genome has a 5' methylated cap and 3' poly-A and functions directly as
mRNA. Entry of
the CoV into a human cell occurs via endocytosis and membrane fusion; and
replication occurs
in the cell's cytoplasm. CoV are transmitted by aerosols of respiratory
secretions, by the faecal-
oral route, and by mechanical transmission. Most virus growth occurs in
epithelial cells.
Occasionally the liver, kidneys, heart or eyes may be infected, as well as
other cell types such
as macrophages. In cold-type respiratory infections, growth appears to be
localized to the
epithelium of the upper respiratory tract. Coronavirus infection is very
common and occurs
worldwide. The incidence of infection is strongly seasonal, with the greatest
incidence in
children in winter. Adult infections are less common. The number of
coronavirus serotypes and
the extent of antigenic variation is unknown. Re-infections appear to occur
throughout life,
implying multiple serotypes (at least four are known) and/or antigenic
variation, hence the
prospects for immunization against all serotypes with a single vaccine is
highly unlikely. SARS
is a type of viral pneumonia, with symptoms including fever, a dry cough,
dyspnea (shortness
of breath), headache, and hypoxaemia (low blood oxygen concentration). Typical
laboratory
findings include lymphopaenia (reduced lymphocyte numbers) and mildly elevated
aminotransferase levels (indicating liver damage). Death may result from
progressive
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respiratory failure due to alveolar damage. The typical clinical course of
SARS involves an
improvement in symptoms during the first week of infection, followed by a
worsening during
the second week. A substantial need remains for effective antiviral treatments
(compositions
and methods) against human CoV.
[0018] Oleandrin, and an extract of Nerium oleander have been shown to
prevent the
incorporation of the gp120 envelope glycoprotein of HIV-1 into mature virus
particles and
inhibit viral infectivity in vitro (Singh et al., "Nerium oleander derived
cardiac glycoside
oleandrin is a novel inhibitor of HIV infectivity" in Fitoterapia (2013) 84,32-
39).
[0019] Oleandrin has demonstrated anti-HIV activity but has not been
evaluated against
many viruses. The triterpenes oleanolic acid, betulinic acid and ursolic acid
have been reported
to exhibit differing levels of antiviral activity but have not been evaluated
against many viruses.
Betulinic acid has demonstrated some anti-viral activity against HSV-1 strain
1C, influenza A
H7N1, ECHO 6, and HIV-1. Oleanolic acid has demonstrated some anti-viral
activity against
HIV-1, HEP C, and HCV H strain NS5B. Ursolic acid has demonstrated some anti-
viral activity
against HIV-1, HEP C, HCV H strain NS5B, HSV-1, HSV-2, ADV-3, ADV-8, ADV-11,
HEP
B, ENTV CVB1 and ENTV EV71. The antiviral activity of oleandrin, oleanolic
acid, ursolic
acid and betulinic acid is unpredictable as far as efficacy against specific
viruses. Viruses exist
against which oleandrin, oleanolic acid, ursolic acid and/or betulinic acid
have little to no
antiviral activity, meaning one cannot predic a priori whether oleandrin,
oleanolic acid, ursolic
acid and/or betulinic acid will exhibit antiviral activity against particular
genuses of viruses.
[0020] Barrows et al. ("A screen of FDA-approved drugs for inhibitors of
Zikavirus
infection" in Cell Host Microbe (2016), 20, 259-270) report that digoxin
demonstrates antiviral
activity against Zikavirus but the doses are too high and likely toxic. Cheung
et al. ("Antiviral
activity of lanatoside C against dengue virus infection" in Antiviral Res.
(2014) 111, 93-99)
report that lanatoside C demonstrates antiviral activity against Dengue virus.
[0021] Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus belonging
to the family
Retroviridae and the genus deltaretrovirus. It has a positive-sense RNA genome
that is reverse
transcribed into DNA and then integrated into the cellular DNA. Once
integrated, HTLV-1
continues to exist only as a provirus which can spread from cell to cell
through a viral synapse.
Few, if any, free virions are produced, and there is usually no detectable
virus in the blood
Date Recue/Date Received 2021-04-13
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plasma though the virus is present in genital secretions. HTLV-1 predominately
infects CD4+
T-lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL) ¨a rare, yet
aggressive
hematological malignancy with high rates of therapy-resistance and generally
poor clinical
outcomes, in addition to several autoimmune/inflammatory conditions, including
infectious
dermatitis, rheumatoid arthritis, uveitis, keratoconjunctivitis, sicca
syndrome, Sjogren's
syndrome, and HAM/TSP, among others. HAM/TSP is clinically characterized by
chronic
progressive spastic paraparesis, urinary incontinence, and mild sensory
disturbance. While
ATLL is etiologically linked to viral latency, oncogenic transformation, and
the clonal
expansion of HTLV-1-infected cells, the inflammatory diseases, such as HTLV-1-
associated
myelopathy/tropical spastic paraparesis (HAM/TSP), are caused by autoimmune
and/or
immunopathological responses to proviral replication and the expression of
viral antigens.
HAM/TSP is a progressive neuroinflammatory disease that results in the
deterioration and
demyelination of the lower spinal cord. HTLV-1-infected circulating T-cells
invade the central
nervous system (CNS) and cause an immunopathogenic response against virus and
possibly
components of the CNS. Neural damage and subsequent degeneration can cause
severe
disability in patients with HAM/TSP. The persistence of proviral replication
and the
proliferation of HTLV-1-infected cells in the CNS leads to a cytotoxic T-cell
response targeted
against viral antigens, and which may be responsible for the autoimmune
destruction of nervous
tissues.
[0022] Even though cardiac glycosides have been demonstrated to exhibit
some antiviral
activity against a few viruses, the specific compounds exhibit very different
levels of antiviral
activity against different viruses, meaning that some exhibit very poor
antiviral activity and
some exhibit better antiviral activity when evaluated against the same
virus(es).
[0023] A need remains for improved pharmaceutical compositions containing
oleandrin,
oleanolic acid, ursolic acid, betulinic acid or any combination thereof that
are therapeutically
active against specific viral infections.
SUMMARY OF THE INVENTION
[0024] The invention provides a pharmaceutical composition and method for
treating and/or
preventing viral infection in a mammalian subject. The invention also provides
a
Date Recue/Date Received 2021-04-13
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pharmaceutical composition and method for treating viral infection, e.g. Viral
hemorrhagic
fever (VHF) infection, in a mammalian subject. The invention also provides a
method of
treating viral infection in mammals by administration of the pharmaceutical
composition. The
inventors have succeeded in preparing antiviral compositions that exhibit
sufficient antiviral
activity to justify their use in treating viral infection in humans and
animals. The inventors have
developed corresponding treatment methods employing particular dosing
regimens. The
invention also provides a prophylactic method of treating a subject at risk of
contracting a viral
infection, the method comprising chronically administering to the subject one
or more doses of
an antiviral composition on a recurring basis over an extended treatment
period prior to the
subject contracting the viral infection, thereby preventing the subject from
contracting the viral
infection; wherein the antiviral composition comprises oleandrin.
[0025] In some embodiments, the antiviral composition is administered to
subjects having
virally infected cells, wherein the cells exhibit an elevated ratio of alpha-3
to alpha-1 isoforms
of Na,K-ATPase.
[0026] In some embodiments, the viral infection is caused by any of the
following virus
families: Arenaviridae, Arterviridae, Bunyaviridae, Filoviridae, Flaviviridae,
Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae (in particular,
Deltaretrovirus genus), Coronaviridae, or Togaviridae. In some embodiments,
the viral
infection is caused by (+)-ss-envRNAV or (-)-ss-envRNAV.
[0027] Some embodiments of the invention are directed to compositions for
and methods of
treating Filovirus infection, Flavivirus infection, Henipavirus infection,
alphavirus infection, or
Togavirus infection. Viral infections that can be treated include, at least,
Ebolavirus,
Marburgvirus, Alphavirus, Flavivirus, Yellow Fever, Dengue Fever, Japanese
Enchephalitis,
West Nile Viruses, Zikavirus, Venezuelan Equine Encephalomyelitis
(encephalitis) (VEE)
virus, Chikungunya virus, Western Equine Encephalomyelitis (encephalitis)
(WEE) virus,
Eastern Equine Encephalomyelitis (encephalitis) (EEE) virus, Tick-borne
Encephalitis,
Kyasanur Forest Disease, Alkhurma Disease, Omsk Hemorrhagic Fever, Hendra
virus, Nipah
virus, Deltaretrovirus genus, HTLV-1 virus, and species thereof.
[0028] Some embodiments of the invention are directed to compositions for
and methods of
treating viral infections from viruses of the Arenaviridae family,
Arterviridae, Bunyaviridae
Date Recue/Date Received 2021-04-13
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family, Filoviridae family, Flaviviridae family (Flavivirus genus),
Orthomyxyoviridae family,
Paramyxoviridae family, Rhabdoviridae family, Retroviridae family
(Deltaretrovirus genus),
Coronaviridae family, (+)-ss-envRNAV, (-)-ss-envRNAV, or Togaviridae family.
[0029] Some embodiments of the invention are directed to compositions for
and methods of
treating viral infections from viruses of the Henipavirus genus, Ebolavirus
genus, Flavivirus
genus, Marburgvirus genus, Deltaretrovirus genus, Coronavirus (CoV), or
Alphavirus genus.
[0030] In some embodiments, the (+)-ss-envRNAV is a virus selected from the
group
consisting of Coronaviridae family, Flaviviridae family, Togaviridae family,
and Arterviridae
family.
[0031] In some embodiments, the (+)-ss-envRNAV is a coronavirus that is
pathogenic to
humans. In some embodiments, the coronavirus spike protein binds to ACE2
(angiotensin
converting enzyme 2) receptors in human tissue. In some embodiments, the
coronavirus is
selected from the group consisting of SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-
2),
CoV 229E, CoV NL63, CoV 0C43, CoV HKU1, and CoV HKU20.
[0032] In some embodiments, the (+)-ss-envRNAV is a virus selected from the
group
consisting of flavivirus, Yellow Fever virus, Dengue Fever virus, Japanese
Encephalitis virus,
West Nile virus, Zikavirus, Tick-borne Encephalitis virus, Kyasanur Forest
Disease virus,
Alkhurma Disease virus, Omsk Hemorrhagic Fever virus, and Powassan virus.
[0033] In some embodiments, the (+)-ss-envRNAV is a Togaviridae family
virus selected
from the group consisting of arborvirus, eastern equine encephalomyelitis
virus (EEEV),
western equine encephalomyelitis virus (WEEV), Venezuelan equine
encephalomyelitis virus
(VEEV), Chikungunya virus (CHIKV), O'nyong'nvirus (ONNV), Pogosta disease
virus,
Sindbis virus, Ross River fever virus (RRV) and Semliki Forest virus.
[0034] In some embodiments, the (-)-(ss)-envRNAV is a virus selected from
the group
consisting of Arenaviridae family, Bunyaviridae family (Bunyavirales order),
Filoviridae
family, Orthomyxoviridae family, Paramyxoviridae family, or Rhabdoviridae
family.
[0035] In some embodiments, Arenaviridae family virus is selected from the
group
consisting of Lassa virus, aseptic meningitis, Guanarito virus, Junin virus,
Lujo virus, Machupo
virus, Sabia virus and Whitewater Arroyo virus.
Date Recue/Date Received 2021-04-13
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100361 In some embodiments, Bunyaviridae family virus is selected from the
group
consisting of Hantavirus, and Crimean-Congo hemorrhagic fever orthonairovirus.
[0037] In some embodiments, Paramyxoviridae family virus is selected from
the group
consisting of Mumps virus, Nipah virus, Hendra virus, respiratory syncytial
virus (RSV),
human parainfluenza virus (HPIV), and Newcastle disease virus (NDV).
[0038] In some embodiments, Orthomyxoviridae family virus is selected from
the group
consisting of influenza virus (A through C), Isavirus, Thogotovirus,
Quaranjavirus, H1N1 virus,
H2N2 virus, H3N2 virus, H1N2 virus, Spanish flu virus, Asian flu virus, Hong
Kong Flu virus,
and Russian flu virus.
[0039] In some embodiments, Rhabdoviridae family virus is selected from the
group
consisting of rabies virus, vesiculovirus, Lyssavirus, and Cytorhabdovirus.
[0040] The invention also provides embodiments for the treatment of HTLV-1-
associated
condition or neuro-inflammatory disease. In some embodiments, the HTLV-1-
associated
condition or neuro-inflammatory disease is selected from the group consisting
of
myelopathy/tropical spastic paraparesis (HAM/TSP), adult T-cell
leukemia/lymphoma
(ATLL), autoimmune condition, inflammatory condition, infectious dermatitis,
rheumatoid
arthritis, uveitis, keratoconjunctivitis, sicca syndrome, Sjogren's syndrome,
and Strongyloides
stercoralis.
[0041] The invention also provides a method of inhibiting the infectivity
of HTLV-1
particles released into the culture supernatants of treated cells and also
reducing the intercellular
transmission of HTLV-1 by inhibiting the Env-dependent formation of
virological synapses,
the method comprising administering to a subject in need thereof an effective
amount of the
antiviral composition.
[0042] In some embodiments, the invention provides an antiviral composition
comprising
(consisting essentially of): a) specific cardiac glycoside(s); b) plural
triterpenes; or c) a
combination of specific cardiac glycoside(s) and plural triterpenes.
[0043] One aspect of the invention provides a method of treating viral
infection in a subject
by chronic administration to the subject of an antiviral composition. The
subject is treated by
chronically administering to the subject a therapeutically effective amount
(therapeutically
relevant dose) of the composition, thereby providing relief of symptoms
associated with the
Date Recue/Date Received 2021-04-13
- 12 -
viral infection or amelioration of the viral infection. Administration of the
composition to the
subject can begin immediately after infection or any time within one day to 5
days after
infection or at the earliest time after definite diagnosis of infection with
virus. The virus can be
any virus described herein.
[0044] Accordingly, the invention also provides a method of treating viral
infection in a
mammal, the method comprising administering to the mammal one or more
therapeutically
effective doses of the antiviral composition. One or more doses are
administered on a daily,
weekly or monthly basis. One or more doses per day can be administered. The
virus can be
any virus described herein.
[0045] The invention also provides a method of treating viral infection in
a subject in need
thereof, the method comprising:
determining whether or not the subject has a viral infection;
indicating administration of antiviral composition;
administering an initial dose of antiviral composition to the subject
according to a prescribed
initial dosing regimen for a period of time;
periodically determining the adequacy of subject's clinical response and/or
therapeutic
response to treatment with antiviral composition; and
if the subject's clinical response and/or therapeutic response is adequate,
then continuing
treatment with antiviral composition as needed until the desired clinical
endpoint is
achieved; or
if the subject's clinical response and/or therapeutic response are inadequate
at the initial dose
and initial dosing regimen, then escalating or deescalating the dose until the
desired
clinical response and/or therapeutic response in the subject is achieved.
[0046] Treatment of the subject with antiviral composition is continued as
needed. The dose
or dosing regimen can be adjusted as needed until the patient reaches the
desired clinical
endpoint(s) such as a reduction or alleviation of specific symptoms associated
with the viral
infection. Determination of the adequacy of clinical response and/or
therapeutic response can
be conducted by a clinician familiar with viral infections.
[0047] The individual steps of the methods of the invention can be
conducted at separate
facilities or within the same facility.
Date Recue/Date Received 2021-04-13
- 13 -
[0048] The invention provides alternate embodiments, for all the
embodiments described
herein, wherein the oleandrin is replaced with digoxin or used in combination
with digoxin.
The methods of the invention may employ oleandrin, digoxin, or a combination
of oleandrin
and digoxin. Accordingly, oleandrin, digoxin, oleandrin-containing
composition, digoxin-
containing composition, or oleandrin- and digoxin-containing composition may
be used in the
methods of the invention. Cardiac glycoside can be taken to mean oleandrin,
digoxin or a
combination thereof. A cardiac glycoside-containing composition comprises
oleandrin,
digoxin or a combination thereof.
[0049] The invention also provides a method of treating coronavirus
infection, in particular
an infection of coronavirus that is pathogenic to humans, e.g. SARS-CoV-2
infection, the
method comprising chronically administering to a subject, having said
infection, therapeutically
effective doses of cardiac glycoside (cardiac glycoside-containing
composition).
[0050] The invention also provides a dual pathway method of treating
coronavirus infection,
in particular an infection of coronavirus that is pathogenic to humans, e.g.
SARS-CoV-2
infection, the method comprising chronically administering to a subject,
having said infection,
therapeutically effective doses of cardiac glycoside (cardiac glycoside-
containing
composition), thereby inhibiting viral replication of said coronavirus and
reducing the
infectivity of progeny virus of said coronavirus.
[0051] The invention also provides a method of treating coronavirus
infection, in particular
SARS-CoV-2 infection, by repeatedly administering (through any of the modes of
administration discussed herein) to a subject, having said infection, plural
therapeutically
effective doses of cardiac glycoside (cardiac glycoside-containing
composition). One or more
doses may be administered per day for one or more days per week and optionally
for one or
more weeks per month and optionally for one or more months per year.
[0052] The invention also provides a method of treating coronavirus
infection in a human,
the method comprising administering to the subject 1-10 doses of cardiac
glycoside (cardiac
glycoside-containing composition) per day for a treatment period of 2 days to
about 2 months.
Two to eight, two to six, or four doses can be administered daily during the
treatment period.
Doses can be administered for 2 days to about 60 days, 2 days to about 45
days, 2 days to about
30 days, 2 days to about 21 days, or 2 days to about 14 days. Said
administering can be through
Date Recue/Date Received 2021-04-13
- 14 -
any of the modes of administration discussed herein. Systemic administration
that provides
therapeutically effective plasma levels of oleandrin and/or digoxin in said
subject is preferred.
[0053] In some embodiments, one or more doses of oleandrin are administered
per day for
plural days until the viral infection is cured. In some embodiments, one or
more doses of cardiac
glycoside (cardiac glycoside-containing composition) are administered per day
for plural days
and plural weeks until the viral infection is cured. One or more doses can be
administered in a
day. One, two, three, four, five, six or more doses can be administered per
day.
[0054] In some embodiments, the concentration of oleandrin and/or digoxin
in the plasma
of a treated infected subject, e.g. with coronavirus infection, is about 10
microg/mL or less,
about 5 microg/mL or less, about 2.5 microg/mL or less, about 2 microg/mL or
less, or about 1
microg/mL or less. In some embodiments, the concentration of oleandrin and/or
digoxin in the
plasma of a treated subject with coronavirus infection is about 0.0001
microg/mL or more,
about 0.0005 microg/mL or more, about 0.001 microg/mL or more, about 0.0015
microg/mL
or more, about 0.01 microg/mL or more, about 0.015 microg/mL or more, about
0.1 microg/mL
or more, about 0.15 microg/mL or more, about 0.05 microg/mL or more, or about
0.075
microg/mL or more. In some embodiments, the concentration of oleandrin and/or
digoxin in
the plasma of a treated infected subject is about 10 microg/mL to about 0.0001
microg/mL,
about 5 microg/mL to about 0.0005 microg/mL, about 1 microg/mL to about 0.001
microg/mL,
about 0.5 microg/mL to about 0.001 microg/mL, about 0.1 microg/mL to about
0.001
microg/mL, about 0.05 microg/mL to about 0.001 microg/mL, about 0.01 microg/mL
to about
0.001 microg/mL, about 0.005 microg/mL to about 0.001 microg/mL. The invention
includes
all combinations and selections of the plasma concentration ranges set forth
herein.
[0055] The antiviral composition can be administered chronically, i.e. on a
recurring basis,
such as daily, every other day, every second day, every third day, every
fourth day, every fifth
day, every sixth day, weekly, every other week, every second week, every third
week, monthly,
bimonthly, semi-monthly, every other month every second month, quarterly,
every other
quarter, trimesterly, seasonally, semi-annually and/or annually. The treatment
period one or
more weeks, one or more months, one or more quarters and/or one or more years.
An effective
dose of cardiac glycoside (cardiac glycoside-containing composition) is
administered one or
more times in a day.
Date Recue/Date Received 2021-04-13
- 15 -
[0056] In some embodiments, the subject is administered 140 microg to 315
microg per day
of cardiac glycoside. In some embodiments, a dose comprises 20 microg to 750
microg, 12
microg to 300 microg, or 12 microg to 120 microg of cardiac glycoside. The
daily dose of
cardiac glycoside can range from 20 microg to 750 microg, 0.01 microg to 100
mg, or 0.01
microg to 100 microg of cardiac glycoside/day. The recommended daily dose of
oleandrin,
present in the SCF extract, is generally about 0.25 to about 50 microg twice
daily or about 0.9
to 5 microg twice daily or about every 12 hours. The dose can be about 0.5 to
about 100
microg/day, about 1 to about 80 microg/day, about 1.5 to about 60 microg/day,
about 1.8 to
about 60 microg/day, about 1.8 to about 40 microg/day. The maximum tolerated
dose can be
about 100 microg/day, about 80 microg/day, about 60 microg/day, about 40
microg/day, about
38.4 microg/day or about 30 microg/day of oleander extract containing
oleandrin and the
minimum effective dose can be about 0.5 microg/day, about 1 microg/day, about
1.5
microg/day, about 1.8 microg/day, about 2 microg/day, or about 5 microg/day.
Suitable doses
comprising cardiac glycoside and triterpene can be about 0.05-0.5 mg/kg/day,
about 0.05-0.35
mg/kg/day, about 0.05-0.22 mg/kg/day, about 0.05-0.4 mg/kg/day, about 0.05-0.3
mg/kg/day,
about 0.05-0.5 microg/kg/day, about 0.05-0.35 microg/kg/day, about 0.05-0.22
microg/kg/day,
about 0.05-0.4 microg/kg/day, or about 0.05-0.3 microg/kg/day. In some
embodiments, the
dose of oleandrin is about 1 mg to about 0.05 mg, about 0.9 mg to about 0.07
mg, about 0.7 mg
to about 0.1 mg, about 0.5 mg to about 0.1 mg, about 0.4 mg to about 0.1 mg,
about 0.3 mg to
about 0.1 mg, about 0.2 mg. The invention includes all combinations of the
doses set forth
herein.
[0057] In some embodiments, the cardiac glycoside is administered in at
least two dosing
phases: a loading phase and a maintenance phase. The loading phase is
continued until about
achievement of steady state plasma level of cardiac glycoside. The maintenance
phase begins
at either the initiation of therapy or after about completion of the loading
phase. Dose titration
can occur in the loading phase and/or the maintenance phase.
[0058] All dosing regimens, dosing schedules, and doses described herein
are contemplated
as being suitable; however, some dosing regimens, dosing schedules, and doses
may be more
suitable for some subject than for others. The target clinical endpoints are
used to guide said
dosing.
Date Recue/Date Received 2021-04-13
- 16 -
[0059] The composition can be administered systemically.
Modes of systemic
administration include parenteral, buccal, enteral, intramuscular, subdermal,
sublingual,
peroral, pulmonary, or oral. The composition can also be administered via
injection or
intravenously. The composition may also be administered by two or more routes
to the same
subject. In some embodiments, the composition is administered by a combination
of any two
or more modes of administration selected from the group consisting of
parenteral, buccal,
enteral, intramuscular, subdermal, sublingual, peroral, pulmonary, and oral.
[0060] The invention also provides a sublingual dosage form comprising
oleandrin and
liquid carrier. The invention also provides a method of treating viral
infection, in particular
coronavirus infection, e.g. as defined herein, comprising sublingually
administering plural
doses of an oleandrin-containing (digoxin-containing) composition to a subject
having said
viral infection. One or more doses can be administered per day for two or more
days per week
and for one or more weeks per month, optionally for one or months per year.
[0061] In some embodiments, the antiviral composition comprises oleandrin
(or digoxin or
a combination of oleandrin and digoxin) and oil. The oil can comprise medium
chain
triglycerides. The antiviral composition can comprise one, two or more
oleandrin-containing
extracts and one or more pharmaceutical excipients.
[0062] If present in the antiviral composition, additional cardiac
glycoside can be further
included: odoroside, neritaloside, or oleandrigenin. In some embodiments, the
composition
further comprises a) one or more triterpenes; b) one or more steroids; c) one
or more triterpene
derivatives; d) one or more steroid derivatives; or e) a combination thereof.
In some
embodiments, the composition comprises cardiac glycoside and a) two or three
triterpenes; b)
two or three triterpene derivatives; c) two or three triterpene salts; or d) a
combination thereof.
In some embodiments, the triterpene is selected from the group consisting of
oleanolic acid,
ursolic acid, betulinic acid and salts or derivatives thereof.
[0063] Some embodiments of the invention include those wherein a
pharmaceutical
composition comprises at least one pharmaceutical excipient and the antiviral
composition. In
some embodiments, the antiviral composition comprises: a) at least one cardiac
glycoside and
at least one triterpene; b) at least one cardiac glycoside and at least two
triterpenes; c) at least
one cardiac glycoside and at least three triterpenes; d) at least two
triterpenes and excludes
Date Recue/Date Received 2021-04-13
- 17 -
cardiac glycoside; e) at least three triterpenes and excludes cardiac
glycoside; or f) at least one
cardiac glycoside, e.g. oleandrin, digoxin. As used herein, the generic terms
triterpene and
cardiac glycoside also encompass salts and derivatives thereof, unless
otherwise specified.
[0064] The cardiac glycoside can be present in a pharmaceutical composition
in pure form
or as part of an extract containing one or more cardiac glycosides. The
triterpene(s) can be
present in a pharmaceutical composition in pure form or as part of an extract
containing
triterpene(s). In some embodiments, the cardiac glycoside is present as the
primary therapeutic
component, meaning the component primarily responsible for antiviral activity,
in the
pharmaceutical composition. In some embodiments, the triterpene(s) is/are
present as the
primary therapeutic component(s), meaning the component(s) primarily
responsible for
antiviral activity, in the pharmaceutical composition.
[0065] In some embodiments, an oleandrin-containing extract is obtained by
extraction of
plant material. The extract can comprise a hot-water extract, cold-water
extract, supercritical
fluid (SCF) extract, subcritical liquid extract, organic solvent extract, or
combination thereof of
the plant material. In some embodiments, the extract has been (biomass)
prepared by subcritical
liquid extraction of Nerium plant mass (biomass) using, as the extraction
fluid, subcritical liquid
carbon dioxide, optionally comprising alcohol. In some embodiments, the
oleandrin-containing
composition comprises two or more different types of oleandrin-containing
extracts.
[0066] Embodiments of the invention include those wherein the oleandrin-
containing
biomass (plant materia) is Nerium sp., Nerium oleander, Nerium oleander L
(Apocynaceae),
Nerium odourum, white oleander, pink oleander, Thevetia sp., Thevetia
peruviana, yellow
oleander, Thevetia nerifolia, Agrobacterium tumefaciens , cell culture
(cellular mass) of any of
said species, or a combination thereof. In some embodiments, the biomass
comprises leaves,
stems, flowers, bark, fruits, seeds, sap, and/or pods.
[0067] In some embodiments, the extract comprises at least one other
pharmacologically
active agent, obtained along with the cardiac glycoside during extraction,
that contributes to the
therapeutic efficacy of the cardiac glycoside when the extract is administered
to a subject. In
some embodiments, the composition further comprises one or more other non-
cardiac glycoside
therapeutically effective agents, i.e. one or more agents that are not cardiac
glycosides. In some
Date Recue/Date Received 2021-04-13
- 18 -
embodiments, the composition further comprises one or more antiviral
compound(s). In some
embodiments, the antiviral composition excludes a pharmacologically active
polysaccharide.
[0068] In some embodiments, the extract comprises one or more cardiac
glycosides and one
or more cardiac glycoside precursors (such as cardenolides, cardadienolides
and
cardatrienolides, all of which are the aglycone constituents of cardiac
glycosides, for example,
digitoxin, acetyl digitoxins, digitoxigenin, digoxin, acetyl digoxins,
digoxigenin, medigoxin,
strophanthins, cymarine, ouabain, or strophanthidin). The extract may further
comprise one or
more glycone constituents of cardiac glycosides (such as glucoside,
fructoside, and/or
glucuronide) as cardiac glycoside presursors. Accordingly, the antiviral
composition may
comprise one or more cardiac glycosides and two more cardiac glycoside
precursors selected
from the group consisting of one or more aglycone constituents, and one or
more glycone
constituents. The extract may also comprise one or more other non-cardiac
glycoside
therapeutically effective agents obtained from Nerium sp. or Thevetia sp.
plant material.
[0069] In some embodiments, a composition containing oleandrin (OL),
oleanolic acid
(OA), ursolic acid (UA) and betulinic acid (BA) is more efficacious than pure
oleandrin, when
equivalent doses based upon oleandrin content are compared.
[0070] In some embodiments, the molar ratio of total triterpene content (OA
+ UA + BA) to
oleandrin ranges from about 15:1 to about 5:1, or about 12:1 to about 8:1, or
about 100:1 to
about 15:1, or about 100:1 to about 50:1, or about 100:1 to about 75:1, or
about 100:1 to about
80:1, or about 100:1 to about 90:1, or about 10:1.
[0071] In some embodiments, the molar ratios of the individual triterpenes
to oleandrin
range as follows: about 2-8 (OA) : about 2-8 (UA) : about 0.1-1 (BA) : about
0.5-1.5 (OL); or
about 3-6 (OA) : about 3-6 (UA) : about 0.3-8 (BA) : about 0.7-1.2 (OL); or
about 4-5 (OA) :
about 4-5 (UA) : about 0.4-0.7 (BA) : about 0.9-1.1 (OL); or about 4.6 (OA) :
about 4.4 (UA) :
about 0.6 (BA) : about 1 (OL).
[0072] In some embodiments, the other therapeutic agent, such as that
obtained by extraction
of Nerium sp. or Thevetia sp. plant material, is not a polysaccharide obtained
during preparation
of the extract, meaning it is not an acidic homopolygalacturonan or
arabinogalaturonan. In
some embodiments, the extract excludes another therapeutic agent and/or
excludes an acidic
homopolygalacturonan or arabinogalaturonan obtained during preparation of the
extract.
Date Recue/Date Received 2021-04-13
- 19 -
[0073] In
some embodiments, the other therapeutic agent, such as that obtained by
extraction
of Nerium sp. or Thevetia sp. plant material, is a polysaccharide obtained
during preparation of
the extract, e.g. an acidic homopolygalacturonan or arabinogalaturonan. In
some embodiments,
the extract comprises another therapeutic agent and/or comprises an acidic
homopolygalacturonan or arabinogalaturonan obtained during preparation of the
extract from
said plant material.
[0074] In
some embodiments, the extract comprises oleandrin and at least one other
compound selected from the group consisting of cardiac glycoside, glycone,
aglycone, steroid,
triterpene, polysaccharide, saccharide, alkaloid, fat, protein, neritaloside,
odoroside, oleanolic
acid, ursolic acid, betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-
ene-313,28-diol), 28-
norurs-12-en-3 P-ol, urs-12-en-313-ol, 313,313-hydroxy-12-oleanen-28-oic acid,
313,20a-
dihydroxyurs-21 -en-28-oi c acid, 3 13,27-dihydroxy-12-ursen-28-oi c acid,
313,13 I3-dihydroxyurs-
11 -en-28-oic acid, 3 I3,12a-dihydroxyoleanan-28,1313-olide, 3 I3,27-dihydroxy-
12-oleanan-28-
oic acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, caffeic
acid, L-quinic
acid, 4-coumaroyl-CoA, 3-0-caffeoylquinic acid, 5- 0-caffeoylquinic acid,
cardenolide B-1,
cardenolide B-2, oleagenin, neridiginoside, nerizoside, odoroside-H, 3-beta-0-
(D-diginosyl)-
5-beta, 14 beta -di hydroxy-card-20(22)-en ol i de pectic polysaccharide
composed of gal acturonic
acid, rhamnose, arabinose, xylose, and galactose, polysaccharide with MW in
the range of
17000-120000 D, or MW about 35000 D, about 3000 D, about 5500 D, or about
12000 D,
cardenolide monoglycoside, cardenolide N-1, cardenolide N-2, cardenolide N-3,
cardenolide
N-4, pregnane, 4,6-diene- 3,12,20-trione, 20R-hydroxypregna-4,6-diene-3,12-
dione,
16beta,17b eta-epoxy-12b eta-hydroxypregna-4,6-di ene-3,20-di one,
12beta-hydroxypregna-
4,6,16-triene-3,20-dione (neridienone A), 20S,21-dihydroxypregna-4,6-diene-
3,12-dione
(neridienone B), neriucoumaric acid, isoneriucoumaric acid, oleanderoic acid,
oleanderen,
8alpha-methoxylabdan-18-oic acid, 12-ursene, kaneroside, neriumoside, 313-0-(D-
diginosy1)-
2a- hydroxy-8,1413-epoxy-513-carda-16:17, 20: 22- dienolide, 313-0-(D-
diginosyl)-2u,1413-
dihydroxy-5f3- carda-16:17,20:22-dienolide,
313,27-dihydroxy-urs-18-en-13,28-olide,
313,22a,28-trihydroxy-25-nor-lup-1(10),20(29)-di en-2-one, cis-karenin (313-
hydroxy-28-Z-p-
coumaroyloxy-urs-12-en-27-oic acid), trans-karenin (3-13-hydroxy-28-E-p-
coumaroyloxy-urs-
12-en-27-oic acid), 3beta-hydroxy-5alpha-carda-14(15),20(22)-dienolide
(beta-
Date Recue/Date Received 2021-04-13
- 20 -
anhydroepi di gitoxi genin), 3 beta-0-(D-digitalosyl)-21-hydroxy-5beta-carda-
8,14,16,20(22)-
tetraenolide (neriumogenin-A-3beta-D-digitaloside), proceragenin, neridienone
A, 3beta,27-
dihydroxy-12-ursen-28-oic acid, 3beta,13beta-dihydroxyurs-11-en-28-oic acid,
3beta-
hydroxyurs-12-en-28-aldehyde, 28- orurs-12-en-3beta-ol, urs-12-en-3beta-ol,
urs-12-ene-
3beta,28-diol, 3beta,27-dihydroxy-12-oleanen-28-oic acid, (20S, 24R)-
epoxydammarane-
3beta,25-diol, 20beta,28-epoxy-28a1pha-methoxytaraxasteran-3beta-ol,
20beta,28-
epoxytaraxaster-21-en-3beta-ol, 28-nor-urs-12-ene-3beta,17 b eta-di ol, 3beta-
hydroxyurs-12-
en-28-aldehyde, alpha-neriursate, beta-neriursate, 3alpha-acetophenoxy-urs-12-
en-28-oic acid,
3beta-acetophenoxy-urs-12-en-28-oic acid, oleanderolic acid, kanerodione,
30-P-
hydroxyphenoxy-11a-methoxy-12a-hydroxy-20-ursen-28-oic acid, 28-hydroxy-20(29)-
lupen-
3,7-dione, kanerocin, 3alpha-hydroxy-urs-18,20-dien-28-oic acid, D-sarmentose,
D-diginose,
neridiginoside, nerizoside, isoricinoleic acid,
gentiobiosylnerigoside,
genti obi osylb e aumontosi de, genti obi osylol e andrin,
folinerin, 1213-hydroxy-513-carda-
8,14,16,20(22)-tetraenolide, 813-hydroxy-digitoxigenin, A16-813- hydroxy-
digitoxigenin, A16-
neriagenin, uvaol, ursolic aldehyde, 27(p-coumaroyloxy)ursolic acid,
oleanderol, 16-anhydro-
deacteyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, adigoside,
adynerin, alpha-
amyrin, beta-sitosterol, campestrol, caoutchouc, capric acid, caprylic acid,
choline, cornerin,
cortenerin, deacetyloleandrin, diacetyl-nerigoside, foliandrin,
pseudocuramine, quercetin,
quercetin-3-rhamnoglucoside, quercitrin, rosaginin, rutin, stearic acid,
stigmasterol,
strospeside, urehitoxin, and uzarigenin. Additional components that may be
present in the
extract are disclosed by Gupta et al. (IJPSR (2010(, 1(3), 21-27).
[0075]
Oleandrin may also be obtained from extracts of suspension cultures derived
from
Agrobacterium tumefaciens-transformed calli. Hot water, organic solvent,
aqueous organic
solvent, or supercritical fluid extracts of agrobacterium may be used
according to the invention.
[0076]
Oleandrin may also be obtained from extracts of Nerium oleander microculture
in
vitro, whereby shoot cultures can be initiated from seedlings and/or from
shoot apices of the
Nerium oleander cultivars, e.g. Splendens Giganteum, Revanche or Alsace, or
other cultivars.
Hot water, organic solvent, aqueous organic solvent, or supercritical fluid
extracts of
microcultured Nerium oleander may be used according to the invention.
Date Recue/Date Received 2021-04-13
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[0077] The extract may also be obtained by extraction of cellular mass
(such as is present in
cell culture) of any of said plant species.
[0078] The invention also provides use of a cardiac glycoside in the
manufacture of a
medicament for the treatment of viral infection in a subject. In some
embodiments, the
manufacture of such a medicament comprises: providing one or more antiviral
compounds of
the invention; including a dose of antiviral compound(s) in a pharmaceutical
dosage form; and
packaging the pharmaceutical dosage form. In some embodiments, the manufacture
can be
conducted as described in PCT International Application No. PCT/US06/29061.
The
manufacture can also include one or more additional steps such as: delivering
the packaged
dosage form to a vendor (retailer, wholesaler and/or distributor); selling or
otherwise providing
the packaged dosage form to a subject having a viral infection; including with
the medicament
a label and a package insert, which provides instructions on use, dosing
regimen,
administration, content and toxicology profile of the dosage form. In some
embodiments, the
treatment of viral infection comprises: determining that a subject has a viral
infection;
indicating administration of pharmaceutical dosage form to the subject
according to a dosing
regimen; administering to the subject one or more pharmaceutical dosage forms,
wherein the
one or more pharmaceutical dosage forms is administered according to the
dosing regimen.
[0079] The pharmaceutical composition can further comprise a combination of
at least one
material selected from the group consisting of a water soluble (miscible) co-
solvent, a water
insoluble (immiscible) co-solvent, a surfactant, an antioxidant, a chelating
agent, and an
absorption enhancer.
[0080] The solubilizer is at least a single surfactant, but it can also be
a combination of
materials such as a combination of: a) surfactant and water miscible solvent;
b) surfactant and
water immiscible solvent; c) surfactant, antioxidant; d) surfactant,
antioxidant, and water
miscible solvent; e) surfactant, antioxidant, and water immiscible solvent; f)
surfactant, water
miscible solvent, and water immiscible solvent; or g) surfactant, antioxidant,
water miscible
solvent, and water immiscible solvent.
[0081] The pharmaceutical composition optionally further comprises a) at
least one liquid
carrier; b) at least one emulsifying agent; c) at least one solubilizing
agent; d) at least one
dispersing agent; e) at least one other excipient; or f) a combination
thereof.
Date Recue/Date Received 2021-04-13
- 22 -
[0082] In some embodiments, the water miscible solvent is low molecular
weight (less than
6000) PEG, glycol, or alcohol. In some embodiments, the surfactant is a
pegylated surfactant,
meaning a surfactant comprising a poly(ethylene glycol) functional group.
[0083] The invention includes all combinations of the aspects, embodiments
and
sub-embodiments of the invention disclosed herein.
BRIEF DESCRIPTION OF THE FIGURES
[0084] The following figures form part of the present description and
describe exemplary
embodiments of the claimed invention. The skilled artisan will, in light of
these figures and the
description herein, be able to practice the invention without undue
experimentation.
[0085] FIGS. 1-2 depict charts summarizing the in vitro dose response
antiviral activity of
various compositions against Ebolavirus.
[0086] FIGS. 3-4 depict charts summarizing the in vitro dose response
antiviral activity of
various compositions against Marburgvirus.
[0087] FIG. 5 depicts a chart summarizing the in vitro dose response
antiviral activity of
oleandrin against Zikavirus (SIKV strain PRVABC59) in Vero E6 cells.
[0088] FIG. 6 depicts a chart summarizing the in vitro dose response
antiviral activity of
digoxin against Zikavirus (SIKV strain PRVABC59) in Vero E6 cells.
[0089] FIG. 7 depicts a chart summarizing the in vitro dose response
antiviral activity of
various compositions (oleandrin, digoxin and PBI-05204) against Ebolavirus in
Vero E6 cells.
[0090] FIG. 8 depicts a chart summarizing the in vitro dose response
antiviral activity of
various compositions (oleandrin, digoxin and PBI-05204) against Marburgvirus
in Vero E6
cells.
[0091] FIG. 9 depicts a chart summarizing the in vitro cellular viability
of Vero E6 cells in
the presence of various compositions (oleandrin, digoxin and PBI-05204).
[0092] FIGS. 10A and 10B depict charts summarizing the ability of
compositions (oleandrin
and PBI-05204) to inhibit Ebolavirus in Vero E6 cells shortly after exposure
to virus: FIG. 10A-
2 hr post-infection; FIG. 10B- 24 hr post-infection.
Date Recue/Date Received 2021-04-13
- 23 -
[0093] FIGS. 11A and 11B depict charts summarizing the ability of
compositions (oleandrin
and PBI-05204) to inhibit Marburgvirus in Vero E6 cells shortly after exposure
to virus: FIG.
11A- 2 hr post-infection; FIG. 11B- 24 hr post-infection.
[0094] FIGS. 12A and 12B depict charts summarizing the ability of
compositions (oleandrin
and PBI-05204) to inhibit the product of infectious progeny by virally
infected Vero E6 cells
having been exposed to oleandrin: FIG. 12A- Ebolavirus; FIG. 12B-
Marburgvirus.
[0095] FIGS. 13A and 13B depict charts summarizing the in vitro dose
response antiviral
activity of various compositions (oleandrin, digoxin and PBI-05204) against
Venezuelen
Equine Encephalomyelits virus (FIG. 13A) and Western Equine Encephalomyelitis
virus (FIG.
13B) in Vero E6 cells.
[0096] FIG. 14 depicts a chart summarizing the effect that vehicle control,
oleandrin, or
extract of N. oleander have upon HTLV-1 replication or the release of newly-
synthesized virus
particles as determined by quantitation of HTLV-1 pl9Gag (see Examples 19 and
20). Untreated
(UT) cells are shown for comparison. All the data is representative of at
least three independent
experiments. The data represent the mean of the experiments standard
deviation (error bars).
[0097] FIG. 15 depicts a chart summarizing the relative cytotoxicity of the
Vehicle control,
oleandrin, and N. oleander extract against the HTLV-1+ SLB1 lymphoma T-cell-
line. All the
data is representative of at least three independent experiments. The data
represent the mean
of the experiments standard deviation (error bars).
[0098] FIGS. 16A-16F depict representative micrographs of the Annexin V-
FITC (green)
and PI (red)-staining results with DIC phase-contrast in the merged images are
shown. The
individual Annexin V-FITC and PI fluorescent channel images are also provided.
Scale bar, 20
[0099] FIG. 17 depicts a chart summarizing the effect that vehicle control,
oleandrin, or
extract of N. oleander have upon HTLV-1 replication or the release of newly-
synthesized virus
particles from oleandrin-treated HTLV-1+ lymphoma T-cells.
[00100] FIG. 18 depicts a chart summarizing the relative cytotoxicity of
vehicle control,
oleandrin, or extract of N. oleander upon treated huPBMCs.
1001011 FIG. 19 depicts a chart summarizing the relative inhibition of HTLV-1
transmision
in co-culture assays huPBMCs containing vehicle control, oleandrin, or extract
of N. oleander.
Date Recue/Date Received 2021-04-13
- 24 -
[00102] FIG. 20 depicts representative micrographs of a GFP-expressing HTLV-1+
SLB1 T-
cell-line: fluorescence-microscopy (top panels) and immunoblotting (lower
panels).
[00103] FIG. 21 depicts representative micrographs of virological synapses
between
huPBMCs and the mitomycin C-treated HTLV-1+ SLB1/pLenti-GFP lymphoblasts
(green
cells).
[00104] FIG. 22 depicts a chart of the averaged data with standard deviation
(error bars) from
quantitation of the micrographs of FIG. 21.
[00105] FIGS. 23A-23D depict charts of log of SARS-CoV-2 viral titer (PFU/mL)
versus
time (h) for VERO E6 cells infected with SARS-CoV-2 virus treated with
oleandrin (red bars)
or control vehicle (incubation medium) (black bars) at 24 hours and 48 hours
after "treatment"
(Example 28). Cells were pretreated with oleandrin prior to infection. After
an initial 2 h
incubation post infection, the infected cells were washed to remove
extracellular virus and
oleandrin. Then, the recovered infected cells were treated as follows. The
infected cells were
treated with oleandrin (FIG. 23A: 1 microg/mL in 0.1% aqueous DMSO with RPMI
1640
culture medium as the aqueous component; FIG. 23C: 0.1 microg/mL in 0.01%
aqueous DMSO
with RPMI 1640) or just control vehicle (FIG. 23B: 0.1% aqueous DMSO with RPMI
1640;
FIG. 23D: 0.01% aqueous DMSO with RPMI 1640), and the viral titer was
measured.
[00106] FIG. 24A depicts a dual-y-axis chart of percent inhibition of viral
replication (Y1,
left axis) and Vero-E6 cell count (Y2, right axis: an expression of potential
cellular toxicity of
oleandrin against said cells) versus concentration of oleandrin (microg/mL) in
the culture
medium at 24 h post-infection (Example 29). FIG. 24B is for the cultures of
FIG. 24A but taken
at 48 h post-infection.
[00107] FIG. 25 depicts a chart of percent of Vero-E6 cells (cell titer)
versus concentration
of oleandrin (microg/mL) in the culture medium at 24 h after continuous
exposure of the cells
to the indicated concentrations of oleandrin (Example 30).
[00108] FIGS. 26A-26B depict charts of log of SARS-CoV-2 viral titer (PFU/mL)
versus
concentration of oleandrin in the culture medium for VERO CCL-81 cells
(ceropithecus
aethiops kidney normal cells; https://www.atcc.org/products/all/CCL-81.aspx)
infected with
SARs-CoV-2 virus and then treated with oleandrin (blue circles) or control
vehicle (incubation
Date Recue/Date Received 2021-04-13
- 25 -
medium) (red squares) at 24 hours (FIG. 26A) and 48 hours (FIG. 26B) after
"treatment"
(Example 31).
[00109] For the samples of FIGS. 26A and 26B, the fold reduction in viral
titer was
determined at 24 hours (FIG. 26C) and 48 hours (FIG. 26D).
[00110] FIGS. 27A-27D depict charts of log of SARS-CoV-2 viral titer (PFU/mL)
versus
time (h) for VERO E6 cells infected with SARS-CoV-2 virus treated with
oleandrin (blue
circles) or control vehicle (incubation medium) (red squares) at 24 hours and
48 hours after
"treatment" (Example 28). Cells were pretreated with oleandrin prior to
infection. After an
initial 2 h incubation post infection, the infected cells were washed to
remove extracellular virus
and oleandrin. Then, the recovered infected cells were treated as follows. The
infected cells
were treated with oleandrin (FIG. 27A: 0.005 microg/mL in aqueous DMSO
(0.005%) with
RPMI 1640 culture medium as the aqueous component; FIG. 27B: 0.01 microg/mL in
aqueous
DMSO (0.01%) with RPMI 1640; FIG. 27C: 0.05 microg/mL in aqueous DMSO (0.05%)
with
RPMI 1640; FIG. 27B: 0.1 microg/mL in aqueous DMSO (0.1%) with RPMI 1640), and
the
viral titer was measured.
[00111] FIGS. 28A and 28B depict charts of log of SARS-CoV-2 viral titer
(PFU/mL) versus
concentration of oleandrin in the culture medium for VERO 81 cells infected
with SARS-CoV-
2 virus and then treated with oleandrin (dark blue circles (Exp. 2) and light
blue circles (Exp.
3)) or control vehicle (incubation medium) (dark red squares (Exp. 2) and
light red squares
(Exp. 3)) at 24 hours (FIG. 28A) and 48 hours (FIG. 28B) after "treatment".
Exp. 2 and Exp.
3 are merely duplicate runs of the assay.
[00112] FIGS. 29A and 29B depict bar graphs of the viral titer versus
oleandrin concentration
in the culture medium, wherein the viral titer was measured at 24 h (FIG. 29A)
and at 48 h
(FIG. 29B) post-infection. For some samples, cells were treated, before and
after (2 h)
infection, with oleandrin (solid blue bars) or just DMSO control vehicle
(solid red bars). For
other samples, cells were treated with oleandrin (hashed blue bars: 12 h post-
infection;
hollowed blue bars: 24 h post-infection) or just DMSO control vehicle (hashed
red bars: 12 h
post infection; hollowed red bars: 24 h post infection).
[00113] FIGS. 30A and 30B depict charts for evaluation of the anti-COVID-19
activity of the
dual extract combination composition (PBI-A). For FIG. 30B, the tg/m1
(oleandrin
Date Recue/Date Received 2021-04-13
- 26 -
concentration) designation assumes that the PBI-A was supplied as 1mg/m1
(oleandrin
concentration) solution. The viral titer (Logio (PFU/mL)) versus Logio
dilution factor (FIG.
30A) or versus Logio concentration of oleandrin (FIG. 30B) was determined.
FIG. 30A is for
the data treatment pre-infection assay of Example 31, and FIG. 30B is for the
treatment post-
infection assay of Example 34.
DETAILED DESCRIPTION OF THE INVENTION
[00114] The invention provides a method of treating viral infection in a
subject by chronic or
acute administration of one or more effective doses of antiviral composition
(or pharmaceutical
composition comprising the antiviral composition and at least one
pharmaceutical excipient) to
the subject. The composition is administered according to a dosing regimen
best suited for the
subject, the suitability of the dose and dosing regimen to be determined
clinically according to
conventional clinical practices and clinical treatment endpoints for viral
infection.
[00115] As used herein, the term "subject" is taken to mean warm blooded
animals such as
mammals, for example, cats, dogs, mice, guinea pigs, horses, bovine cows,
sheep, and humans.
[00116] As used herein, a subject at risk of viral infection is: a) a subject
living in a
geographical area within which mosquitos, in particular Aedes species (Aedes
egypti, Aedes
albopictus) mosquitos, live; b) a subject living with or near a person or
people having viral
infection; c) a subject having sexual relations with a person having a viral
infection; d) a subject
living in a geographical area within which ticks, in particular Ixodes species
(Ixodes marx,
Ixodes scapularis, or Ixodes cooke species) ticks, live; e) a subject living
in a geographical area
within which fruit bats live; f) subjects living in a tropical region; g)
subjects living in Africa;
h) subjects in contact with bodily fluids of other subjects having a viral
infection; i) a child; or
j) a subject with a weakened immune system. In some embodiments, the subject
is a female, a
female capable of getting pregnant, or a pregnant female.
[00117] A subject treated according to the invention will exhibit a
therapeutic response. By
"therapeutic response" is meant that a subject suffering from the viral
infection will enjoy at
least one of the following clinical benefits as a result of treatment with a
cardiac glycoside:
reduction of the active viral titre in the subject's blood or plasma,
eradication of active virus
from the subject's blood or plasma, amelioration of the infection, reduction
in the occurrence
Date Recue/Date Received 2021-04-13
- 27 -
of symptoms associated with the infection, partial or full remission of the
infection or increased
time to progression of the infection, and/or reduction in the infectivity of
the virus causing said
viral infection. The therapeutic response can be a full or partial therapeutic
response.
[00118] As used herein, "time to progression" is the period, length or
duration of time after
viral infection is diagnosed (or treated) until the infection begins to
worsen. It is the period of
time during which the level of infection is maintained without further
progression of the
infection, and the period of time ends when the infection begins to progress
again. Progression
of a disease is determined by "staging" a subject suffering from the infection
prior to or at
initiation of therapy. For example, the subject's health is determined prior
to or at initiation of
therapy. The subject is then treated with antiviral composition, and the viral
titer is monitored
periodically. At some later point in time, the symptoms of the infection may
worsen, thus
marking progression of the infection and the end of the "time to progression".
The period of
time during which the infection did not progress or during which the level or
severity of the
infection did not worsen is the "time to progression".
[00119] A dosing regimen includes a therapeutically relevant dose (or
effective dose) of one
or more cardiac glycosides, and/or triterpene(s), administered according to a
dosing schedule.
A therapeutically relevant dose, therefore, is a therapeutic dose at which a
therapeutic response
of the viral infection to treatment with antiviral composition is observed and
at which a subject
can be administered the antiviral composition without an excessive amount of
unwanted or
deleterious side effects. A therapeutically relevant dose is non-lethal to a
subject, even though
it may cause some side effects in the patient. It is a dose at which the level
of clinical benefit
to a subject being administered the antiviral composition exceeds the level of
deleterious side
effects experienced by the subject due to administration of the antiviral
composition or
component(s) thereof. A therapeutically relevant dose will vary from subject
to subject
according to a variety of established pharmacologic, pharmacodynamic and
pharmacokinetic
principles. However, a therapeutically relevant dose (relative, for example,
to oleandrin) will
typically be about about 25 micrograms, about 100 micrograms, about 250
micrograms, about
500 micrograms or about 750 micrograms of cardiac glycoside/day or it can be
in the range of
about 25-750 micrograms of cardiac glycoside per dose, or might not exceed
about 25
micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms
or about
Date Recue/Date Received 2021-04-13
- 28 -
750 micrograms of cardiac glycoside/day. Another example of a therapeutically
relevant dose
(relative, for example, to triterpene either individually or together) will
typically be in the range
of about 0.1 micrograms to 100 micrograms, about 0.1 mg to about 500 mg, about
100 to about
1000 mg per kg of body weight, about 15 to about 25 mg/kg, about 25 to about
50 mg/kg, about
50 to about 100 mg/kg, about 100 to about 200 mg/kg, about 200 to about 500
mg/kg, about 10
to about 750 mg/kg, about 16 to about 640 mg/kg, about 15 to about 750 mg/kg,
about 15 to
about 700 mg/kg, or about 15 to about 650 mg/kg of body weight. It is known in
the art that
the actual amount of antiviral composition required to provide a target
therapeutic result in a
subject may vary from subject to subject according to the basic principles of
pharmacy.
[00120] Treatment with digoxin can be conducted using two or more dosing
phases: loading
phase and maintenance phase. The loading phase can employ the following dosing
regimen
until steady state plasma levels of digoxin are achieved, and the maintenance
phase can employ
the following dosing regimen after completion of the loading phase.
Human age Oral Loading phase Oral maintenance phase dose, mcg/kg/day
dose, mcg/kg/day
Premature 20 to 30 or 15-25 4.7 to 7.8 2.3 to 3.9 Twice Daily
Full-Term 25 to 35 or 20-30 7.5 to 11.3 3.8 to 5.6 Twice Daily
1 to 24 35 to 60 or 30-50 11.3 to 18.8 5.6 to 9.4 Twice Daily
months
2 to 5 years 30 to 45 or 25-35 9.4 to 13.1 4.7 to 6.6 Twice Daily
to 10 years 20 to 35 or 15-30 5.6 to 11.3 2.8 to 5.6 Twice
Daily
Over 10 years 10 to 15 or 8-12 3.0 to 4.5 or 3.0 to 4.5 Once Daily
2.4 to 3.6 or
3.4 to 5.1
[00121] A therapeutically relevant dose can be administered according to any
dosing regimen
typically used in the treatment of viral infection. A therapeutically relevant
dose can be
administered once, twice, thrice or more daily. It can be administered every
other day, every
third day, every fourth day, every fifth day, semiweekly, weekly, biweekly,
every three weeks,
every four weeks, monthly, bimonthly, semimonthly, every three months, every
four months,
Date Recue/Date Received 2021-04-13
- 29 -
semiannually, annually, or according to a combination of any of the above to
arrive at a suitable
dosing schedule. For example, a therapeutically relevant dose can be
administered one or more
times daily (up to 10 times daily for the highest dose) for one or more weeks.
[00122] Example 15 provides a detailed description of an in vitro assay used
to evaluate the
efficacy of compositions containing oleandrin (as sole active), AnvirzelTM
(hot water extract of
Nerium oleander) and PHI-05204 (supercritical fluid (SCF) extract of Nerium
oleander) for the
treatment of Ebolavirus (FIGS. 1-2) and Marburgvirus (FIGS. 3-4) infection,
both of which are
Filoviruses.
[00123] The hot-water extract can be administered orally, sublingually,
subcutaneously, and
intramuscularly. One embodiment is available under the tradename ANVIRZELTM
(Nerium
Biotechnology, Inc., San Antonio, TX; Salud Integral Medical Clinic,
Tegucigalpa, Honduras;
www.saludintegral.com; www.anvirzel.com) as a liquid dosage form. For
sublingual
administration, a typical dosing regimen is 1.5 ml per day or three doses of
0.5 ml in one day.
For administration by injection, a typical dosing regimen is about 1 to about
2 ml/day, or about
0.1 to about 0.4 ml/m2/day for about 1 week to about 6 months or longer, or
about 0.4 to about
0.8 ml/m2/day for about 1 week to about 6 months or longer, or about 0.8 to
about 1.2 ml/m2/day
for about 1 week to about 6 months or longer. Higher dosing can be used
because the maximum
tolerated dose of ANVIRZELTM is much higher. ANVIRZELTM comprises oleandrin,
oleandrigenin, polysaccharides extracted (hot water extraction) from Nerium
oleander.
Commercially available vials comprise about 150 mg of oleander extract as a
freeze-dried
powder (prior to reconstitution with water before administration) which
comprises about 200
to about 900 microg of oleandrin, about 500 to about 700 microg of
oleandrigenin, and
polysaccharides extracted from Nerium oleander. Said vials may also include
pharmaceutical
excipients such as at least one osmotic agent, e.g. mannitol, sodium chloride,
at least one
buffering agent, e.g. sodium ascorbate with ascorbic acid, at least one
preservative, e.g.
propylparaben, methylparaben.
[00124] The experiments were set up by adding the compositions to cells at 40
microg/mL,
then adding virus and incubating for lhr. Upon addition of the virus to the
cells, the final
concentration of the compositions is 20 microg/mL. Compositions containing
different
amounts of oleandrin can be adjusted according to the concentration of
oleandrin they contain
Date Recue/Date Received 2021-04-13
- 30 -
and converted that to molarity. FIGS. 1-4 depict the efficacy based on the
oleandrin content of
the extracts. OL on its own is efficacious. PBI-05204, the SCF extract of
Nerium oleander
comprising OL, OA, UA and BA, is substantially more efficacious than OL on its
own.
AnvirzelTM, the hot water extract of Nerium oleander, is more efficacious than
OL on its own.
Both extracts clearly exhibit efficacy in the nanomolar range. The percentage
of oleandrin in
the PBI-05204 extract (1.74%) is higher than in AnvirzelTM (0.459%, 4.59
microg/mg). At the
highest dose of PBI-05204, it completely inhibited EBOV and MARV infection,
whereas
AnvirzelTM did not exhibit complete inhibition, because at a dose higher than
20 microg/mL
with AnvirzelTM, toxicity is observed. The data demonstrate highest antiviral
activity against
Ebolavirus and Marburgvirus for PBI-05204. The combination of triterpenes in
PBI-05204
increased the antiviral activity of oleandrin.
[00125] Example 6 provides a detailed description of an in vitro assay used to
evaluate the
efficacy of the cardiac glycosides for the treatment of Zikavirus (a
flavivirus) infection. Vero
E6 cells were infected with Zika virus (ZIKV strain PRVABC59) at an MOI of 0.2
in the
presence of oleandrin (FIG. 5) or digoxin (FIG. 6). The cells were incubated
with virus and the
cardiac glycoside for 1 hr, after which the inoculum and non-absorbed cardiac
glycoside (if
any) was removed. The cells were immersed in fresh medium and incubated for 48
hr, after
which they were fixed with formalin and stained for ZIKV infection. The data
demonstrate
antiviral activity against Zikavirus for both cardiac glycosides; however,
oleandrin exhibited
higher (almost 8-fold greater) antiviral activity than digoxin.
[00126] Example 14 provides a detailed description of an assay used to
evaluate the antiviral
activity of test compositions against Zikavirus and Dengue virus. The data
indicate that
oleandrin demonstrates efficacy against Zikavirus and Dengue virus.
[00127] FIG. 7 a chart summarizing the in vitro dose response antiviral
activity of various
compositions (oleandrin, digoxin and PBI-05204) against Ebolavirus (EBOV) in
Vero E6 cells.
FIG. 8 depicts a chart summarizing the in vitro dose response antiviral
activity of various
compositions (oleandrin, digoxin and PBI-05204) against Marburgvirus (MARV) in
Vero E6
cells. FIG. 9 depicts a chart summarizing the in vitro cellular viability of
Vero E6 cells in the
presence of various compositions (oleandrin, digoxin and PBI-05204). For FIGS.
7-8, the host
cells were exposed to the compositions prior to infection with virus. Vero E6
cells were infected
Date Recue/Date Received 2021-04-13
-31 -
with EBOV/Kik (FIG. 7, M01=1) or MARV/Ci67 (FIG. 8, MOI=1) in the presence of
oleandrin, digoxin or PBI-05204, an oleandrin-containing plant extract. After
lhr, inoculum
and compounds were removed and fresh medium added to cells. 48hr later, cells
were fixed and
immunostained to detect cells infected with EBOV or MARV. Infected cells were
enumerated
using an Operetta.
[00128] In order to ensure that false positives, in terms of antiviral
activity, were not being
observed, cellular viability in the presence of the compositions was tested.
For the data in FIG.
9, Vero E6 cells were treated with compound as above. ATP levels were measured
by CellTiter-
Glo as a measurement of cell viability. It was determined that oleandrin,
digoxin, and PBI-
05204 did not reduce cellular viability, meaning that the antiviral activity
detailed in other
figures herein is not due to false positives caused by cellular toxicity of
the individual
compounds.
[00129] Accordingly, the invention provides a method of treating viral
infection in a mammal
or host cell, the method comprising: administering an antiviral composition to
the mammal or
host cell prior to contraction of said viral infection, whereby upon viral
infection of said
mammal or host cell, the antiviral composition reduces the viral titre and
ameliorates, reduces
or eliminates the viral infection.
[00130] The antiviral composition and method of the invention are also useful
in treating viral
infection that has occurred prior to administration of the antiviral
composition. Vero E6 cells
were infected with EBOV (FIGS. 10A, 10B) or MARV (FIGS. 11A, 11B). At 2hr post-
infection (FIGS. 10A, 11A) or 24hr post-infection (FIGS. 10B, 11B), oleandrin
or PBI-05204
was added to cells for lhr, then discarded and cells were returned to culture
medium.
[00131] FIGS. 10A and 10B depict charts summarizing the ability of
compositions (oleandrin
and PBI-05204) to inhibit Ebolavirus in Vero E6 cells shortly after exposure
to virus: FIG. 10A-
2 hr post-infection; FIG. 10B- 24 hr post-infection. When the antiviral
composition is
administered within two hours (or within up to 12 hours) after viral
infection, the viral titre
antiviral composition provides effective treatment and reduces the EBOV viral
titre. Even after
24 hours, the viral composition is effective; however, its efficacy is lower
as time after initial
viral infection increases. The same evaluations were conducted on MARV. FIGS.
11A and
11B depict charts summarizing the ability of compositions (oleandrin and PBI-
05204) to inhibit
Date Recue/Date Received 2021-04-13
- 32 -
Marburgvirus in Vero E6 cells shortly after exposure to virus: FIG. 11A- 2 hr
post-infection;
FIG. 11B- 24 hr post-infection. When the antiviral composition is administered
within two
hours (or within up to 12 hours) after viral infection, the viral titre
antiviral composition
provides effective treatment and reduces the MARV viral titre. Even after 24
hours, the viral
composition is effective; however, its efficacy is lower as time after initial
viral infection
increases.
[00132] Given that the antiviral activity of the composition herein is reduced
for a single
generation of virus-infected cells, e.g. within 24 hours post-infection, we
evaluated whether the
antiviral composition is capable of inhibiting viral propagation, meaning
inhibiting production
of infectious progeny. Vero E6 cells were infected with EBOV or MARV in the
presence of
oleandrin or PBI-05204 and incubated for 48hr. Supernatants from infected cell
cultures were
passaged onto fresh Vero E6 cells, incubated for lhr, then discarded. Cells
containing passaged
supernatant were incubated for 48hr. Cells infected with EBOV (B) or MARV (C)
were
evaluated as described herein. Control infection rates were 66% for EBOV and
67% for
MARV. The antiviral composition of the invention inhibited production of
infectious progeny.
[00133] Accordingly, the antiviral composition of the invention: a) can be
administered
prophylactically before viral infection to inhibit viral infection after
exposure to virus; b) can
be administered after viral infection to inhibit or reduce viral replication
and production of
infectious progeny; or c) a combination of a) and b).
[00134] Antiviral activity of the antiviral composition against Togaviridae
alphavirus was
evaluated using VEE virus and WEE virus in Vero E6 cells. FIGS. 13A and 13B
depict charts
summarizing the in vitro dose response antiviral activity of various
compositions (oleandrin,
digoxin and PBI-05204) against Venezuelan Equine Encephalomyelitis virus (FIG.
13A) and
Western Equine Encephalomyelitis virus (FIG. 13B) in Vero E6 cells. Vero E6
cells were
infected with Venezuelan equine encephalitis virus (FIG. 13A, MOI=0.01) or
Western equine
encephalitis virus (FIG. 13B, MOI=0.1) for 18hr in the presence or absence of
indicated
compounds. Infected cells were detected as before and enumerated on an
Operetta. The
antiviral composition of the invention was found to be efficacious.
[00135] Accordingly, the invention provides a method of treating a viral
infection, caused by
a Arenaviridae family virus, Filoviridae family virus, Flaviviridae family
virus (Flavivirus
Date Recue/Date Received 2021-04-13
- 33 -
genus), Retroviridae family virus, Deltaretrovirus genus virus, Coronaviridae
family virus,
Paramyxoviridae family virus, or Togaviridae family virus, in a subject or
host cell, the method
comprising administering an effective amount of the antiviral composition,
thereby exposing
the virus to the antiviral composition and treating said viral infection.
[00136] We evaluated use of oleandrin and the extract described herein for the
treatment of
HTLV-1 (human T-cell leukemia virus type-1; an enveloped retrovirus;
Deltaretrovirus genus)
infection. To determine whether the purified oleandrin compound, or an extract
of N. oleander,
could inhibit HTLV-1 proviral replication and/or the production and release of
p 19Gag-
containing virus particles, the virus-producing HTLV-1-transformed SLB1
lymphoma T-cell-
line was treated with increasing concentrations of oleandrin or a N. oleander
extract, or the
sterile vehicle control (20% DMSO in MilliQ-treated ddH20) and then incubated
for 72 hrs at
37 C under 10% CO2. The cells were later pelleted by centrifugation and the
relative levels of
extracellular p19Gag-containing virus particles released into the culture
supernatants were
quantified by performing Anti-HTLV-1 pl9G1g ELISAs (Zeptometrix).
[00137] FIG. 14 depicts data for quantitation of HTLV-1 pl9Gag expressed by
HTLV-1+
SLB1 lymphoma T-cell-line treated for 72 hrs with the vehicle control (1.5 1,
7.5 1, or 15 IA),
or increasing concentrations (10 tg/m1, 50 tg/ml, and 100 tg/m1) of the
oleandrin compound
or an extract of N. oleander (Example 19 and 20). Viral replication and the
release of
extracellular particles into the culture supernatants were quantified by
performing Anti-HTLV-
1 pl9G1g ELISAs (Zeptometrix). Oleandrin does not significantly inhibit HTLV-1
replication
or the release of newly-synthesized virus particles. We determined that
neither the extract nor
oleandrin alone significantly inhibit viral replication or the release of
p19Gag-containing
particles into the supernatants of the cultures. We, thus, expected no further
antiviral activity;
however, we unexpectedly found that the collected virus particles from treated
cells exhibited
reduced infectivity on primary human peripheral blood mononuclear cells
(huPBMCs). Unlike
HIV-1, extracellular HTLV-1 particles are poorly infectious and viral
transmission typically
occurs via direct intercellular interactions across a virological synapse.
[00138] The invention thus provides a method of producing HTLV-1 virus
particles with
reduced infectivity, the method comprising treating HTLV-1 virus particles
with the antiviral
composition of the invention to provide said HTLV-1 virus particles with
reduced infectivity.
Date Recue/Date Received 2021-04-13
- 34 -
[00139] To ensure that the antiviral activity observed was not an artifact due
to potential
cytotoxicity of the antiviral composition to HTLV-1+ SLB1 lymphoblast, we then
assessed the
cytotoxicity of the different dilutions of the purified oleandrin compound and
N. oleander
extract in treated HTLV-1+ SLB1 lymphoblast cultures (Example 21). SLB1 T-
cells were
treated with increasing concentrations (10, 50, and 100 g/m1) of oleandrin or
a N. oleander
extract for 72 hrs as described herein. As a negative control, the cells were
also treated with
increasing amounts (1.5, 7.5, and 15 IA) of the vehicle solution which
corresponded to the
volumes used in the drug-treated cultures. Cyclophosphamide (501,1M; Sigma-
Aldrich)-treated
cells were included as a positive control for apoptosis. Then, the samples
were washed and
stained with Annexin V-FITC and propidium iodide (PI) and analyzed by confocal
fluorescence-microscopy. The relative percentages of Annexin V-FITC and/or PI-
positive
cells were quantified by fluorescence-microscopy and counting triplicate
visual fields using a
20x objective lens.
[00140] The results (FIG. 15 and FIGS. 16A-16F) indicate that the lowest
concentration (10
g/m1) of oleandrin and the N. oleander extract did not induce significant
cytotoxicity/apoptosis. However, the higher concentrations (about 50 and about
100 g/m1) of
the crude phytoextract induced notably more apoptosis than did the oleandrin
compound. This
is consistent with the fact that oleandrin represents about 1.23% of the N.
oleander extract. The
cytotoxicity caused by oleandrin was not significantly higher than the Vehicle
control in treated
HTLV-1+ SLB1 cells.
[00141] We then investigated whether oleandrin or a N. oleander extract could
inhibit virus
transmission from a Green Fluorescent Protein (GFP)-expressing HTLV-1+
lymphoma T-cell-
line to huPBMCs in co-culture assays (Example 20). For these studies, HTLV-1+
SLB1
lymphoma T-cells were treated with increasing concentrations of either the
oleandrin
compound or N oleander extract, or the Vehicle control for 72 hrs in 96-well
microtiter plates,
and then the virus-containing supernatants were collected and used to directly
infect primary
cultured, human peripheral blood mononuclear cells (huPBMCs) in vitro.
Following 72 hrs, the
relative levels of extracellular p19Gag-containing virus particles released
into the culture
supernatants, as a result of direct infection, were quantified by performing
Anti-HTLV-1 pl9Gag
ELISAs.
Date Recue/Date Received 2021-04-13
- 35 -
[00142] The HTLV-1+ SLB1 lymphoma T-cell-line was treated with the Vehicle
control, or
increasing concentrations (10 m/ml, 50 m/ml, and 100 m/m1) of the N. oleander
extract or
oleandrin compound for 72 hrs and then the virus-containing supernatents were
collected and
used to directly infect primary huPBMCs. The vehicle control, N. oleander
extract, or oleandrin
were also included in the culture media for the huPBMCs. After 72 hrs, the
culture supernatants
were collected and the relative amounts of extracellular virus particles
produced were quantified
by performing Anti-HTLV-1 pl9G1g ELISAs.
[00143] The data (FIG. 17) indicate that the even lowest concentration (10
tg/m1) of both
oleandrin and the N. oleander extract inhibited the infectivity of newly-
synthesized p 1 9Gag-
containing virus particles released into the culture supernatants of treated
cells, relative to a
comparable amount of the vehicle control. Both oleandrin and the crude extract
inhibited the
formation of virological synapses and the transmission of HTLV-1 in vitro.
Extracellular virus
particles produced by oleandrin-treated HTLV-1+ lymphoma T-cells exhibit
reduced infectivity
on primary huPBMCs. Importantly, oleandrin exhibits antiviral activity against
enveloped
viruses by reducing the incorporation of the envelope glycoprotein into mature
particles, which
represents a unique stage of the retroviral infection cycle.
1001441 To ensure that the antiviral activity observed was not an artifact due
to potential
cytotoxicity of the antiviral composition to treated huPBMCs, we also
investigated (Example
21) the cytotoxicity of purified oleandrin and the N. oleander extract,
compared to the vehicle
negative control, in treated huPBMCs. Primary buffy-coat huPBMCs were isolated
and
stimulated with phytohemagglutinin (PHA) and cultured in the presence of
recombinant human
interleukin-2 (hIL-2). The cells were then treated for 72 hrs with increasing
concentrations of
oleandrin or a N. oleander extract, or with increasing volumes of the Vehicle.
The samples were
subsequently stained with Annexin V-FITC and PI and the relative percentages
of apoptotic
(i.e., Annexin V-FITC and/or PI-positive) cells per field were quantified by
confocal
fluorescence-microscopy and counting in-triplicate.
[00145] Cytotoxic effects of the Vehicle control, N. oleander extract, and the
oleandrin
compound were assessed by treating primary huPBMCs for 72 hrs, and then the
cultures were
stained with Annexin V-FITC and PI. The relative percentages of apoptotic
(i.e., Annexin V-
FITC and/or PI-positive) cells were quantified by fluorescence-microscopy and
counting
Date Recue/Date Received 2021-04-13
- 36 -
triplicate visual fields using a 20x objective lens. The total numbers of
cells were determined
using DIC phase-contrast microscopy. Cyclophosphamide (50 04)-treated cells
were included
as a positive control for apoptosis. NA indicates the number of cells in this
sample was too low
for accurate assessment due to higher toxicity.
[00146] The data (FIG. 18) indicate oleandrin exhibited moderate cytotoxicity
(e.g., 35-37%
at the lowest concentration) in huPBMCs as compared to the vehicle control. By
contrast, the
N. oleander extract was significantly cytotoxic and induced high levels of
programmed cell-
death even at the lowest concentration. The huPBMCs were somewhat more
sensitive to
purified oleandrin than the HTLV-1+ SLB1 lymphoblasts; however, the huPBMCs
were
drastically more sensitive to the crude N. oleander extract which also
contains other cytotoxic
compounds such as the triterpenes described herein.
[00147] We also investigated (Example 22) whether oleandrin or the N. oleander
extract
could interfere with the transmission of HTLV-1 particles to target huPBMCs in
co-culture
experiments. For these studies, the virus-producing HTLV-1+ SLB1 T-cell-line
was treated
with mitomycin C and then with increasing amounts of oleandrin, N. oleander
extract, or the
Vehicle control for either 15 min or 3 hrs. The SLB1 cells were washed 2X with
serum-free
medium and equivalent numbers of huPBMCs were then added to each well, and the
samples
were co-cultured for 72 hrs in complete medium at 37 C under 10% CO2 in a
humidified
incubator. The relative intercellular transmission of HTLV-1 was assessed by
performing Anti-
HTLV-1 pl9G1g ELISAs to measure the levels of extracellular virus released
into the culture
supernatants.
[00148] Primary huPBMCs were co-cultured with mitomycin C-treated HTLV-1+ SLB1
lymphoma T-cells which were pre-treated for either 15 min or 3 hrs with the
Vehicle control,
or increasing concentrations (10 tg/ml, 50 tg/ml, and 100 tg/m1) of the N.
oleander extract or
oleandrin compound. The vehicle control, extract, and compound were also
present in the co-
culture media. After 72 hrs, the supernatants were collected, and the amounts
of extracellular
virus particles released were quantified by performing Anti-HTLV-1 pl9Gag
ELISAs.
[00149] The results depicted in FIG. 19 demonstrate that both oleandrin and
the N. oleander
extract inhibited the transmission of HTLV-1 as compared to the vehicle
control; although,
Date Recue/Date Received 2021-04-13
- 37 -
there were no differences observed between the 15 min and 3 hrs of pre-
treatment of the HTLV-
1+ SLB1 cells
[00150] We also investigated whether oleandrin inhibits virological synapse-
formation and
the transmission of HTLV-1 in co-culture assays (Example 22). A GFP-expressing
HTLV-1+
SLB1 T-cell-line was generated by transducing SLB1 lymphoma T-cells with a
pLenti-6.2/V5-
DEST-GFP vector with selection on blasticidin (5 pg/m1; Life Technologies) for
two weeks.
The GFP-positive clones were screened by fluorescence-microscopy (FIG. 20 top
panels) and
immunoblotting (FIG. 20 lower panels) and expanded and repeatedly passaged.
The DIC phase-
contrast image is provided for comparison.
[00151] The formation of virological synapses between huPBMCs and the
mitomycin C-
treated HTLV-1+ SLB1/pLenti-GFP lymphoblasts (green cells) that had been pre-
treated for 3
hrs with the Vehicle control or increasing amounts (10 pg/ml, 50 pg/ml, and
100 pg/m1) of the
N. oleander extract or oleandrin compound were visualized by fluorescence-
microscopy (FIG.
21). Virus transmission was assessed by quantifying the relative percentages
of infected (i.e.,
HTLV-1 gp21-positive, red) huPBMCs (GFP-negative) in 20 visual fields (n=20)
by
fluorescence-microscopy using a 20x objective lens (see arrows in the Vehicle
control panels).
The fluorescence-microscopy data was quantified (FIG. 22). The data confirm
that the antiviral
composition inhibits virological synapse-formation and the transmission of
HTLV-1 in co-
culture assays.
1001521 The invention, thus, also provides a method of inhibiting (reducing)
the infectivity
of HTLV-1 particles released into the culture supernatants of treated cells
and also reducing the
intercellular transmission of HTLV-1 by inhibiting the Env-dependent formation
of virological
synapses, the method comprising treating virus-infected cells (in vitro or in
vivo) with an
effective amount of the antiviral composition.
[00153] Antiviral activity of the compositions herein was evaluated against
rhinovirus
infection. Rhinovirus is of the Picomaviridae family and Enterovirus genus. It
is not enveloped
and is an ss-RNA virus of (+) polarity. Oleandrin was found to be inactive
against rhinovirus
in the concentrations and assays employed herein, because it did not inhibit
viral replication.
[00154] CoV infection can be treated in vivo as detailed in Example 26,
wherein the antiviral
composition is administered to a subject as monotherapy or combination
therapy. Efficacy of
Date Recue/Date Received 2021-04-13
- 38 -
oleandrin against CoV was established in vivo according to Example 27. In a
small portion of
orange juice, a child was administered 0.25 ml of reconstituted ANVIRZELTM.
Then every 12
hours, the child was administered 0.5 ml of reconstituted ANVIRZELTM for a
period of about
2-3 days. The infant recuperated from COVID-19 infection.
[00155] Further proof of the efficacy of oleandrin (oleandrin-containing
composition) against
coronavirus, e.g. SARS-CoV-2 (COVID-19), was obtained through in vitro
evaluation
according to Example 28, wherein Vero cells were pretreated with oleandrin and
then infected
with SARS-CoV-2. Following infection of the cells, the extracellular virus and
oleandrin was
washed away, and the infected cells were then treated with oleandrin (FIG.
23A: 1 microg/mL
in 0.1% v/v aqueous DMSO; FIG. 23C: 0.1 microg/mL in 0.01% v/v aqueous DMSO)
or just
aqueous DMSO as control vehicle (FIG. 23B: 0.1% v/v aqueous DMSO; FIG. 23D:
0.01% v/v
aqueous DMSO). The results indicate that a) oleandrin pretreatment caused a
1368-fold
reduction in virus load at the 24-h time and a 369-fold reduction at the 48-h
time point; b)
oleandrin is efficacious over the entire concentration range of about 0.1 to
about 1.0 microg/mL
with the higher dose being slightly better than the lower dose so it is very
likely that oleandrin
is efficacious at even lower concentrations, e.g. 0.01 to 0.1 microg/mL; c)
oleandrin should be
administered repeatedly, since a single dose is not sufficient to fully stop
viral replication; and
d) using just 30 min preincubation of Vero cells with oleandrin is only
slightly effective at
reducing initial viral infection and does not appear to impact infectivity of
progeny virions. The
results also indicated that oleandrin at concentrations of 0.1 and 1.0
microg/mL is not overly
toxic to Vero cells. The results further indicate that oleandrin inhibits
infectivity of progeny
virus by a) about 1 logio without continuous drug treatment; and b) about >3
logio with
continuous drug treatments (without toxicity).
[00156] In order to determine whether oleandrin directly inhibits viral
replication, Vero-E6
cells were infected with SARS-CoV-2 virus and treated with oleandrin at
various concentrations
according to Example 29. The results are depicted in FIGS. 24A and 24B. At the
24 h time
point (FIG. 24A), in wells treated with oleandrin only during the absorption
phase (Pre-
treatment data), antiviral activity was observed with an estimated IC50 of
0.625 microg/mL. In
wells treated with oleandrin for the duration of the assay (duration data),
oleandrin significantly
limited virus entry and/or viral replication even in the presence of high
amounts of inoculating
Date Recue/Date Received 2021-04-13
- 39 -
virus. At the 48-h time point (FIG. 24B), in wells treated with oleandrin only
during the
absorption phase (Pre-treatment data), minimal antiviral activity was observed
by the end of
the time period. In wells treated with oleandrin for the duration of the assay
(duration data),
oleandrin significantly limited viral infection. Potential methods of action
include inhibition of
viral replication, assembly, and/or egress.
[00157] To ensure that the observed antiviral activity of oleandrin against
SARS-CoV-2 was
not due to cellular toxicity of oleandrin against Vero-E6 cells, the cell
titer was determined at
the 24-h (FIG. 24A) and 48-h (FIG. 24B) time points. At concentrations of
oleandrin of 1.0
microg/mL or higher, cellular toxicity appeared and potentially interfered
with the assay;
however, at concentrations of oleandrin of 0.625 microg/mL or lower,
interference of cellular
toxicity was substantially reduced, thereby confirming the strong antiviral
activity of oleandrin
even at very low concentrations. Additional evidence of the extent of toxicity
of oleandrin
against Vero-E6 cells was observed in the assay of Example 30 (FIG. 25). At an
oleandrin
concentration of 0.625 microg/mL, about 80% of the Vero cells remained viable
at the 24 h
time point, and even less toxicity was observed at lower concentrations. It
should be understood
that toxicity of oleandrin against Vero-E6 cells does not suggest that
oleandrin is toxic to
humans. This measure of toxicity is simply used to determine the potential
impact of
background cell death when measuring antiviral activity.
[00158] Oleandrin thus possesses at least a dual mechanism (pathway) for
treating viral
infection, in particular coronavirus infection, e.g. SARS-CoV-2 infection: a)
direct inhibition
of viral replication; and b) reduction of infectivity of progeny virus.
[00159] Moreover, oleandrin possesses antiviral activity even at very low
doses and oleandrin
exhibits a substantial prophylactic effect. This was demonstrated according to
Example 31,
wherein VERO CCL-81 cells were infected with SARS-CoV-2. Cells were pretreated
with
oleandrin prior to infection. After an initial 2 h incubation post infection,
the infected cells were
washed to remove extracellular virus and oleandrin. Then, the recovered
infected cells were
treated as follows. The infected cells were treated with oleandrin (various
concentration in
aqueous DMSO with RPMI 1640 culture medium as the aqueous component) or just
control
vehicle (aqueous DMSO with RPMI 164), and the viral titer was measured at 24
hours (FIG.
26A) and 48 hours (FIG. 26B) after "treatment". In the absence of oleandrin,
SARS-CoV-2
Date Recue/Date Received 2021-04-13
-40 -
reached high (approximately 6 logio plaque-forming units (pfu)/m1) titers by
the 24-hour
timepoint and maintained that titer at the later timepoint: it consistently
remained either at or
below the limit of detection for the assay. Oleandrin concentrations of 1
microg/mL to 0.05
microg/mL provided substantial reduction in viral titer even in just 24 hours.
The two higher
doses reduced the viral titer essentially to or below the limit of detection,
and no cellular toxicity
was observed at any of the oleandrin concentrations tested. The fold reduction
in viral titer was
calculated for these samples. The fold reduction (FIGS. 26C and 26D) in viral
titer ranged from
about 1,000-fold to about 40,000-fold was observed at the 48-h time point and
about 1,000-fold
to about 20,000-fold at the 24 h time point. Even though, the 10 ng/ml dose,
which had no
significant effect compared to its DMSO control at 24 hours post-infection, it
did result in a
significant reduction in titer at 48 hours post-infection. Importantly, the
reduction attributable
to oleandrin increased for the highest concentrations when measured at 48
hours compared to
24 hours. The increased prophylactic efficacy of oleandrin over time (24 vs.
48 hours) was
reflected in its ECso values, calculated at 11.98ng/m1 at 24 hours post-
infection and 7.07ng/m1
at 48 hours post-infection.
[00160] The Vero 81 cells described above were subjected to genome analysis
determine
whether the inhibition of SARS-CoV-2 was at the level of total or infectious
particle production.
RNA was extracted from the cell culture supernatants of the prophylactic
study, and genomic
equivalents were quantified via qRT-PCR (Example 39). The prophylactic effect
of oleandrin,
initially observed via infectious assay, was confirmed at the level of genome
equivalents. At
24 hours-post infection, oleandrin significantly decreased SARS-CoV-2 genomes
in the
supernatant at the four highest doses. The prophylactic effect of oleandrin,
initially observed
via infectious assay, was confirmed at the level of genome equivalents. At 24
hours-post
infection, oleandrin significantly decreased SARS-CoV-2 genomes in the
supernatant at the
four highest doses.
[00161] Additional studies were conducted to determine the dose response of
COVID-19
infection to oleandrin (FIGS. 27A-27B) at 24 h and 48 h post infection. A dose
response was
observed, wherein increasing the concentration of oleandrin in the culture
medium provided a
greater reduction of the viral titer; however, even the lowest concentration
tested (0.05
microg/mL) resulted in a titer reduction at 24 h and an even greater titer
reduction at 48 h post
Date Recue/Date Received 2021-04-13
- 41 -
infection. The highest dose resulted in a greater than 1,000-fold reduction in
infectious SARS-
CoV-2 titer, with the 0.5 jig/m1 and 100 ng/ml doses causing greater than 100-
fold reductions,
and the 50ng/m1 dose resulting in a 78-fold reduction.
[00162] FIGS. 28A and 28B depict the results of duplicate studies, each
conducted in
triplicate, to determine the dose-response of COVID-19 to treatment with
varying
concentrations (0.005 to 1 microg/mL) of oleandrin in the culture medium.
Substantial antiviral
activity was observed even 24 h and 48 h post-infection in Vero 81 cells for
concentrations
above 0.01 microg/mL. Even at a very low concentration of 0.05 microg/mL a
large reduction
in viral titer was observed.
[00163] In order to determine the antiviral efficacy of oleandrin post-
infection, a study
according to Example 34 was conducted. The Vero 81 cells were not pretreated
with oleandrin
prior to infection. Instead, the cells were infected with COVID-19 virus and
then treated with
oleandrin (at the indicated concentrations) at 12 h and 24 h post-infection.
The viral titer was
then measured at 24 h (FIG. 29A) and 48 h (FIG. 29B) post-infection. The data
demonstrate
that even with just a single treatment, oleandrin is able to exert antiviral
activity for at least 12,
at least 24 h, or at least 36 h post infection. It is important to note that
this assay is a time-
compressed assay as compared to human viral infection. The 24 h time point
would be
equivalent to about 5 to 7 days post-infection in a human, and the 48 h time
point would be
equivalent to about 10 to 14 days post-infection in a human.
[00164] The assays of Examples 31 and 34 were repeated using the dual extract
combination
composition (PBI-A, containing 1% wt of the ethanolic extract 1% wt of Example
36 dissolved
in DMSO (98% wt)). FIG. 30A details the results for evaluation of the dual
extract combination
composition according to the assay of Example 31, and FIG. 30B details the
results for
evaluation of the dual extract (1% wt) according to the assay of Example 34.
The data in FIG.
30A demonstrates relative antiviral (anti-COVID-19) efficacy of PBI-A based on
relative
dilution of the original stock solution. The data in FIG. 30B is based upon
the relative
concentration of oleandrin (.1.g/mL) in the assay solution. The dotted line in
each graph depicts
the lowest concentration of virus that can be detected using the CFU (virus
colony forming unit)
assay.
Date Recue/Date Received 2021-04-13
-42 -
[00165] Based upon the results in FIGS. 30A and 30B, the dual extract
combination
composition is effective as an antiviral agent against COVID-19 at
concentrations including
0.05 through 1.0 tg/m1 which is the same range as that observed with pure
oleandrin.
[00166] It is also important to observe that the concentrations of oleandrin
evaluated in the
assays are clinically relevant in terms of dosing and plasma concentration.
[00167] Proof of the safety of the oleandrin-containing composition was
further provided by
in vitro cellular assay for determining the release of lactate dehydrogenase
after exposure of
said cells to solutions containing different concentrations of oleandrin. It
was determined that
up to concentrations of 1 microg/mL, there was no additional toxicity over
control vehicle.
[00168] The efficacy of oleandrin (oleandrin-containing composition, oleandrin-
containing
extract) in treating COVID-19 viral infection was further established by
administration of
oleandrin-containing sublingual dosage form (Example 32 or 37) to subjects
according to
Example 35 under the Expanded Access program of the FDA. Subjects ranging in
ages from
18 to 78 y of age were administered four 15 microg doses of oleandrin (as the
dual extract
composition) per day spaced at about 6 h intervals or three 15 mg doses per
day spaced at about
8 h intervals. Prior to initiation of treatment, subjects' clinical status
and/or viral titer were
observed. Some subjects were on palliative care or hospice care. Clinical
status and/or viral
titers were determined periodically during the treatment period of one to two
weeks, ten to
fourteen days. The following results were observed after initiation of
treatment.
Age Initial Clinical Presentation Results after initiation of treatment
(y)
78 Female; sent home with pneumonia After 36 h, resolution of symptoms
began
after 14 d hospital stay; labored to lessen. After one week, subject was
breathing, fatigue productive cough, fully recovered.
on oxygen
51 Female; fever, cough, headaches, Complete resolution of symptoms
within
body aches and pains, three days
Date Recue/Date Received 2021-04-13
-43 -
Age Initial Clinical Presentation Results after initiation of treatment
(y)
18 Male; Fever 103.0, migraine, muscle After 2 d, symptoms lessened. After
4 d,
ache, neck/shoulder pain, confusion, almost complete resolution of symptoms
bloodshot eyes, no smell, sore
throat, shortness of breath
35 Female; 35 days of symptomology; After 2 d, overall about 90%
Fatigue, aches, tight chest and improvement in symptoms
burning when breathing
18 Male; Asymptomatic/positive for After 4 days, viral load below
detection
Covid. Viral load 7500-10,000 limit.
18 Male; fever, migraine, breathing After 36 h, overall about 90%
problems, head/neck pain, bedridden improvement in symptoms
39 Male; fever, achiness, diarrhea. Took first dose within 24 h of
initial
symptoms. Symptoms resolved within 24
h of first dose.
41 Male; bedridden, tight chest, fever, Almost complete resolution of
symptoms
sore throat, severe cough. within 48 h
47 Female; 29 days of low grade Was able to reunite with family within
symptoms: fever, fatigue, tight chest one week
42 Female; 14 d of symptoms: fever, About 90% improvement within 5 days
fatigue, headaches
27 Female; 3 d of symptoms: achiness, After 2 days and just 2 doses per
day,
cough, fever, loss of smell and taste almost complete recovery
[00169] Additional in vivo studies under the Expanded Access program of the
FDA were
conducted in a second group of human subjects exhibiting different levels of
COVID-19-
associated symptomology. Prior to initiation of treatment, subjects' clinical
status and/or viral
titer were determined to confirm COVID-19 infection. Some subjects exhibited
moderate to
severe symptomology. Subjects ranging in ages were administered four 15 microg
doses of
Date Recue/Date Received 2021-04-13
-44 -
oleandrin (as the dual extract composition) per day spaced at about 6 h
intervals. Clinical status
and/or viral titers were determined periodically during the treatment period
of one to two weeks,
ten to fourteen days. All subjects recovered completely from COVID-19
infection within five
to twelve days after initiation of treatment.
[00170] Oleandrin has also been shown to produce a strong anti-inflammatory
response,
which may be of benefit in preventing hyper-inflammatory responses to
infection with SARS-
CoV-2.
[00171] The invention thus provides a method of treating COVID-19 viral
infection
comprising administered plural doses of cardiac glycoside (cardiac glycoside-
containing
composition, or cardiac glycoside-containing extract) to a subject having said
infection. The
plural doses can be divided as one or more doses per day for two or more days
per week,
optionally for one or more weeks per month and further optionally for one or
more months per
year. The preferred cardiac glycoside is oleandrin.
[00172] The invention thus provides a method of treating coronavirus
infection, in particular
an infection of coronavirus that is pathogenic to humans, e.g. SARS-CoV-2
infection, the
method comprising chronically administering to a subject, having said
infection, therapeutically
effective doses of cardiac glycoside (cardiac glycoside-containing
composition). Chronic
administration can be achieved by repeatedly administering one or more
(plural) therapeutically
effective doses of cardiac glycoside (cardiac glycoside-containing
composition). One or more
doses may be administered per day for one or more days per week and optionally
for one or
more weeks per month and optionally for one or more months per year.
[00173] Accordingly, the invention provides a method of treating viral, e.g.
CoV, infection
in a subject (in particular a human subject) in need thereof comprising
administering to the
subject one or more doses of antiviral composition comprising a) oleandrin;
orb) oleandrin and
one or more other compounds extracted from Nerium species. The oleandrin may
be present
as part of an extract of Nerium species, which extract may be a a)
supercritical fluid extract; b)
hot-water extract; c) organic solvent extract; d) aqueous organic solvent
extract; e) extract using
supercritical fluid, optionally plus at least one organic solvent (extraction
modifier); f) extract
using subcritical liquid, optionally plus at least one organic solvent
(extraction modifier); or g)
any combination of any two or more of said extracts.
Date Recue/Date Received 2021-04-13
-45 -
[00174] PBI-05204 (as described herein and in US 8187644 B2 to Addington,
which issued
May 29, 2012, US 7402325 B2 to Addington, which issued July 22, 2008, US
8394434 B2 to
Addington et al, which issued Mar. 12, 2013) comprises cardiac glycoside
(oleandrin, OL) and
triterpenes (oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA))
as the primary
pharmacologically active components. The molar ratio of OL to total triterpene
is about 1:(10-
96). The molar ratio of OA:UA:BA is about 7.8:7.4:1. The combination of OA, UA
and BA
in PBI-05204 increases the antiviral activity of oleandrin when compared on an
OL equimolar
basis. PBI-04711 is a fraction of PBI-05204, but it does not contain cardiac
glycoside (OL).
The molar ratio of OA:UA:BA in PBI-04711 is about 3:2.2:1. PBI-04711 also
possesses
antiviral activity. Accordingly, an antiviral composition comprising OL, OA,
UA, and BA is
more efficacious than a composition comprising OL as the sole active
ingredient based upon an
equimolar content of OL. In some embodiments, the molar ratios of the
individual triterpenes
to oleandrin range as follows: about 2-8 (OA) : about 2-8 (UA) : about 0.1-1
(BA) : about 0.5-
1.5 (OL); or about 3-6 (OA) : about 3-6 (UA) : about 0.3-8 (BA) : about 0.7-
1.2 (OL); or about
4-5 (OA) : about 4-5 (UA) : about 0.4-0.7 (BA) : about 0.9-1.1 (OL); or about
4.6 (OA) : about
4.4 (UA) : about 0.6 (BA) : about 1 (OL).
[00175] Antiviral compositions comprising oleandrin as the sole antiviral
agent are within
the scope of the invention. Antiviral compositions comprising digoxin as the
sole antiviral agent
are within the scope of the invention.
[00176] Antiviral compositions comprising oleandrin and plural triterpenes as
the antiviral
agents are within the scope of the invention. In some embodiments, the
antiviral composition
comprises oleandrin, oleanolic acid (free acid, salt, derivative or prodrug
thereof), ursolic acid
(free acid, salt, derivative or prodrug thereof), and betulinic acid (free
acid, salt, derivative or
prodrug thereof). The molar ratios of the compounds is as described herein.
[00177] Antiviral compositions comprising plural triterpenes as the primary
active
ingredients (meaning excluding steroid, cardiac glycoside and
pharmacologically active
components) are also within the scope of the invention. As noted above, PBI-
04711 comprises
OA, UA and BA as the primary active ingredients, and it exhibits antiviral
activity. In some
embodiments, a triterpene-based antiviral composition comprises OA, UA and BA,
each of
Date Recue/Date Received 2021-04-13
-46 -
which is independently selected upon each occurrence from its free acid form,
salt form,
deuterated form and derivative form.
[00178] PBI-01011 is an improved triterpene-based antiviral composition
comprising OA,
UA and BA, wherein the molar ratio of OA:UA:BA is about 9-12 : up to about 2 :
up to about
2, or about 10 : about 1: about 1, or about 9-12 : about 0.1-2 : about 0.1-2,
or about 9-11 : about
0.5-1.5: about 0.5-1.5, or about 9.5-10.5 : about 0.75-1.25 : about 0.75-1.25,
or about 9.5-10.5
: about 0.8-1.2 : about 0.8-1.2, or about 9.75-10.5 : about 0.9-1.1 : about
0.9-1.1.
[00179] In some embodiments, an antiviral composition comprises at least
oleanolic acid
(free acid, salt, derivative or prodrug thereof) and ursolic acid (free acid,
salt, derivative or
prodrug thereof) present at a molar ratio of OA to UA as described herein. OA
is present in
large molar excess over UA.
[00180] In some embodiments, an antiviral composition comprises at least
oleanolic acid
(free acid, salt, derivative or prodrug thereof) and betulinic acid (free
acid, salt, derivative or
prodrug thereof) present at a molar ratio of OA to BA as described herein. OA
is present in
large molar excess over BA.
[00181] In some embodiments, an antiviral composition comprises at least
oleanolic acid
(free acid, salt, derivative or prodrug thereof), ursolic acid (free acid,
salt, derivative or prodrug
thereof), and betulinic acid (free acid, salt, derivative or prodrug thereof)
present at a molar
ratio of OA to UA to BA as described herein. OA is present in large molar
excess over both UA
and BA.
[00182] In some embodiments, a triterpene-based antiviral composition excludes
cardiac
glycoside.
[00183] In general, a subject having Arenaviridae infection, Arternviridae
infection,
Filoviridae infection, Flaviviridae infection (Flavivirus genus),
Deltaretrovirus genus,
Coronaviridae, Paramyxoviridae, Orthomyxoviridae, or Togaviridae infection is
treated as
follows. The subject is evaluated to determine whether said subject is
infected with said virus.
Administration of antiviral composition is indicated. Initial doses of
antiviral composition are
administered to the subject according to a prescribed dosing regimen for a
period of time (a
treatment period). The subject's clinical response and level of therapeutic
response are
determined periodically. If the level of therapeutic response is too low at
one dose, then the
Date Recue/Date Received 2021-04-13
-47 -
dose is escalated according to a predetermine dose escalation schedule until
the desired level of
therapeutic response in the subject is achieved. Treatment of the subject with
antiviral
composition is continued as needed. The dose or dosing regimen can be adjusted
as needed
until the patient reaches the desired clinical endpoint(s) such as cessation
of the infection itself,
reduction in infection-associated symptoms, and/or a reduction in the
progression of the
infection.
[00184] If a clinician intends to treat a subject having viral infection with
a combination of a
antiviral composition and one or more other therapeutic agents, and it is
known that the viral
infection, which the subject has, is at least partially therapeutically
responsive to treatment with
said one or more other therapeutic agents, then the present method invention
comprises:
administering to the subject in need thereof a therapeutically relevant dose
of antiviral
composition and a therapeutically relevant dose of said one or more other
therapeutic agents,
wherein the antiviral composition is administered according to a first dosing
regimen and the
one or more other therapeutic agents is administered according to a second
dosing regimen. In
some embodiments, the first and second dosing regimens are the same. In some
embodiments,
the first and second dosing regimens are different.
[00185] The antiviral composition(s) of the invention can be administered as
primary
antiviral therapy, adjunct antiviral therapy, or co-antiviral therapy. Methods
of the invention
include separate administration or coadministration of the antiviral
composition with at least
one other known antiviral composition, meaning the antiviral composition of
the invention can
be administered before, during or after administration of a known antiviral
composition
(compound(s)) or of a composition for treating symptoms associated with the
viral infection.
For example, medications used to treat inflammation, vomiting, nausea,
headache, fever,
diarrhea, nausea, hives, conjunctivitis, malaise, muscle pain, joint pain,
seizure, or paralysis can
be administered with or separately from the antiviral composition of the
invention.
[00186] The one or more other therapeutic agents can be administered at doses
and according
to dosing regimens that are clinician-recognized as being therapeutically
effective or at doses
that are clinician-recognized as being sub-therapeutically effective. The
clinical benefit and/or
therapeutic effect provided by administration of a combination of antiviral
composition and one
or more other therapeutic can be additive or synergistic, such level of
benefit or effect being
Date Recue/Date Received 2021-04-13
-48 -
determined by comparison of administration of the combination to
administration of the
individual antiviral composition component(s) and one or more other
therapeutic agents. The
one or more other therapeutic agents can be administered at doses and
according to dosing
regimens as suggested or described by the Food and Drug Administration, World
Health
Organization, European Medicines Agency (E.M.E.A.), Therapeutic Goods
Administration
(TGA, Australia), Pan American Health Organization (PAHO), Medicines and
Medical
Devices Safety Authority (Medsafe, New Zealand) or the various Ministries of
Health
worldwide.
[00187] Exemplary other therapeutic agents that can be included in the
antiviral composition
of the invention for the treatment of viral infection include antiretroviral
agent, interferon alpha
(IFN-a), zidovudine, lamivudine, cyclosporine A, CHOP with arsenic trioxide,
sodium
valproate, methotrexate, azathioprine, one or more symptom alleviating
drug(s), steroid sparing
drug, corticosteroid, cyclophosphamide, immunosuppressant, anti-inflammatory
agent, Janus
kinase inhibitor, tofacitinib, calcineurin inhibitor, tacrolimus, mTOR
inhibitor, sirolimus,
everolimus, IMDH inhibitor, azathioprine, leflunomide, mycophenolate,
biologic, abatacept,
adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab,
ixekizumab,
natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab,
monoclonal
antibody, basiliximab, daclizumab, polyclonal antibody, nucleoside analogs,
reverse
transcriptase inhibitor, emtricitabine, telbivudine, abacavir, adefovir,
didanosine, emtricitabine,
entecavir, stavudine, tenofovir, azithromycin, macrolide-type antibiotic,
protease inhibitor,
interferon, immune response modifier, mRNA synthesis inhibitor, protein
synthesis, inhibitor,
thiazolide, CYP3A4 inhibitor, heterocyclic biguanidine, CCR5 receptor
inhibitor, and
combinations thereof. Therapies studied also include plasmapheresis and/or
radiation.
Antibodies to specific viruses may also be administered to a subject treated
with the antiviral
composition of the invention. Plasma obtained from the blood of survivors of a
first viral
infection can be administered to other subjects having the same type of viral
infection, said
other subjects also being administered the antiviral composition of the
invention. For example,
the plasma from a survivor of COVID-19 infection may be administered to
another subject
having a COVID-19 infection, said other subject also being administered the
antiviral
composition of the invention.
Date Recue/Date Received 2021-04-13
-49 -
[00188] Example 5 provides an exemplary procedure for the treatment of
Zikavirus infection
in a mammal. Example 12 provides an exemplary procedure for the treatment of
Filovirus
infection (Ebolavirus, Marburgvirus) in a mammal. Example 13 provides an
exemplary
procedure for the treatment of Flavivirus infection (Yellow Fever, Dengue
Fever, Japanese
Enchephalitis, West Nile Viruses, Zikavirus, Tick-borne Encephalitis, Kyasanur
Forest
Disease, Alkhurma Disease, Omsk Hemorrhagic Fever, Powassan virus infection)
in a
mammal. Example 25 provides an exemplary procedure for the treatment of
Deltaretrovirus
genus (HTLV-1) infection.
[00189] The antiviral compound(s) (triterpene(s), cardiac glycoside(s), etc.)
present in the
pharmaceutical composition can be present in their unmodified form, salt form,
derivative form
or a combination thereof. As used herein, the term "derivative" is taken to
mean: a) a chemical
substance that is related structurally to a first chemical substance and
theoretically derivable
from it; b) a compound that is formed from a similar first compound or a
compound that can be
imagined to arise from another first compound, if one atom of the first
compound is replaced
with another atom or group of atoms; c) a compound derived or obtained from a
parent
compound and containing essential elements of the parent compound; or d) a
chemical
compound that may be produced from first compound of similar structure in one
or more steps.
For example, a derivative may include a deuterated form, oxidized form,
dehydrated,
unsaturated, polymer conjugated or glycosilated form thereof or may include an
ester, amide,
lactone, homolog, ether, thioether, cyano, amino, alkylamino, sulfhydryl,
heterocyclic,
heterocyclic ring-fused, polymerized, pegylated, benzylidenyl, triazolyl,
piperazinyl or
deuterated form thereof.
[00190] As used herein, the term "oleandrin" is taken to mean all known forms
of oleandrin
unless otherwise specified. Oleandrin can be present in racemic, optically
pure or optically
enriched form. Nerium oleander plant material can be obtained, for example,
from commercial
plant suppliers such as Aldridge Nursery, Atascosa, Texas.
[00191] The supercritical fluid (SCF) extract can be prepared as detailed in
US 7,402,325,
US 8394434, US 8187644, or PCT International Publication No. WP 2007/016176
A2.
Extraction can be conducted with supercritical carbon dioxide in the presence
or absence of a
modifier (organic solvent) such as ethanol.
Date Recue/Date Received 2021-04-13
- 50 -
[00192] Other extracts containing cardiac glycoside, especially oleandrin, can
be prepared by
various different processes. An extract can be prepared according to the
process developed by
Dr. Huseyin Ziya Ozel (U.S. Patent No. 5,135,745) describes a procedure for
the preparation
of a hot water extract. The aqueous extract reportedly contains several
polysaccharides with
molecular weights varying from 2KD to 30KD, oleandrin, oleandrigenin,
odoroside and
neritaloside. The polysaccharides reportedly include acidic
homopolygalacturonans or
arabinogalaturonans. U.S. Patent No. 5,869,060 to Selvaraj et al. discloses
hot water extracts of
Nerium species and methods of production thereof, e.g. Example 2. The
resultant extract can
then be lyophilized to produce a powder. U.S. Patent No. 6,565,897 (U.S.
Pregrant Publication
No. 20020114852 and PCT International Publication No. WO 2000/016793 to
Selvaraj et al.)
discloses a hot-water extraction process for the preparation of a
substantially sterile extract.
Erdemoglu et al. (I Ethnopharmacol. (2003) Nov. 89(1), 123-129) discloses
results for the
comparison of aqueous and ethanolic extracts of plants, including Nerium
oleander, based upon
their anti-nociceptive and anti-inflammatory activities. Organic solvent
extracts of Nerium
oleander are disclosed by Adome et al. (Afr. Health Sci. (2003) Aug. 3(2), 77-
86; ethanolic
extract), el-Shazly et al. (.1 Egypt Soc. Parasitol. (1996), Aug. 26(2), 461-
473; ethanolic
extract), Begum et al. (Phytochemistry (1999) Feb. 50(3), 435-438; methanolic
extract), Zia et
al. (I Ethnolpharmacol. (1995) Nov. 49(1), 33-39; methanolic extract), and
Vlasenko et al.
(Farmatsiia. (1972) Sept.-Oct. 21(5), 46-47; alcoholic extract). U.S. Pregrant
Patent
Application Publication No. 20040247660 to Singh et al. discloses the
preparation of a protein
stabilized liposomal formulation of oleandrin for use in the treatment of
cancer. U.S. Pregrant
Patent Application Publication No. 20050026849 to Singh et al. discloses a
water soluble
formulation of oleandrin containing a cyclodextrin. U.S. Pregrant Patent
Application
Publication No. 20040082521 to Singh et al. discloses the preparation of
protein stabilized
nanoparticle formulations of oleandrin from the hot-water extract.
[00193] Oleandrin may also be obtained from extracts of suspension cultures
derived from
Agrobacterium tumefaciens-transformed calli (Ibrahim et al., "Stimulation of
oleandrin
production by combined Agrobacterium tumefaciens mediated transformation and
fungal
elicitation in Nerium oleander cell cultures" in Enz. Microbial Techno.
(2007), 41(3), 331-336).
Date Recue/Date Received 2021-04-13
-51 -
Hot water, organic solvent, aqueous organic solvent, or supercritical fluid
extracts of
agrobacterium may be used according to the invention.
[00194] Oleandrin may also be obtained from extracts of Nerium oleander
microculture in
vitro, whereby shoot cultures can be initiated from seedlings and/or from
shoot apices of the
Nerium oleander cultivars Splendens Giganteum, Revanche or Alsace, or other
cultivars (Vila
et al., "Micropropagation of Oleander (Nerium oleander L.)" in HortScience
(2010), 45(1), 98-
102). Hot water, organic solvent, aqueous organic solvent, or supercritical
fluid extracts of
microcultured Nerium oleander may be used according to the invention.
[00195] The extracts also differ in their polysaccharide and carbohydrate
content. The hot
water extract contains 407.3 glucose equivalent units of carbohydrate relative
to a standard
curve prepared with glucose while analysis of the SCF CO2 extract found
carbohydrate levels
that were found in very low levels that were below the limit of quantitation.
The amount of
carbohydrate in the hot water extract of Nerium oleander was, however, at
least 100-fold greater
than that in the SCF CO2 extract. The polysaccharide content of the SCF
extract can be 0%,
<0.5%, <0.1%, <0.05%, or <0.01% wt. In some embodiments, the SCF extract
excludes
polysaccharide obtained during extraction of the plant mass.
Nerium oleander preparation Polysaccharide content
(tg glucose equivalents/ mg of plant
extract)
Hot water extract 407.3 6.3
SCF CO2 extract BLQ (below limit of quantitation)
[00196] The partial compositions of the SCF CO2 extract and hot water extract
were
determined by DART TOF-MS (Direct Analysis in Real Time Time of Flight Mass
Spectrometry) on a JEOL AccuT0E-DART mass spectrometer (JEOL USA, Peabody, MA,
USA).
[00197] The SCF extract of Nerium species or Thevetia species is a mixture of
pharmacologically active compounds, such as oleandrin and triterpenes. The
extract obtained
by the SCF process is a substantially water-insoluble, viscous semi-solid
(after solvent is
Date Recue/Date Received 2021-04-13
- 52 -
removed) at ambient temperature. The SCF extract comprises many different
components
possessing a variety of different ranges of water solubility. The extract from
a supercritical
fluid process contains by weight a theoretical range of 0.9% to 2.5% wt of
oleandrin or 1.7% to
2.1% wt of oleandrin or 1.7% to 2.0% wt of oleandrin. SCF extracts comprising
varying amount
of oleandrin have been obtained. In one embodiment, the SCF extract comprises
about 2% by
wt. of oleandrin. The SCF extract contains a 3-10 fold higher concentration of
oleandrin than
the hot-water extract. This was confirmed by both HPLC as well as LC/MS/MS
(tandem mass
spectrometry) analyses.
[00198] The SCF extract comprises oleandrin and the triterpenes oleanolic
acid, betulinic acid
and ursolic acid and optionally other components as described herein. The
content of oleandrin
and the triterpenes can vary from batch to batch; however, the degree of
variation is not
excessive. For example, a batch of SCF extract (PBI-05204) was analyzed for
these four
components and found to contain the following approximate amounts of each.
Oleandrin Oleanolic acid Ursolic acid Betulinic acid
Content of 20 73 69 9.4
component
(mg/g of SCF
extract)
Content of 2 7.3 6.9 0.94
component (%
wt WRT g of
SCT extract)
Content of 34.7 160 152 20.6
component
(mmole/g of
SCF extract)
Molar ratio of 1 4.6 4.4 0.6
component
WRT oleandrin
WRT denotes "with respect to".
Date Recue/Date Received 2021-04-13
- 53 -
[00199] The content of the individual components may vary by +25%, +20%, +15%,
+10%
or +5% relative to the values indicated. Accordingly, the content of oleandrin
in the SCF extract
would be in the range of 20 mg 5 mg (which is +25% of 20 mg) per mg of SCF
extract.
[00200] Oleandrin, oleanolic acid, ursolic acid, betulinic acid and
derivatives thereof can also
be purchased from Sigma-Aldrich (www.sigmaaldrich.com; St. Louis, MO, USA).
Digoxin is
commercially available from HIKMA Pharmaceuticals International LTD (NDA
N012648,
elixir, 0.05 mg/mL; tablet, 0.125 mg, 0.25 mg), VistaPharm Inc. (NDA A213000,
elixir, 0.05
mg/mL), Sandoz Inc. (NDA A040481, injectable, 0.25 mg/mL), West-Ward
Pharmaceuticals
International LTD (NDA A083391, injectable, 0.25 mg/mL), Covis Pharma BV (NDA
N009330, 0.1 mg/mL, 0.25 mg/mL), Impax Laboratories (NDA A078556, tablet,
0.125 mg,
0.25 mg), Jerome Stevens Pharmaceuticals Inc. (NDA A076268, tablet, 0.125 mg,
0.25 mg),
Mylan Pharmaceuticals Inc. (NDA A040282, tablet, 0.125 mg, 0.25 mg), Sun
Pharmaceutical
Industries Inc. (NDA A076363, tablet, 0.125 mg, 0.25 mg), Concordia
Pharmaceuticals Inc.
(NDA A020405, tablet, 0.0625, 0.125 mg, 0.1875 mg, 0.25 mg, 0.375 mg, 0.5 mg,
LANOXIN),
GlaxoSmithKline LLC (NDA 018118, capsule, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg,
LANOXICAPS).
[00201] As used herein, the individually named triterpenes can independently
be selected
upon each occurrence in their native (unmodified, free acid) form, in their
salt form, in
derivative form, prodrug form, or a combination thereof. Compositions
containing and methods
employing deuterated forms of the triterpenes are also within the scope of the
invention.
[00202] Oleanolic acid derivatives, prodrugs and salts are disclosed in US
20150011627 Al
to Gribble et al. which published Jan. 8, 2015, US 20140343108 Al to Rong et
al which
published Nov. 20, 2014, US 20140343064 Al to Xu et al. which published Nov.
20, 2014, US
20140179928 Al to Anderson et al. which published June 26, 2014, US
20140100227 Al to
Bender et al. which published April 10, 2014, US 20140088188 Al to Jiang et
al. which
published Mar. 27, 2014, US 20140088163 Al to Jiang et al. which published
Mar. 27, 2014,
US 20140066408 Al to Jiang et al. which published Mar. 6, 2014, US 20130317007
Al to
Anderson et al. which published Nov. 28, 2013, US 20130303607 Al to Gribble et
al. which
published Nov. 14, 2013, US 20120245374 to Anderson et al. which published
Sep. 27, 2012,
Date Recue/Date Received 2021-04-13
- 54 -
US 20120238767 Al to Jiang et al. which published Sep. 20, 2012, US
20120237629 Al to
Shode et al. which published Sept. 20, 2012, US 20120214814 Al to Anderson et
al. which
published Aug. 23, 2012, US 20120165279 Al to Lee etal. which published June
28, 2012, US
20110294752 Al to Arntzen et al. which published Dec. 1, 2011, US 20110091398
Al to
Majeed et al. which published April 21, 2011, US 20100189824 Al to Arntzen et
al. which
published July 29, 2010, US 20100048911 Al to Jiang et al. which published
Feb. 25, 2010,
and US 20060073222 Al to Amtzen et al. which published April 6, 2006.
[00203] Ursolic acid derivatives, prodrugs and salts are disclosed in US
20150011627 Al to
Gribble et al. which published Jan. 8, 2015, US 20130303607 Al to Gribble et
al. which
published Nov. 14, 2013, US 20150218206 Al to Yoon et al. which published Aug.
6, 2015,
US 6824811 to Fritsche et al. which issued Nov. 30, 2004, US 7718635 to Ochiai
et al. which
issued May 8, 2010, US 8729055 to Lin et al. which issued May 20, 2014, and US
9120839 to
Yoon et al. which issued Sep. 1, 2015.
[00204] Betulinic acid derivatives, prodrugs and salts are disclosed in US
20150011627 Al
to Gribble et al. which published Jan. 8, 2015, US 20130303607 Al to Gribble
et al. which
published Nov. 14, 2013, US 20120237629 Al to Shode et al. which published
Sept. 20, 2012,
US 20170204133 Al to Regueiro-Ren et al. which published July 20, 2017, US
20170096446
Al to Nitz et al. which published April 6, 2017, US 20150337004 Al to
Parthasaradhi Reddy
etal. which published Nov. 26, 2015, US 20150119373 Al to Parthasaradhi Reddy
etal. which
published April 30, 2015, US 20140296546 Al to Yan et al. which published Oct.
2, 2014, US
20140243298 Al to Swidorski et al. which published Aug. 28, 2014, US
20140221328 Al to
Parthasaradhi Reddy etal. which published Aug. 7, 2014, US 20140066416 Al fp
Leunis et al.
which published March 6, 2014, US 20130065868 Al to Durst et al. which
published March
14, 2013, US 20130029954 Al to Regueiro-Ren et al. which published Jan. 31,
2013, US
20120302530 Al to Zhang etal. which published Nov. 29,2012, US 20120214775 Al
to Power
etal. which published Aug. 23, 2012, US 20120101149 Al to Honda etal. which
published
April 26, 2012, US 20110224182 to Bullock et al. which published Sep. 15,
2011, US
20110313191 Al to Hemp et al. which published Dec. 22, 2011, US 20110224159 Al
to
Pichette et al. which published Sep. 15, 2011, US 20110218204 to Parthasaradhi
Reddy et al.
which published Sep. 8, 2011, US 20090203661 Al to Safe et al. which published
Aug. 13,
Date Recue/Date Received 2021-04-13
- 55 -
2009, US 20090131714 Al to Krasutsky et al. which published May 21, 2009, US
20090076290 to Krasutsky et al. which published March 19, 2009, US 20090068257
Al to
Leunis et al. which published March 12, 2009, US 20080293682 to Mukherjee et
al. which
published Nov. 27, 2008, US 20070072835 Al to Pezzuto et al. which published
March 29,
2007, US 20060252733 Al to Jansen et al. which published Nov. 9, 2006, and US
2006025274
Al to O'Neill et al. which published Nov. 9, 2006.
[00205] The antiviral composition can be formulated in any suitable
pharmaceutically
acceptable dosage form. Parenteral, otic, ophthalmic, nasal, inhalable,
buccal, sublingual,
enteral, topical, oral, peroral, and injectable dosage forms are particularly
useful. Particular
dosage forms include a solid or liquid dosage forms. Exemplary suitable dosage
forms include
tablet, capsule, pill, caplet, troche, sache, solution, suspension,
dispersion, vial, bag, bottle,
injectable liquid, i. v. (intravenous), i.m. (intramuscular) or i.p.
(intraperitoneal) administrable
liquid and other such dosage forms known to the artisan of ordinary skill in
the pharmaceutical
sciences.
[00206] Since viral infection may affect multiple organs simultaneously and
cause multiple
organ failure, it may be advantageous to administer the composition by more
than one route.
For example, COVID-19 is known to affect the lungs, heart, gastrointestinal
tract, and brain.
Accordingly, the cardiac glycoside-containing composition can be
advantageously
administered as an inhalable composition and a peroral composition, a
sublingual composition
and a peroral composition, an inhalable composition and a sublingual
composition, an inhalable
composition and a parenteral composition, a sublingual composition and a
parenteral
composition, a peroral composition and a parenteral composition, or other such
combination.
[00207] Suitable dosage forms containing the antiviral composition can be
prepared by
mixing the antiviral composition with pharmaceutically acceptable excipients
as described
herein or as described in Pi et al. ("Ursolic acid nanocrystals for
dissolution rate and
bioavailability enhancement: influence of different particle size" in Curr.
Drug Deliv. (Mar
2016), 13(8), 1358-1366), Yang et al. ("Self-microemulsifying drug delivery
system for
improved oral bioavailability of oleanolic acid: design and evaluation" in
Int. J. Nanomed.
(2013), 8(1), 2917-2926), Li et al. (Development and evaluation of optimized
sucrose ester
stabilized oleanolic acid nanosuspensions prepared by wet ball milling with
design of
Date Recue/Date Received 2021-04-13
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experiments" in Biol. Pharm. Bull. (2014), 37(6), 926-937), Zhang et al.
("Enhancement of oral
bioavailability of triterpene through lipid nanospheres: preparation,
characterization, and
absorption evaluation" in J. Pharm. Sci. (June 2014), 103(6), 1711-1719),
Godugu et al.
("Approaches to improve the oral bioavailability and effects of novel
anticancer drugs berberine
and betulinic acid" in PLoS One (Mar 2014), 9(3):e89919), Zhao et al.
("Preparation and
characterization of betulin nanoparticles for oral hypoglycemic drug by
antisolvent
precipitation" in Drug Deliv. (Sep 2014), 21(6), 467-479), Yang et al.
("Physicochemical
properties and oral bioavailability of ursolic acid nanoparticles using
supercritical anti-solvent
(SAS) process" in Food Chem. (May 2012), 132(1), 319-325), Cao et al.
("Ethylene glycol-
linked amino acid diester prodrugs of oleanolic acid for PEPT1-mediated
transport: synthesis,
intestinal permeability and pharmacokinetics" in Mol. Pharm. (Aug. 2012),
9(8), 2127-2135),
Li et al. ("Formulation, biological and pharmacokinetic studies of sucrose
ester-stabilized
nanosuspensions of oleanolic acid" in Pharm. Res. (Aug 2011), 28(8), 2020-
2033), Tong et al.
("Spray freeze drying with polyvinylpyrrolidone and sodium caprate for
improved dissolution
and oral bioavailablity of oleanolic acid, a BCS Class IV compound" in Int. J.
Pharm. (Feb
2011), 404(1-2), 148-158), Xi et al. (Formulation development and
bioavailability evaluation
of a self-nanoemulsified drug delivery system of oleanolic acid" in AAPS
PharmSciTech
(2009), 10(1), 172-182), Chen et al. ("Oleanolic acid nanosuspensions:
preparation, in-vitro
characterization and enhanced hepatoprotective effect" in J. Pharm. Pharmacol.
(Feb 2005),
57(2), 259-264).
[00208] Suitable dosage forms can also be made according to US 8187644 B2 to
Addington,
which issued May 29, 2012, US 7402325 B2 to Addington, which issued July 22,
2008, US
8394434 B2 to Addington et al, which issued Mar. 12, 2013. Suitable dosage
forms can also
be made as described in Examples 13-15.
[00209] An effective amount or therapeutically relevant amount of antiviral
compound
(cardiac glycoside, triterpene or combinations thereof) is specifically
contemplated. By the term
"effective amount", it is understood that a pharmaceutically effective amount
is contemplated.
A pharmaceutically effective amount is the amount or quantity of active
ingredient which is
enough for the required or desired therapeutic response, or in other words,
the amount, which
is sufficient to elicit an appreciable biological response when, administered
to a patient. The
Date Recue/Date Received 2021-04-13
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appreciable biological response may occur as a result of administration of
single or multiple
doses of an active substance. A dose may comprise one or more dosage forms. It
will be
understood that the specific dose level for any patient will depend upon a
variety of factors
including the indication being treated, severity of the indication, patient
health, age, gender,
weight, diet, pharmacological response, the specific dosage form employed, and
other such
factors.
[00210] The desired dose for oral administration is up to 5 dosage forms
although as few as
one and as many as ten dosage forms may be administered as a single dose.
Exemplary dosage
forms can contain 0.01-100 mg or 0.01-100 microg of the antiviral composition
per dosage
form, for a total 0.1 to 500 mg (1 to 10 dose levels) per dose. Doses will be
administered
according to dosing regimens that may be predetermined and/or tailored to
achieve specific
therapeutic response or clinical benefit in a subject.
[00211] The cardiac glycoside can be present in a dosage form in an amount
sufficient to
provide a subject with an initial dose of oleandrin of about 20 to about 100
microg, about 12
microg to about 300 microg, or about 12 microg to about 120 microg. A dosage
form can
comprise about 20 of oleandrin to about 100 microg, about 0.01 microg to about
100 mg or
about 0.01 microg to about 100 microg oleandrin, oleandrin extract or extract
of Nerium
oleander containing oleandrin.
[00212] The antiviral can be included in an oral dosage form. Some embodiments
of the
dosage form are not enteric coated and release their charge of antiviral
composition within a
period of 0.5 to 1 hours or less. Some embodiments of the dosage form are
enteric coated and
release their charge of antiviral composition downstream of the stomach, such
as from the
jejunum, ileum, small intestine, and/or large intestine (colon). Enterically
coated dosage forms
will release antiviral composition into the systemic circulation within 1-10
hr after oral
administration.
[00213] The antiviral composition can be included in a rapid release,
immediate release,
controlled release, sustained release, prolonged release, extended release,
burst release,
continuous release, slow release, or pulsed release dosage form or in a dosage
form that exhibits
two or more of those types of release. The release profile of antiviral
composition from the
dosage form can be a zero order, pseudo-zero, first order, pseudo-first order
or sigmoidal release
Date Recue/Date Received 2021-04-13
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profile. The plasma concentration profile for triterpene in a subject to which
the antiviral
composition is administered can exhibit one or more maxima.
[00214] Based on human clinical data it is anticipated that 50% to 75% of an
administered
dose of oleandrin will be orally bioavailable therefore providing about 10 to
about 20 microg,
about 20 to about 40 microg, about 30 to about 50 microg, about 40 to about 60
microg, about
50 to about 75 microg, about 75 to about 100 microg of oleandrin per dosage
form. Given an
average blood volume in adult humans of 5 liters, the anticipated oleandrin
plasma
concentration will be in the range of about 0.05 to about 2 ng/ml, about 0.005
to about 10
ng/mL, about 0.005 to about 8 ng/mL, about 0.01 to about 7 ng/mL, about 0.02
to about 7
ng/mL, about 0.03 to about 6 ng/mL, about 0.04 to about 5 ng/mL, or about 0.05
to about 2.5
ng/mL. The recommended daily dose of oleandrin, present in the SCF extract, is
generally
about 0.2 microg to about 4.5 microg/kg body weight twice daily. The dose of
oleandrin can
be about 0.2 to about 1 microg/kg body weight/day, about 0.5 to about 1.0
microg/kg body
weight/day, about 0.75 to about 1.5 microg/kg body weight/day, about 1.5 to
about 2.52
microg/kg body weight/day, about 2.5 to about 3.0 microg/kg body weight/day,
about 3.0 to
4.0 microg/kg body weight/day or about 3.5 to 4.5 microg oleandrin/kg body
weight/day. The
maximum tolerated dose of oleandrin can be about about 3.5 microg/kg body
weight/day to
about 4.0 microg/kg body weight/day. The minimum effective dose can be about
0.5
microg/day, about 1 microg/day, about 1.5 microg/day, about 1.8 microg/day,
about 2
microg/day, or about 5 microg/day.
[00215] The antiviral composition can be administered at low to high dose due
to the
combination of triterpenes present and the molar ratio at which they are
present. A
therapeutically effective dose for humans is about 100-1000 mg or about 100-
1000 microg of
antiviral composition per Kg of body weight. Such a dose can be administered
up to 10 times
in a 24-hour period. Other suitable dosing ranges are specified below.
Date Recue/Date Received 2021-04-13
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Composition Oleandrin Oleanolic Ursolic Betulinic Suitable
(moles) acid acid acid dose
(moles) (moles) (moles)
A 0.5-1.5 4-6 - - 0.05 to 0.5
mg/kg/day
B 0.5-1.5 4-6 4-6 - 0.05 to 0.35
mg/kg/day
C 0.5-1.5 4-6 4-6 0.1-1 0.05 to 0.22
(PBI-05204) mg/kg/day
D 0.5-1.5 - 4-6 - 0.05 to 0.4
mg/kg/day
E 0.5-1.5 - - 0.1-1 0.05 to 0.4
mg/kg/day
AA About 1 - - 0.3-0.7 0.05 to 0.4
mg/kg/day
AB About 1 About 4.7 - - 0.05 to 0.5
mg/kg/day
AC About 1 About 4.7 About 4.5 - 0.05 to 0.4
mg/kg/day
AD About 1 About 4.7 About 4.5 About 0.6 0_05 to 0_22
(PBI-05204) mg/kg/day
AE About 1 - About 4.5 - 0.05 to 0.4
mg/kg/day
AF About 1 - - About 0.6 0.05 to 0.3
mg/kg/day
All values are approximate, meaning "about" the specified value.
[00216] It should be noted that a compound herein might possess one or more
functions in a
composition or formulation of the invention. For example, a compound might
serve as both a
surfactant and a water miscible solvent or as both a surfactant and a water
immiscible solvent.
Date Recue/Date Received 2021-04-13
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[00217] A liquid composition can comprise one or more pharmaceutically
acceptable liquid
carriers. The liquid carrier can be an aqueous, non-aqueous, polar, non-polar,
and/or organic
carrier. Liquid carriers include, by way of example and without limitation, a
water miscible
solvent, water immiscible solvent, water, buffer and mixtures thereof.
[00218] As used herein, the terms "water soluble solvent" or "water miscible
solvent", which
terms are used interchangeably, refer to an organic liquid which does not form
a biphasic
mixture with water or is sufficiently soluble in water to provide an aqueous
solvent mixture
containing at least five percent of solvent without separation of liquid
phases. The solvent is
suitable for administration to humans or animals. Exemplary water soluble
solvents include,
by way of example and without limitation, PEG (poly(ethylene glycol)), PEG 400
(poly(ethylene glycol having an approximate molecular weight of about 400),
ethanol, acetone,
alkanol, alcohol, ether, propylene glycol, glycerin, triacetin, poly(propylene
glycol), PVP
(poly(vinyl pyrrolidone)), dimethylsulfoxide, N,N-dimethylformamide,
formamide, N,N-
dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-
pyrrolidone),
pharmasolve (N-methyl-2-pyrrolidone).
[00219] As used herein, the terms "water insoluble solvent" or "water
immiscible solvent",
which terms are used interchangeably, refer to an organic liquid which forms a
biphasic mixture
with water or provides a phase separation when the concentration of solvent in
water exceeds
five percent. The solvent is suitable for administration to humans or animals.
Exemplary water
insoluble solvents include, by way of example and without limitation,
medium/long chain
triglycerides, oil, castor oil, corn oil, vitamin E, vitamin E derivative,
oleic acid, fatty acid, olive
oil, softisan 645 (Diglyceryl Caprylate / Caprate / Stearate / Hydroxy
stearate adipate), miglyol,
captex (Captex 350: Glyceryl Tricaprylate/ Caprate/ Laurate triglyceride;
Captex 355: Glyceryl
Tricaprylate/ Caprate triglyceride; Captex 355 EP / NF: Glyceryl Tricaprylate/
Caprate medium
chain triglyceride).
[00220] Suitable solvents are listed in the "International Conference on
Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)
guidance
for industry Q3C Impurities: Residual Solvents" (1997), which makes
recommendations as to
what amounts of residual solvents are considered safe in pharmaceuticals.
Exemplary solvents
are listed as class 2 or class 3 solvents. Class 3 solvents include, for
example, acetic acid,
Date Recue/Date Received 2021-04-13
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acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butlymethyl ether,
cumene, ethanol,
ethyl ether, ethyl acetate, ethyl formate, formic acid, heptane, isobutyl
acetate, isopropyl
acetate, methyl acetate, methyl-1 -butanol, methylethyl ketone, methylisobutyl
ketone, 2-
methyl-1 -propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, or propyl
acetate.
[00221] Other materials that can be used as water immiscible solvents in the
invention
include: Captex 100: Propylene Glycol Dicaprate; Captex 200: Propylene Glycol
Dicaprylate/
Dicaprate; Captex 200 P: Propylene Glycol Dicaprylate/ Dicaprate; Propylene
Glycol
Dicaprylocaprate; Captex 300: Glyceryl Tricaprylate/ Caprate; Captex 300 EP /
NF: Glyceryl
Tricaprylate/ Caprate Medium Chain Triglycerides; Captex 350: Glyceryl
Tricaprylate/
Caprate/ Laurate; Captex 355: Glyceryl Tricaprylate/ Caprate; Captex 355 EP /
NF: Glyceryl
Tricaprylate/ Caprate Medium Chain Triglycerides; Captex 500: Triacetin;
Captex 500 P:
Triacetin (Pharmaceutical Grade); Captex 800: Propylene Glycol Di (2-
Ethythexanoate);
Captex 810 D: Glyceryl Tricaprylate/ Caprate/ Linoleate; Captex 1000: Glyceryl
Tricaprate;
Captex CA: Medium Chain Triglycerides; Captex MCT-170: Medium Chain
Triglycerides;
Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP/NF: Glyceryl Monooleate;
Capmul
MCM: Medium Chain Mono- & Diglycerides; Capmul MCM C8: Glyceryl Monocaprylate;
Capmul MCM C10: Glyceryl Monocaprate; Capmul PG-8: Propylene Glycol
Monocaprylate;
Capmul PG-12: Propylene Glycol Monolaurate; Caprol 10G100: Decaglycerol
Decaoleate;
Caprol 3G0: Triglycerol Monooleate; Caprol ET: Polyglycerol Ester of Mixed
Fatty Acids;
Caprol MPGO: Hexaglycerol Dioleate; Caprol PGE 860: Decaglycerol Mono-,
Dioleate.
[00222] As used herein, a "surfactant" refers to a compound that comprises
polar or charged
hydrophilic moieties as well as non-polar hydrophobic (lipophilic) moieties;
i.e., a surfactant is
amphiphilic. The term surfactant may refer to one or a mixture of compounds. A
surfactant
can be a solubilizing agent, an emulsifying agent or a dispersing agent. A
surfactant can be
hydrophilic or hydrophobic.
[00223] The hydrophilic surfactant can be any hydrophilic surfactant suitable
for use in
pharmaceutical compositions. Such surfactants can be anionic, cationic,
zwitterionic or non-
ionic, although non-ionic hydrophilic surfactants are presently preferred. As
discussed above,
these non-ionic hydrophilic surfactants will generally have HLB values greater
than about 10.
Mixtures of hydrophilic surfactants are also within the scope of the
invention.
Date Recue/Date Received 2021-04-13
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[00224] Similarly, the hydrophobic surfactant can be any hydrophobic
surfactant suitable for
use in pharmaceutical compositions. In general, suitable hydrophobic
surfactants will have an
HLB value less than about 10. Mixtures of hydrophobic surfactants are also
within the scope of
the invention.
[00225] Examples of additional suitable solubilizer include: alcohols and
polyols, such as
ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene
glycol, butanediols
and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol,
transcutol, dimethyl
isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol,
hydroxypropyl
methylcellulose and other cellulose derivatives, cyclodextrins and
cyclodextrin derivatives;
ethers of polyethylene glycols having an average molecular weight of about 200
to about 6000,
such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, available
commercially from BASF
under the trade name Tetraglycol) or methoxy PEG (Union Carbide); amides, such
as 2-
pyrrolidone, 2-piperidone, caprolactam, N-alkylpyrrolidone, N-
hydroxyalkylpyrrolidone, N-
alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, and
polyvinypyrrolidone; esters,
such as ethyl propionate, tributylcitrate,acetyl triethylcitrate, acetyl
tributyl citrate,
triethylcitrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin,
propylene glycol
monoacetate, propylene glycol diacetate, caprolactone and isomers thereof,
valerolactone and
isomers thereof, butyrolactone and isomers thereof; and other solubilizers
known in the art,
such as dimethyl acetamide, dimethyl isosorbide (Arias lve DMI (ICI)), N-
methyl pyrrolidones
(Pharmasolve (ISP)), monooctanoin, diethylene glycol nonoethyl ether
(available from
Gattefosse under the trade name Transcutol), and water. Mixtures of
solubilizers are also within
the scope of the invention.
[00226] Except as indicated, compounds mentioned herein are readily available
from
standard commercial sources.
[00227] Although not necessary, the composition or formulation may further
comprise one
or more chelating agents, one or more preservatives, one or more antioxidants,
one or more
adsorbents, one or more acidifying agents, one or more alkalizing agents, one
or more
antifoaming agents, one or more buffering agents, one or more colorants, one
or more
electrolytes, one or more salts, one or more stabilizers, one or more tonicity
modifiers, one or
more diluents, or a combination thereof.
Date Recue/Date Received 2021-04-13
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[00228] The composition of the invention can also include oils such as fixed
oils, peanut oil,
sesame oil, cottonseed oil, corn oil and olive oil; fatty acids such as oleic
acid, stearic acid and
isostearic acid; and fatty acid esters such as ethyl oleate, isopropyl
myristate, fatty acid
glycerides and acetylated fatty acid glycerides. The composition can also
include alcohol such
as ethanol, isopropanol, hexadecyl alcohol, glycerol and propylene glycol;
glycerol ketals such
as 2,2-dimethy1-1,3-dioxolane-4-methanol; ethers such as poly(ethylene glycol)
450; petroleum
hydrocarbons such as mineral oil and petrolatum; water; a pharmaceutically
suitable surfactant,
suspending agent or emulsifying agent; or mixtures thereof.
[00229] It should be understood that the compounds used in the art of
pharmaceutical
formulation generally serve a variety of functions or purposes. Thus, if a
compound named
herein is mentioned only once or is used to define more than one term herein,
its purpose or
function should not be construed as being limited solely to that named
purpose(s) or function(s).
[00230] One or more of the components of the formulation can be present in its
free base,
free acid or pharmaceutically or analytically acceptable salt form. As used
herein,
"pharmaceutically or analytically acceptable salt" refers to a compound that
has been modified
by reacting it with an acid as needed to form an ionically bound pair.
Examples of acceptable
salts include conventional non-toxic salts formed, for example, from non-toxic
inorganic or
organic acids. Suitable non-toxic salts include those derived from inorganic
acids such as
hydrochloric, hydrobromic, sulfuric, sulfonic, sulfamic, phosphoric, nitric
and others known to
those of ordinary skill in the art. The salts prepared from organic acids such
as amino acids,
acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, pamoic,
maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,
2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,
isethionic, and others
known to those of ordinary skill in the art. On the other hand, where the
pharmacologically
active ingredient possesses an acid functional group, a pharmaceutically
acceptable base is
added to form the pharmaceutically acceptable salt. Lists of other suitable
salts are found in
Remington 's Pharmaceutical Sciences, 17t1i. ed., Mack Publishing Company,
Easton, PA, 1985,
p. 1418.
[00231] The phrase "pharmaceutically acceptable" is employed herein to refer
to those
compounds, materials, compositions, and/or dosage forms which are, within the
scope of sound
Date Recue/Date Received 2021-04-13
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medical judgment, suitable for use in contact with tissues of human beings and
animals and
without excessive toxicity, irritation, allergic response, or any other
problem or complication,
commensurate with a reasonable benefit/risk ratio.
[00232] A dosage form can be made by any conventional means known in the
pharmaceutical
industry. A liquid dosage form can be prepared by providing at least one
liquid carrier and
antiviral composition in a container. One or more other excipients can be
included in the liquid
dosage form. A solid dosage form can be prepared by providing at least one
solid carrier and
antiviral composition. One or more other excipients can be included in the
solid dosage form.
[00233] A dosage form can be packaged using conventional packaging equipment
and
materials. It can be included in a pack, bottle, via, bag, syringe, envelope,
packet, blister pack,
box, ampoule, or other such container.
[00234] The composition of the invention can be included in any dosage form.
Particular
dosage forms include a solid or liquid dosage forms. Exemplary suitable dosage
forms include
tablet, capsule, pill, caplet, troche, sache, and other such dosage forms
known to the artisan of
ordinary skill in the pharmaceutical sciences.
[00235] In view of the above description and the examples below, one of
ordinary skill in the
art will be able to practice the invention as claimed without undue
experimentation. The
foregoing will be better understood with reference to the following examples
that detail certain
procedures for the preparation of embodiments of the present invention. All
references made
to these examples are for the purposes of illustration. The following examples
should not be
considered exhaustive, but merely illustrative of only a few of the many
embodiments
contemplated by the present invention.
[00236] Vero CCL81 cells were used for the prophylactic and therapeutic assays
(ATCC,
Manassas, VA). Plaque assays were performed in Vero E6 cells, kindly provided
by Vineet
Menachery (UTMB, Galveston, TX). The cells were maintained in a 37 C incubator
with 5%
CO2. Cells were propagated utilizing a Dulbecco's Modified Eagle Medium
(Gibco, Grand
Island, NY) supplemented with 5% fetal bovine serum (FBS) (Atlanta
Biologicals,
Lawrenceville, GA) and 1% penicillium/streptomycin (Gibco, Grand Island, NY).
Maintenance media reduced the FBS to 2%, but was otherwise identical. SARS-CoV-
2, strain
USA WA1/2020 (Genbank accession MT020880), was provided by the World Reference
Date Recue/Date Received 2021-04-13
- 65 -
Center for Emerging Viruses and Arboviruses. All studies utilized a NextGen
sequenced Vero
passage 4 stock of SARS-CoV-2.
Example 1
Supercritical fluid extraction of powdered oleander leaves
Method A. With carbon dioxide.
[00237] Powdered oleander leaves were prepared by harvesting, washing, and
drying
oleander leaf material, then passing the oleander leaf material through a
comminuting and
dehydrating apparatus such as those described in U.S. Patent Nos. 5,236,132,
5,598,979,
6,517,015, and 6,715,705. The weight of the starting material used was 3.94
kg.
[00238] The starting material was combined with pure CO2 at a pressure of 300
bar (30 MPa,
4351 psi) and a temperature of 50 C (122 F) in an extractor device. A total of
197 kg of CO2
was used, to give a solvent to raw material ratio of 50:1. The mixture of CO2
and raw material
was then passed through a separator device, which changed the pressure and
temperature of the
mixture and separated the extract from the carbon dioxide.
[00239] The extract (65 g) was obtained as a brownish, sticky, viscous
material having a nice
fragrance. The color was likely caused by chlorophyll and other residual
chromophoric
compounds. For an exact yield determination, the tubes and separator were
rinsed out with
acetone and the acetone was evaporated to give an addition 9 g of extract. The
total extract
amount was 74 g. Based on the weight of the starting material, the yield of
the extract was
1.88%. The content of oleandrin in the extract was calculated using high
pressure liquid
chromatography and mass spectrometry to be 560.1 mg, or a yield of 0.76%.
Method B. With mixture of carbon dioxide and ethanol
[00240] Powdered oleander leaves were prepared by harvesting, washing, and
drying
oleander leaf material, then passing the oleander leaf material through a
comminuting and
dehydrating apparatus such as those described in U.S. Patent Nos. 5,236,132,
5,598,979,
6,517,015, and 6,715,705. The weight of the starting material used was 3.85
kg.
[00241] The starting material was combined with pure CO2 and 5% ethanol as a
modifier at
a pressure of 280 bar (28 MPa, 4061 psi) and a temperature of 50 C (122 F) in
an extractor
Date Recue/Date Received 2021-04-13
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device. A total of 160 kg of CO2 and 8 kg ethanol was used, to give a solvent
to raw material
ratio of 43.6 to 1. The mixture of CO2, ethanol, and raw material was then
passed through a
separator device, which changed the pressure and temperature of the mixture
and separated the
extract from the carbon dioxide.
[00242] The extract (207 g) was obtained after the removal of ethanol as a
dark green, sticky,
viscous mass obviously containing some chlorophyll. Based on the weight of the
starting
material, the yield of the extract was 5.38%. The content of oleandrin in the
extract was
calculated using high pressure liquid chromatography and mass spectrometry to
be 1.89 g, or a
yield of 0.91%.
Example 2
Hot-water extraction of powdered oleander leaves_
(comparative example)
[00243] Hot water extraction is typically used to extract oleandrin and other
active
components from oleander leaves. Examples of hot water extraction processes
can be found in
U.S. Patent Nos. 5,135,745 and 5,869,060.
[00244] A hot water extraction was carried out using 5 g of powdered oleander
leaves. Ten
volumes of boiling water (by weight of the oleander starting material) were
added to the
powdered oleander leaves and the mixture was stirred constantly for 6 hours.
The mixture was
then filtered and the leaf residue was collected and extracted again under the
same conditions.
The filtrates were combined and lyophilized. The appearance of the extract was
brown. The
dried extract material weighed about 1.44 g. 34.21 mg of the extract material
was dissolved in
water and subjected to oleandrin content analysis using high pressure liquid
chromatography
and mass spectrometry. The amount of oleandrin was determined to be 3.68 mg.
The oleandrin
yield, based on the amount of extract, was calculated to be 0.26%.
Example 3
Preparation of pharmaceutical compositions.
Method A. Cremophor-based drug delivery system
[00245] The following ingredients were provided in the amounts indicated.
Date Recue/Date Received 2021-04-13
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Reagent Percent of Formulation
Name Function (% w/w)
Antiviral composition Active agent 3.7
Vitamin E Antioxidant 0.1
Labrasol Surfactant 9.2
Ethanol Co-solvent 9.6
Cremophor EL Surfactant 62.6
Cremophor RH40 Surfactant 14.7
[00246] The excipients were dispensed into a jar and shook in a New Brunswick
Scientific
C24KC Refrigerated Incubator shaker for 24 hours at 60 C to ensure
homogeneity. The
samples were then pulled and visually inspected for solubilization. Both the
excipients and
antiviral composition were totally dissolved for all formulations after 24
hours.
Method B. GMO/Cremophor-based drug delivery system
[00247] The following ingredients were provided in the amounts indicated.
Reagent Percent of Formulation
Name Function (% w/w)
antiviral composition Active agent 4.7
Vitamin E Antioxidant 0.1
Labrasol Surfactant 8.5
Ethanol Co-solvent 7.6
Cremophor EL Surfactant 56.1
Glycerol Monooleate Surfactant 23.2
[00248] The procedure of Method A was followed.
Method C. Labrasol-based drug delivery system
Date Recue/Date Received 2021-04-13
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[00249] The following ingredients were provided in the amounts
indicated.
Reagent
Name Function Percent of Formulation (% w/w)
antiviral composition Active agent 3.7
Vitamin E Antioxidant 0.1
Labrasol Surfactant 86.6
Ethanol Co-solvent 9.6
[00250] The procedure of Method A was followed.
Method D. Vitamin E-TPG,S' based micelle forming system
[00251] The following ingredients were provided in the amounts indicated.
Component Function Weight % (w/w)
Vitamin E Antioxidant 1.0
Vitamin E TPGS Surfactant 95.2
antiviral composition Active agent 3.8
[00252] The procedure of Method A was followed.
Method E. Multi-component drug delivery system
[00253] The following ingredients were provided in the amounts indicated.
Date Recue/Date Received 2021-04-13
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Component Weight (g) Weight % (w/w)
Vitamin E 10.0 1.0
Cremophor ELP 580.4 55.9
Labrasol 89.0 8.6
Glycerol Monooleate 241.0 23.2
Ethanol 80.0 7.7
antiviral composition 38.5 3.7
Total 1038.9 100
[00254] The procedure of Method A was followed.
Method F. Multi-component drug delivery system
[00255] The following ingredients were provided in the amounts indicated an
included in a
capsule.
Component Tradename Weight % (w/w)
antiviral composition FLAVEX Naturextrakte 0.6
Vitamin E 1.3
Caprylocaproyl Labrasol
polyoxyglycerides Gattefosse 3074TPD 11.1
Lauroyl Gelucire 44/14
polyoxyglycerides Gattefosse 3061TPD 14.6
Polyoxyl 35 Castor Kolliphor
oil BASF Corp. 50251534 72.4
Total 100
[00256] The procedure of Method A was followed.
Example 4
Preparation of enteric coated capsules
Step I. Preparation of liquid-filled capsule
[00257] Hard gelatin capsules (50 counts, 00 size) were filled with a liquid
composition of
Example 3. These capsules were manually filled with 800 mg of the formulation
and then
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sealed by hand with a 50% ethanol/ 50% water solution. The capsules were then
banded by
hand with 22% gelatin solution containing the following ingredients in the
amounts indicated.
Ingredient Wt. (g)
Gelatin 140.0
Polysorbate 80 6.0
Water 454.0
Total 650.0
[00258] The gelatin solution mixed thoroughly and allowed to swell for 1-2
hours. After the
swelling period, the solution was covered tightly and placed in a 55 C oven
and allowed to
liquefy. Once the entire gelatin solution was liquid, the banding was
performed
[00259] Using a pointed round 3/0 artist brush, the gelatin solution was
painted onto the
capsules. Banding kit provided by Shionogi was used. After the banding, the
capsules were
kept at ambient conditions for 12 hours to allow the band to cure.
Step II: Coating of liquid-filled capsule
[00260] A coating dispersion was prepared from the ingredients listed in the
table below.
Ingredient Wt.% Solids % Solids (g) g/Batch
Eudragit L30D55 40.4 60.5 76.5 254.9
TEC 1.8 9.0 11.4 11.4
AlTalc 500V 6.1 30_5 38_5 38_5
Water 51.7 na na 326.2
Total 100.0 100.0 126.4 631.0
[00261] If banded capsules according to Step I were used, the dispersion was
applied to the
capsules to a 20.0 mg/cm2 coating level. The following conditions were used to
coat the
capsules.
Parameters Set-up
Coating Equipment Vector LDCS-3
Batch Size 500 g
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Parameters Set-up
Inlet Air Temp. 40 C
Exhaust Air Temp. 27-30 C
Inlet Air Volume 20-25 CFM
Pan Speed 20 rpm
Pump Speed 9 rpm (3.5 to 4.0 g/min)
Nozzle Pressure 15 psi
Nozzle diameter 1.0 mm
Distance from tablet bed* 2-3 in
* Spray nozzle was set such that both the nozzle and spray path were under the
flow path
of inlet air.
Example 5
Treatment of Zika virus infection in a subject
Method A. Antiviral Composition therapy
[00262] A subject presenting with Zika virus infection is prescribed antiviral
composition,
and therapeutically relevant doses are administered to the subject according
to a prescribed
dosing regimen for a period of time. The subject's level of therapeutic
response is determined
periodically. The level of therapeutic response can be determined by
determining the subject's
Zika virus titre in blood or plasma. If the level of therapeutic response is
too low at one dose,
then the dose is escalated according to a predetermined dose escalation
schedule until the
desired level of therapeutic response in the subject is achieved. Treatment of
the subject with
antiviral composition is continued as needed and the dose or dosing regimen
can be adjusted as
needed until the patient reaches the desired clinical endpoint.
Method B. Combination therapy: antiviral composition with another agent
[00263] Method A, above, is followed except that the subject is prescribed and
administered
one or more other therapeutic agents for the treatment of Zika virus infection
or symptoms
thereof. Then one or more other therapeutic agents can be administered before,
after or with
the antiviral composition. Dose escalation (or de-escalation) of the one or
more other
therapeutic agents can also be done.
Date Recue/Date Received 2021-04-13
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Example 6
In vitro Evaluation of Antiviral Activity Against Zika Virus Infection
Method A. Pure compound
[00264] Vero E6 cells (aso known as Vero C1008 cells, ATTC No. CRL-1586;
https://www.atcc.org/Products/All/CRL-1586.aspx) were infected with ZIKV (Zika
virus strain
PRVAB C59 ; ATCC VR-1843; https://www.atcc org/Products/All/VR-1843 .aspx) at
an M OI
(multiplicity of infection) of 0.2 in the presence of cardiac glycoside. Cells
were incubated with
virus and compound for 1 hr, after which the inoculum and compound were
discarded. Cells
were given fresh medium and incubated for 48hr, after which they were fixed
with formalin
and stained for ZIKV infection. Representative infection rates for oleandrin
(FIG. 1A) and
digoxin (FIG. 1B) as determined by scintigraphy are depicted. Other compounds
are evaluated
under the same conditions and exhibit very varying levels of antiviral
activity against Zika
virus.
Method B. Compound in Extract Form
[00265] An extract containing a target compound being tested is evaluated as
detailed in
Method A, except that the amount of extract is normalized to the amount of
target compound
in the extract. For example, an extract containing 2% wt of oleandrin contains
20 microg of
oleandrin per 1 mg of extract. Accordingly, if the intended amount of
oleandrin for evaluation
is 20 microg, then 1 mg of extract would be used in the assay.
Example 7
Preparation of a tablet comprising antiviral composition
[00266] An initial tabletting mixture of 3% Syloid 244FP and 97%
microcrystalline cellulose
(MCC) was mixed. Then, an existing batch of composition prepared according to
Example 3
was incorporated into the Syloid/MCC mixture via wet granulation. This mixture
is labeled
"Initial Tabletting Mixture) in the table below. Additional MCC was added
extra-granularly to
increase compressibility. This addition to the Initial Tabletting Mixture was
labeled as "Extra-
granular Addition." The resultant mixture from the extra-granular addition was
the same
composition as the "Final Tabletting Mixture."
Date Recue/Date Received 2021-04-13
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Component Weight (g) Weight % (w/w)
Initial Tabletting Mixture
Microcrystalline cellulose 48.5 74.2
Colloidal Silicon Dioxide/Syloid
244FP 1.5 2.3
Formulation from Ex. 3 15.351 23.5
Total 65.351 100.0
Extragranular addition
Component Weight (g) Weight % (w/w)
Initial Tabulating Mixture 2_5 50_0
Microcrystalline cellulose 2.5 50.0
Total 5 100.0
Final Tabletting Mixture:
Abbreviated
Component Weight (g) Weight % (w/w)
Microcrystalline cellulose 4.36 87.11
Colloidal Silicon Dioxide/Syloid
244FP 0.06 1.15
Formulation from Ex. 3 0.59 11.75
Total 5.00 100
Final Tabletting Mixture:
Detailed
Component Weight (g) Weight % (w/w)
Microcrystalline cellulose 4.36 87.11
Colloidal Silicon Dioxide/Syloid
244FP 0.06 1.15
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Vitamin E 0.01 0.11
Cremophor ELP 0.33 6.56
Labrasol 0.05 1.01
Glycerol Monooleate 0.14 2.72
Ethanol 0.05 0.90
SCF extract 0.02 0.44
Total 5.00 100.00
[00267] Syloid 244FP is a colloidal silicon dioxide manufactured by Grace
Davison.
Colloidal silicon dioxide is commonly used to provide several functions, such
as an adsorbant,
glidant, and tablet disintegrant. Syloid 244FP was chosen for its ability to
adsorb 3 times its
weight in oil and for its 5.5 micron particle size.
Example 8
HPLC analysis of solutions containing oleandrin
[00268] Samples (oleandrin standard, SCF extract and hot-water extract) were
analyzed on
HPLC (Waters) using the following conditions: Symmetry C18 column (5.0 m, 150
x4.6 mm
I.D.; Waters); Mobile phase of MeOH:water = 54: 46 (v/v) and flow rate at 1.0
ml/min.
Detection wavelength was set at 217 nm. The samples were prepared by
dissolving the
compound or extract in a fixed amount of HPLC solvent to achieve an
approximate target
concentration of oleandrin. The retention time of oleandrin can be determined
by using an
internal standard. The concentration of oleandrin can be determined/
calibrated by developing
a signal response curve using the internal standard.
Example 9
Preparation of Pharmaceutical Composition
[00269] A pharmaceutical composition of the invention can be prepared any of
the following
methods. Mixing can be done under wet or dry conditions. The pharmaceutical
composition
can be compacted, dried or both during preparation. The pharmaceutical
composition can be
portioned into dosage forms.
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Method A.
[00270] At least one pharmaceutical excipient is mixed with at least one
antiviral compound
disclosed herein.
Method B.
[00271] At least one pharmaceutical excipient is mixed with at least two
antiviral compounds
disclosed herein.
Method C.
[00272] At least one pharmaceutical excipient is mixed with at least one
cardiac glycosides
disclosed herein.
Method D.
[00273] At least one pharmaceutical excipient is mixed with at least two
triterpenes disclosed
herein.
Method E.
[00274] At least one pharmaceutical excipient is mixed with at least one
cardiac glycoside
disclosed herein and at least two triterpenes disclosed herein.
Method D.
[00275] At least one pharmaceutical excipient is mixed with at least three
triterpenes
disclosed herein.
Example 10
Preparation of Triterpene Mixtures
[00276] The following compositions were made by mixing the specified
triterpenes in the
approximate molar ratios indicated.
Triterpene (Approximate Relative Molar Content)
Composition Oleanolic acid (0) Ursolic acid (U) Betulinic acid
(B)
I (A-C) 3 2.2 1
IT (A-C) 7.8 7.4 1
III (A-C) 10 1 1
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Triterpene (Approximate Relative Molar Content)
Composition Oleanolic acid (0) Ursolic acid (U) Betulinic acid
(B)
IV (A-C) 1 10 1
V (A-C) 1 1 10
VI (A-C) 1 1 0
VII (A-C) 1 1 1
VIII (A-C) 10 1 0
IX (A-C) 1 10 0
[00277] For each composition, three different respective solutions were made,
whereby the
total concentration of triterpenes in each solution was approximately 9 M, 18
JIM, or 36 M.
Composition Triterpene (Approximate Content of Each, M)
(total triterpene Oleanolic acid (0) Ursolic acid (U)
Betulinic acid (B)
content, M)
I-A (36) 17.4 12.8 5.8
I-B (18) 8.7 6.4 2.9
I-C (9) 4.4 3.2 1.5
II-A (36) 17.3 16.4 2.2
II-B (18) 8.7 8.2 1.1
IT-C(9) 4.3 4.1 0.6
III-A (36) 30 3 3
III-B (18) 15 1.5 1.5
HI-C (9) 7.5 0.75 0.75
IV-A (36) 3 30 3
IV-B(18) 1.5 15 1.5
IV-C (9) 0.75 7.5 0.75
V-A (36) 3 3 30
V-B (18) 1.5 1.5 15
V-C (9) 0.75 0.75 7.5
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Composition Triterpene (Approximate Content of Each, M)
(total triterpene Oleanolic acid (0) Ursolic acid (U)
Betulinic acid (B)
content, 04)
VI-A (36) 18 18 0
VI-B (18) 9 9 0
VI-C (9) 4.5 4.5 0
VIT-A(36) 12 12 12
VII-B(18) 6 6 6
VII-C (9) 3 3 3
VIII-A (36) 32.7 3.3 0
VIII-B(18) 16.35 1.65 0
VIII-C (9) 8.2 0.8 0
IX-A (36) 3.3 32.7 0
IX-B (18) 1.65 16.35 0
IX-C (9) 0.8 8.2 0
Example 11
Preparation of Antiviral Compositions
[00278] Antiviral compositions can be prepared by mixing the individual
triterpene
components thereof to form a mixture. The triterpene mixtures prepared above
that provided
acceptable antiviral activity were formulated into antiviral compositions.
Antiviral composition with oleanolic acid and ursolic acid
[00279] Known amounts of oleanolic acid and ursolic acid were mixed according
to a
predetermined molar ratio of the components as defined herein. The components
were mixed
in solid form or were mixed in solvent(s), e.g. methanol, ethanol, chloroform,
acetone,
propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF),
dimethylacetamide
(DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resultant
mixture
contained the components in the relative molar ratios as described herein.
Date Recue/Date Received 2021-04-13
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[00280] For a pharmaceutically acceptable antiviral composition, at least one
pharmaceutically acceptable excipient was mixed in with the pharmacologically
active agents.
An antiviral composition is formulated for administration to a mammal.
Antiviral composition with oleanolic acid and betulinic acid
[00281] Known amounts of oleanolic acid and betulinic acid were mixed
according to a
predetermined molar ratio of the components as defined herein. The components
were mixed
in solid form or were mixed in solvent(s), e.g. methanol, ethanol, chloroform,
acetone,
propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF),
dimethylacetamide
(DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resultant
mixture
contained the components in the relative molar ratios as described herein.
[00282] For a pharmaceutically acceptable antiviral composition, at least one
pharmaceutically acceptable excipient was mixed in with the pharmacologically
active agents.
An antiviral composition is formulated for administration to a mammal.
Antiviral composition with oleanolic acid, ursolic acid, and betulinic acid
[00283] Known amounts of oleanolic acid, ursolic acid and betulinic acid were
mixed
according to a predetermined molar ratio of the components as defined herein.
The components
were mixed in solid form or were mixed in solvent(s), e.g. methanol, ethanol,
chloroform,
acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF),
dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures
thereof. The
resultant mixture contained the components in the relative molar ratios as
described herein.
[00284] For a pharmaceutically acceptable antiviral composition, at least one
pharmaceutically acceptable excipient was mixed in with the pharmacologically
active agents.
An antiviral composition is formulated for administration to a mammal.
Antiviral composition with oleadrin, oleanolic acid, ursolic acid, and
betulinic acid
[00285] Known amounts of oleandrin oleanolic acid, ursolic acid and betulinic
acid were
mixed according to a predetermined molar ratio of the components as defined
herein. The
components were mixed in solid form or were mixed in solvent(s), e.g.
methanol, ethanol,
chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide
(DMF),
dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures
thereof. The
resultant mixture contained the components in the relative molar ratios as
described herein.
Date Recue/Date Received 2021-04-13
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[00286] For a pharmaceutically acceptable antiviral composition, at least one
pharmaceutically acceptable excipient was mixed in with the pharmacologically
active agents.
An antiviral composition is formulated for administration to a mammal.
Example 12
Treatment of Filovirus infection in a subject
[00287] Exemplary Filovirus infections include Ebolavirus and Marburgvirus.
Method A. Antiviral Composition therapy
[00288] A subject presenting with Filovirus infection is prescribed antiviral
composition, and
therapeutically relevant doses are administered to the subject according to a
prescribed dosing
regimen for a period of time. The subject's level of therapeutic response is
determined
periodically. The level of therapeutic response can be determined by
determining the subject's
Filovirus titre in blood or plasma. If the level of therapeutic response is
too low at one dose,
then the dose is escalated according to a predetermined dose escalation
schedule until the
desired level of therapeutic response in the subject is achieved. Treatment of
the subject with
antiviral composition is continued as needed and the dose or dosing regimen
can be adjusted as
needed until the patient reaches the desired clinical endpoint.
Method B. Combination therapy: antiviral composition with another agent
[00289] Method A, above, is followed except that the subject is prescribed and
administered
one or more other therapeutic agents for the treatment of Filovirus infection
or symptoms
thereof. Then one or more other therapeutic agents can be administered before,
after or with
the antiviral composition. Dose escalation (or de-escalation) of the one or
more other
therapeutic agents can also be done.
Example 13
Treatment of Flavivirus infection in a subject
[00290] Exemplary Flavivirus infections include Yellow Fever, Dengue Fever,
Japanese
Enchephalitis, West Nile Viruses, Zikavirus, Tick-borne Encephalitis, Kyasanur
Forest
Disease, Alkhurma Disease, Chikungunya virus, Omsk Hemorrhagic Fever, Powassan
virus
infection.
Date Recue/Date Received 2021-04-13
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Method A. Antiviral Composition therapy
[00291] A subject presenting with Flavivirus infection is prescribed antiviral
composition,
and therapeutically relevant doses are administered to the subject according
to a prescribed
dosing regimen for a period of time. The subject's level of therapeutic
response is determined
periodically. The level of therapeutic response can be determined by
determining the subject's
Flavivirus titre in blood or plasma. If the level of therapeutic response is
too low at one dose,
then the dose is escalated according to a predetermined dose escalation
schedule until the
desired level of therapeutic response in the subject is achieved. Treatment of
the subject with
antiviral composition is continued as needed and the dose or dosing regimen
can be adjusted as
needed until the patient reaches the desired clinical endpoint.
Method B. Combination therapy: antiviral composition with another agent
[00292] Method A, above, is followed except that the subject is prescribed and
administered
one or more other therapeutic agents for the treatment of Flavivirus infection
or symptoms
thereof. Then one or more other therapeutic agents can be administered before,
after or with
the antiviral composition. Dose escalation (or de-escalation) of the one or
more other
therapeutic agents can also be done.
Example 14
Evaluation of antiviral activity against Zikavirus and Dengue virus
[00293] A CPE-based antiviral assay was performed by infecting target cells in
the presence
or absence of test compositions, at a range of concentrations. Infection of
target cells by results
in cytopathic effects and cell death. In this type of assay, reduction of CPE
in the presence of
test composition, and the corresponding increase in cell viability, is used as
an indicator of
antiviral activity. For CPE-based assays, cell viability was determined with a
neutral red
readout. Viable cells incorporate neutral red in their lysosomes. Uptake of
neutral red relies on
the ability of live cells to maintain a lower pH inside their lysosomes than
in the cytoplasm, and
this active process requires ATP. Once inside the lysosome, the neutral red
dye becomes
charged and is retained intracellularly. After a 3-hour incubation with
neutral red (0.033%), the
extracellular dye was removed, cells were washed with PBS, and the
intracellular neutral red
was solubilized with a solution of 50% ethanol + 1% acetic acid. The amount of
neutral red in
solution was quantified by reading the absorbance (optical density) of each
well at 490 nm
Date Recue/Date Received 2021-04-13
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[00294] Adherent cell lines were used to evaluate the antiviral activity of
compositions
against a panel of viruses. Compositions were pre-incubated with the target
cells for 30 min
before the addition of virus to the cells. The compositions were present in
the cell culture
medium for the duration of the infection incubation period. For each infection
assay, a viability
assay was set up in parallel using the same concentrations of compositions
(duplicates) to
determine cytotoxicity effects of the compositions in the absence of virus.
[00295] The antiviral activity of test compositions was determined by
comparing infection
levels (for immunostaining-based assay) or viability (for CPE-based assays) of
cells under test
conditions to the infection level or viability of uninfected cells. Cytotoxic
effects were evaluated
in uninfected cells by comparing viability in the presence of inhibitors to
the viability of mock-
treated cells. Cytotoxicity was determined by an XTT viability assay, which
was conducted at
the same timepoint as the readout for the corresponding infection assay.
[00296] Test compositions were dissolved in 100% methanol. Eight
concentrations of the
compositions were generated (in duplicate) by performing 8-fold dilutions,
starting with 50 [tM
as the highest concentration tested. The highest test concentration of
composition (50 [tM)
resulted in a 0.25% final concentration of methanol (v/v%) in the culture
medium. An 8-fold
dilution series of methanol vehicle was included in each assay plate, with
concentrations
minoring the final concentration of methanol in each composition test
condition. When
possible, the EC50 and CC50 of the composition was determined for each assay
using
GraphPad Prism software.
[00297] Antiviral activity was evaluated by the degree of protection against
virus-induced
cytopathic effects (CPE). Cells were challenged with virus in the presence of
different
concentrations of control or compositions. The extent of protection against
CPE was monitored
after 6 days (ZIKV, Zikavirus) or 7 days (DENV, Dengue virus) post infection
by quantifying
cell viability in different test conditions and comparing values with that of
untreated cells and
cells treated with vehicle alone (infection medium).
[00298] Quality controls for the neutralization assay were performed on every
plate to
determine: i) signal to background (S/B) values; ii) inhibition by the known
inhibitors, and iii)
variation of the assay, as measured by the coefficient of variation (C.V.) of
all data points.
Overall variation in the infection assays ranged from 3.4% to 9.5%, and
overall variation in the
Date Recue/Date Received 2021-04-13
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viability assays ranged from 1.4% to 3.2%, calculated as the average of all
C.V. values. The
signal-to-background (S/B) for the infection assays ranged from 2.9 to 11.0,
while the signal-
to-background (S/B) for the viability assays ranged from 6.5 to 29.9.
[00299] Protection of DENV2-induced cytopathic effect (CPE) with Neutral Red
readout:
For the DENV2 antiviral assay, the 08-10381 Montserrat strain was used. Viral
stocks were
generated in C6/36 insect cells. Vero cells (epithelial kidney cells derived
from Cercopithecus
aethiops) were maintained in MEM with 5% FBS (MEM5). For both the infection
and the
viability assays, cells were seeded at 10,000 cells per well in 96-well clear
flat bottom plates
and maintained in MEM5 at 37 C for 24 hours. The day of infection, samples
were diluted 8-
fold in U-bottom plates using MEM with 1% bovine serum albumin (BSA). Test
material
dilutions were prepared at 1.25X the final concentration and 400 were
incubated with the target
cells at 37 C for 30 minutes. Following the test material pre-incubation,
10111 of virus dilutions
prepared in MEM with 1% BSA was added to each well (500 final volume per well)
and plates
were incubated at 37 C in a humidified incubator with 5% CO2 for 3 hours. The
volume of
virus used in the assay was previously determined to produce a signal in the
linear range
inhibited by Ribavirin and compound A3, known inhibitors of DENV2. After the
infection
incubation, cells were washed with PBS, then MEM containing 2% FBS (MEM2) to
remove
unbound virus. Subsequently, 500 of medium containing inhibitor dilutions
prepared at a 1X
concentration in MEM2 was added to each well. The plate was incubated at 37 C
in the
incubator (5% CO2) for 7 days. Controls with no virus ("mock-infected'),
infected cells
incubated with medium alone, infected cells incubated with vehicle alone
(methanol), and wells
without cells (to determine background) were included in the assay plate.
Control wells
containing 5011M Ribavirin and 0.511M compound A3 were also included on the
assay plate.
After 7 days of infection, cells were stained with neutral red to monitor cell
viability. Test
materials were evaluated in duplicates using serial 8-fold dilutions in
infection medium.
Controls included cells incubated with no virus ("mock-infected"), infected
cells incubated with
medium alone, or infected cells in the presence of Ribavirin (0.511M) or A3
(0.511M). A full
duplicate inhibition curve with methanol vehicle only was included on the same
assay plate.
[00300] Protection of ZIKV-induced cytopathic effect (CPE) with Neutral Red
readout: For
the ZIKV antiviral assay, the PLCal ZV strain was used. Vero cells (epithelial
kidney cells
Date Recue/Date Received 2021-04-13
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derived from Cercopithecus aethiops) were maintained in MEM with 5% FBS
(MEM5). For
both the infection and the viability assays, cells were seeded at 10,000 cells
per well in 96-well
clear flat bottom plates and maintained in MEM5 at 37 C for 24 hours. The day
of infection,
samples were diluted 8-fold in U-bottom plates using MEM with 1% bovine serum
albumin
(BSA). Test material dilutions were prepared at 1.25X the final concentration
and 400 were
incubated with the target cells at 37 C for 30 minutes. Following the test
material pre-
incubation, 100 of virus dilutions prepared in MEM with 1% BSA was added to
each well
(500 final volume per well) and plates were incubated at 37 C in a humidified
incubator with
5% CO2 for 3 hours. After the infection incubation, cells were washed with
PBS, then MEM
containing 2% FBS (MEM2) to remove unbound virus. Subsequently, 500 of medium
containing inhibitor dilutions prepared at a 1X concentration in MEM2 was
added to each well.
The plate was incubated at 37 C in the incubator (5% CO2) for 6 days. Controls
with no virus
("mock-infected'), infected cells incubated with medium alone, infected cells
incubated with
vehicle alone (methanol), and wells without cells (to determine background)
were included in
the assay plate. After 6 days of infection, cells were stained with neutral
red to monitor cell
viability. Test materials were evaluated in duplicates using serial 8-fold
dilutions in infection
medium. Controls included cells incubated with no virus ("mock-infected"),
infected cells
incubated with medium alone, or infected cells in the presence of A3 (0.5pM).
A full duplicate
inhibition curve with methanol vehicle only was included on the same assay
plate.
[00301] Analysis of CPE-based viability data: for the neutral red assays, cell
viability was
determined by monitoring the absorbance at 490 nm. The average signal obtained
in wells with
no cells was subtracted from all samples. Then, all data points were
calculated as a percentage
of the average signal observed in the 8 wells of mock (uninfected) cells on
the same assay plate.
Infected cells treated with medium alone reduced the signal to an average of
4.2% (for HRV),
26.9% (for DENV), and 5.1% (for ZIKV) of that observed in uninfected cells.
The signal-to-
background (S/B) for this assay was 2.9 (for DENV), and 7.2 (for ZIKV),
determined as the
viability percentage in "mock-infected" cells compared to that of infected
cells treated with
vehicle only.
[00302] Viability assay (XTT) to assess compound-induced cytotoxicity: Mock-
infected cells
were incubated with inhibitor dilutions (or medium only) using the same
experimental setup
Date Recue/Date Received 2021-04-13
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and inhibitor concentrations as was used in the corresponding infection assay.
The incubation
temperature and duration of the incubation period mirrored the conditions of
the corresponding
infection assay. Cell viability was evaluated with an XTT method. The XTT
assay measures
mitochondrial activity and is based on the cleavage of yellow tetrazolium salt
(XTT), which
forms an orange formazan dye. The reaction only occurs in viable cells with
active
mitochondria. The formazan dye is directly quantified using a scanning multi-
well
spectrophotometer. Background levels obtained from wells with no cells were
subtracted from
all data-points. Controls with methanol vehicle alone (at 7 concentrations
mirroring the final
percent methanol of each Oleandrin test wells) were included in the viability
assay plate. The
extent of viability was monitored by measuring absorbance at 490 nm.
[00303] Analysis of cytotoxicity data: For the XTT assays, cell viability was
determined by
monitoring the absorbance at 490 nm. The average signal obtained in wells with
no cells was
subtracted from all samples. Then, all data points were calculated as a
percentage of the average
signal observed in the 8 wells of mock (uninfected) cells on the same assay
plate. The signal-
to-background (S/B) for this assay was 29.9 (for IVA), 8.7 (for HRV), 6.5 (for
DENV), and 6.7
(for ZIKV), determined as the viability percentage in "mock-infected" cells
compared to the
signal observed for wells without cells.
Example 15
Evaluation of antiviral activity against Filovirus (Ebolavirus and
Marburgvirus)
Method A.
[00304] Vero E6 cells were infected with EBOV/Kik (A, MOI=1) or MARV/Ci67 (B,
MOI=1) in the presence of oleandrin, digoxin or PBI-05204, an oleandrin-
containing plant
extract. After lhr, inoculum and compounds were removed and fresh medium added
to cells.
48hr later, cells were fixed and immunostained to detect cells infected with
EBOV or MARV.
Infected cells were enumerated using an Operetta. C) Vero E6 were treated with
compound as
above. ATP levels were measured by CellTiter-Glo as a measurement of cell
viability.
Method B.
[00305] Vero E6 cells were infected with EBOV (A,B) or MARV (C,D). At 2hr post-
infection (A,C) or 24hr post-infection (B,D), oleandrin or PBI-05204 was added
to cells for lhr,
Date Recue/Date Received 2021-04-13
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then discarded and cells were returned to culture medium. At 48hr post-
infection, infected cells
were analyzed as in Figure 1.
Method C.
[00306] Vero E6 cells were infected with EBOV or MARV in the presence of
oleandrin or
PBI-05204 and incubated for 48hr. Supernatants from infected cell cultures
were passaged onto
fresh Vero E6 cells, incubated for lhr, then discarded (as depicted in A).
Cells containing
passaged supernatant were incubated for 48hr. Cells infected with EBOV (B) or
MARV (C)
were detected as described previously. Control infection rates were 66% for
EBOV and 67%
for MARV.
Example 16
Evaluation of antiviral activity against Togaviri dae virus
(Alphavirus: VEEV and WEEV)
[00307] Vero E6 cells were infected with Venezuelan equine encephalitis virus
(A,
MO1=0.01) or Western equine encephalitis virus (B, MOI=0.1) for 18hr in the
presence or
absence of indicated compounds. Infected cells were detected as described
herein and
enumerated on an Operetta.
Example 17
Treatment of Paramyxoviridae infection in a subject
[00308] Exemplary Paramyxoviridae family viral infections include Henipavirus
genus
infection, Nipah virus infection, or Hendra virus infection.
Method A. Antiviral Composition therapy
[00309] A subject presenting with Paramyxoviridae family infection is
prescribed antiviral
composition, and therapeutically relevant doses are administered to the
subject according to a
prescribed dosing regimen for a period of time. The subject's level of
therapeutic response is
determined periodically. The level of therapeutic response can be determined
by determining
the subject's virus titre in blood or plasma. If the level of therapeutic
response is too low at one
dose, then the dose is escalated according to a predetermined dose escalation
schedule until the
desired level of therapeutic response in the subject is achieved. Treatment of
the subject with
Date Recue/Date Received 2021-04-13
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antiviral composition is continued as needed and the dose or dosing regimen
can be adjusted as
needed until the patient reaches the desired clinical endpoint.
Method B. Combination therapy: antiviral composition with another agent
[00310] Method A, above, is followed except that the subject is prescribed and
administered
one or more other therapeutic agents for the treatment of Paramyxoviridae
family infection or
symptoms thereof. Then one or more other therapeutic agents can be
administered before, after
or with the antiviral composition. Dose escalation (or de-escalation) of the
one or more other
therapeutic agents can also be done.
Example 18
Cell-lines and Isolation of Primary huPBMC's
[00311] The virus-producing HTLV-1-transformed (HTLV-1+) SLB1 lymphoma T-cell -
1 i n e
(Arnold et al., 2008; kindly provided by P. Green, The Ohio State University-
Comprehensive
Cancer Center) was cultured in a humidified incubator at 37 C under 10% CO2 in
Iscove's
Modified Dulbecco's Medium (IMDM; ATCC No. 30-2005), supplemented with 10%
heat-
inactivated fetal bovine serum (FBS; Biowest), 100 U/ml penicillin, 100 g/m1
streptomycin-
sulfate, and 20 ug/m1 gentamycin-sulfate (Life Technologies).
[00312] Primary human peripheral blood mononuclear cells (huPBMCs) were
isolated from
whole blood samples, provided without identifiers by the SMU Memorial Health
Center under
a protocol approved by the SMU Institutional Review Board and consistent with
Declaration
of Helsinki principles. In brief, 2 ml of whole blood was mixed with an equal
volume of sterile
phosphate-buffered saline (PBS), pH 7.4, in polypropylene conical tubes
(Corning) and then
the samples were gently layered over 3 ml of Lymphocyte Separation Medium (MP
Biomedicals). The samples were centrifuged for 30 min at 400 x g in a swinging
bucket rotor
at room temp. The buffy-coat huPBMCs were subsequently aspirated, washed 2X in
RPMI-
1640 medium (ATCC No. 30-2001), and pelleted by centrifugation for 7 min at
260 x g. The
cells were resuspended in RPMI-1640 medium, supplemented with 20% FBS, 100
U/ml
penicillin, 100 g/m1 streptomycin-sulfate, 20 g/ml gentamycin-sulfate, and
50 U/ml
recombinant human interleukin-2 (hu-IL-2; Roche Applied Science), and
stimulated for 24 hrs
with 10 ng/ml phytohemagglutinin (PHA; Sigma-Aldrich) and grown at 37 C under
10% CO2
in a humidified incubator. On the following day, the cells were pelleted by
centrifugation for 7
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min at 260 x g and washed 2X with RPMI-1640 medium to remove the PHA, and then
resuspended and cultured in complete medium, supplemented with antibiotics and
50 U/ml hu-
IL-2.
Example 19
Generation of GFP-expressing HTLV-1+SLB1/pLenti-GFP T-cell Clones
[00313] To generate the GFP-expressing HTLV-1+ SLB1 T-cell clones, 2x106 SLB1
cells
were plated in 60 mm2 tissue-culture dishes (Corning) in IMDM, supplemented
with 10% heat-
inactivated FBS and antibiotics, and then transduced with lentiviral particles
containing a
pLenti-6.2/V5-DEST-green fluorescent protein expression vector which also
carries a
blasticidin-resistance gene. After 6 hrs, the transduced cells were pelleted
by centrifugation for
7 min at 260 x g at room temperature, washed 2X with serum-free TMDM, and
resuspended in
complete medium supplemented with 5 tg/m1 blasticidin (Life Technologies) and
aliquoted
into 96-well microtiter plates (Corning). The cultures were maintained with
blasticidin-
selection for two weeks in a humidified incubator at 37-C and 10% CO2. The GFP-
expressing
lymphoblasts were screened by fluorescence-microscopy, and then plated by
limiting-dilution
in 96-well microtiter plates to obtain homogenous GFP-expressing cell clones.
The resulting
HTLV-1+ SLB1/pLenti-GFP T-lymphocyte clones were expanded and repeatedly
passaged;
and the expression of GFP was confirmed by sodium dodecyl sulfate-
polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblotting using a rabbit polyclonal Anti-
GFP (FL)
antibody (Santa Cruz Biotechnology).
Example 20
Quantitation of Virus Production and Particle Infectivity by Anti-HTLV-1 p 1
9Gag
ELISA' s
[00314] To determine the effects of oleandrin or an extract of N. oleander
upon HTLV-1
proviral replication and the release of newly-synthesized extracellular virus
particles, the
HTLV-1+ SLB1 lymphoma T-cell-line was plated at 2x104 cells per well in 300 1
of complete
medium, supplemented with antibiotics, in 96-well microtiter plates and
incubated at 37 C
under 10% CO2. The purified oleandrin compound and extract of N. oleander
(Phoenix
Biotechnology; see Singh et al., 2013) were resuspended in the Vehicle
solution (20% v/v
dimethyl sulfoxide, DMSO, in MilliQ distilled/deionized H20) at a stock
concentration of 2
Date Recue/Date Received 2021-04-13
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mg/m1 and then sterilized using a luer-lock 0.2 imn syringe filter
(Millipore). The HTLV-1+
SLB1 cells were treated with oleandrin or the N. oleander extract at
concentrations of 10, 50,
and 100 tg/ml, or with increasing amounts (1.5, 7.5, and 15 1) of the Vehicle
control for 72
hrs. The 96-well microtiter plates were then centrifuged for 7 min at 260 x g
at room temp using
an Eppendorf A-2-DWP swinging plate rotor to pellet the cells, and the levels
of extracellular
pl9G1g-containing HTLV-1 particles released into the culture supernatants were
quantified
relative to a p19Gag protein standard by performing colorimetric Anti-p19Gag
enzyme-linked
immunosorbent assays (ELISAs; Zeptometrix). The samples were analyzed with
triplicate
replicates on a Berthold Tristar LB 941 multimode microplate-reader at 450 nm
in absorbance
mode.
[00315] To assess the infectivity of newly-synthesized extracellular HTLV-1
particles
collected from oleandrin-treated cells, 2x104 HTLV-1+ SLB1 T-lymphoblasts were
plated in
300 1_11 of complete medium, supplemented with antibiotics, and the cultures
were treated for
72 hrs with increasing concentrations (10, 50, and 100 g/me of oleandrin or a
N. oleander
extract, or the Vehicle control (1.5, 7.5, and 15 1). Then, 50 1_11 of the
virus-containing
supernatants were used to directly infect huPBMCs plated at a density of 2x104
cells per well
on 96-well microtiter plates in complete medium, supplemented with antibiotics
and hu-IL-2.
The oleandrin compound, N. oleander extract, or Vehicle control were
maintained in the
huPBMCs culture medium to control for possible re-infection events by newly-
produced
particles. After 72 hrs, the relative levels of extracellular p19G1g-
containing HTLV-1 virions
released into the culture supernatants by the infected huPBMCs were quantified
through Anti-
HTLV-1 p19Gag ELISAs.
Example 21
Measuring Cellular Apoptosis
[00316] To assess the relative cytotoxicity of the oleandrin compound, extract
of N. oleander,
or the Vehicle control in treated cell cultures, 2x104 HTLV-1+ SLB1 lymphoma T-
cells or
activated/cultured huPBMCs were plated in 300 1_11 of complete medium,
supplemented with
antibiotics, and maintained at 37 C under 10% CO2 in a humidified incubator.
The cultures
were treated with either increasing concentrations (10, 50, and 100 tg/m1) of
oleandrin or N.
oleander extract, or the Vehicle control (1.5, 7.5, 15 ml) and incubated for
72 hrs.
Date Recue/Date Received 2021-04-13
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Cyclophosphamide (50 0/1; Sigma-Aldrich)-treated cells were included as a
positive control
for apoptosis. The cells were then aspirated and plated on Permanox 8-chamber
tissue-culture
slides (Nalge) that had been pre-treated with a sterile 0.01% solution of Poly-
t-Lysine and
Concanavalin A (1 mg/ml; Sigma-Aldrich). The samples were subsequently stained
using a
microscopy apoptosis detection kit with Annexin V conjugated to fluorescein
isothiocyanate
(Annexin V-FITC) and propidium iodide (PI; BD-Pharmingen), and the relative
percentages of
apoptotic (i.e., Annexin V-FITC and/or PI- positive) cells per field were
quantified in-triplicate
by confocal fluorescence-microscopy using a 20x objective lens. The total
numbers of cells per
field were quantified by microscopy using a DIC phase-contrast filter.
Example 22
HTLV-1 Transmission and Virological Synapse Formation in Co-culture Assays
[00317] As the transmission of HTLV-1 typically occurs through direct contact
between an
infected cell and uninfected target cell across a virological synapse (Igakura
et al., 2003; Pais-
Correia et al., 2010; Gross et al., 2016; Omsland et al., 2018; Majorovits et
al., 2008), we tested
whether oleandrin, a N. oleander extract, or the Vehicle control might
influence the formation
of virological synapses and/or the transmission of infectious HTLV-1 particles
via intercellular
interactions in vitro. For these experiments, 2x104 virus-producing HTLV-1+
SLB1 T-cells
were plated in 96-well microtiter plates and treated with mitomycin C (100
tg/m1) in 300 pi of
complete medium for 2 hrs at 37 C under 10% CO2 (Bryj a et al., 2006). The
culture media was
then removed, the cells were washed 2X with serum-free IMDM, and the cells
were treated for
either 15 min or 3 hrs with increasing amounts (10, 50, and 100 g/m1) of
oleandrin or N.
oleander extract, or the Vehicle control (1.5, 7.5, and 15 1). Alternatively,
2x104 of the GFP-
expressing HTLV-1+ SLB1/pLenti-GFP T-cells were plated on 8-chamber tissue-
culture slides
in 300 p1 of complete medium and treated with mitomycin C, washed 2X with
serum-free
IMDM, and then treated with oleandrin, N. oleander extract, or the Vehicle
control as described
for confocal microscopy experiments. We next aspirated the medium, washed the
HTLV-1+
SLB1 cells 2X with serum-free medium, and added 2x104 huPBMCs to each well in
300 p1 of
RPMI-1640 medium, supplemented with 20% FBS, antibiotics and 50U/m1 hu-IL-2,
and then
co-cultured the cells for another 72 hrs (the cells were co-cultured for 6 hrs
to visualize
virological synapse formation and viral transmission by confocal microscopy
using the
Date Recue/Date Received 2021-04-13
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SLB1/pLenti-GFP lymphoblasts) at 37 C under 10% CO2 in a humidified incubator.
As a
negative control, huPBMCs were cultured alone in the absence of virus-
producing cells. The
oleandrin, N. oleander extract, and Vehicle were maintained in the co-culture
medium. The
relative levels of extracellular p19Gag-containing HTLV-1 particles released
into the co-culture
supernatants as a result of intercellular viral transmission were quantified
by performing Anti-
HTLV-1 pl9Gag ELISAs. Virological synapses formed between the GFP-positive
HTLV-1+
SLB/pLenti-GFP cells and huPBMCs were visualized using immunofluorescence-
confocal
microscopy by staining the fixed samples with an Anti-HTLV-1 gp21Env primary
antibody and
a rhodamine red-conjugated secondary antibody. Diamidino-2-phenyl-indole,
dihydrochloride
(DAPI; Molecular Probes) nuclear-staining was included for comparison and to
visualize
uninfected (i.e., HTLV-1-negative) cells. The intercellular transmission of
HTLV-1 to the
huPBMCs in co-culture assays was quantified by counting the relative
percentages of HTLV-1
gp21Env-positive (and GFP-negative) huPBMCs in 20 visual fields using a 20x
objective lens.
Example 23
Microscopy
[00318] The Annexin V-FITC/PI-stained samples to quantify cellular apoptosis
and
cytotoxicity were visualized by confocal fluorescence-microscopy on a Zeiss
LSM800
instrument equipped with an Airyscan detector and stage CO2 incubator, using a
Plan-
Apochromat 20x/0.8 objective lens and Zeiss ZEN system software (Carl Zeiss
Microscopy).
The formation of virological synapses and viral transmission (i.e., determined
by quantifying
the relative percentages of Anti-HTLV-1 gp21Env-positive huPBMCs) between the
mitomycin
C-treated HTLV-1+ SLB1/pLenti-GFP lymphoblasts and cultured huPBMCs were
visualized
by immunofluorescence-confocal microscopy using a Plan-Apochromat 20x/0.8
objective lens.
The relative fluorescence-intensities of the DAPI, Anti-HTLV-1 gp21Env-
specific (rhodamine
red-positive), and GFP signals were graphically quantified using the Zen 2.5D
analysis tool
(Carl Zeiss Microscopy). The GFP-expressing HTLV-1+ SLB1/pLenti-GFP T-cell
clones were
screened by confocal fluorescence-microscopy on a Nikon Eclipse TE2000-U
inverted
microscope and D-Eclipse confocal imaging system, equipped with 633 nm and 543
nm He/Ne
and 488 nm Ar lasers, using a Plan Fluor 10x/0.30 objective lens and DIC phase-
contrast filter
(Nikon Instruments).
Date Recue/Date Received 2021-04-13
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Example 24
Statistical Analysis
[00319] The statistical significance of experimental data sets was determined
using unpaired
two-tailed Student's t-tests (alpha =0.05) and calculated P-values using the
Shapiro-Wilk
normality test and Graphpad Prism 7.03 software. The P-values were defined as:
0.1234 (ns),
0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). Unless otherwise noted,
error bars
represent the SEM from at least three independent experiments.
Example 25
Treatment of Deltaretrovirus infection in a subject
[00320] Exemplary Deltaretrovirus infections include HTLV-1.
Method A. Antiviral Composition therapy
[00321] A subject presenting with HTLV-1 infection is prescribed antiviral
composition, and
therapeutically relevant doses are administered to the subject according to a
prescribed dosing
regimen for a period of time. The subject's level of therapeutic response is
determined
periodically. The level of therapeutic response can be determined by
determining the subject's
HTLV-1 virus titre in blood or plasma. If the level of therapeutic response is
too low at one
dose, then the dose is escalated according to a predetermined dose escalation
schedule until the
desired level of therapeutic response in the subject is achieved. Treatment of
the subject with
antiviral composition is continued as needed and the dose or dosing regimen
can be adjusted as
needed until the patient reaches the desired clinical endpoint.
Method B. Combination therapy: antiviral composition with another agent
[00322] Method A, above, is followed except that the subject is prescribed and
administered
one or more other therapeutic agents for the treatment of HTLV-1 infection or
symptoms
thereof. Then one or more other therapeutic agents can be administered before,
after or with
the antiviral composition. Dose escalation (or de-escalation) of the one or
more other
therapeutic agents can also be done. Exemplary other therapeutic agents are
described herein.
Example 26
Treatment of CoV infection in a subject
Date Recue/Date Received 2021-04-13
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[00323] Exemplary CoV infections include SARS-CoV, MERS-CoV, COVID-19 (SARS-
CoV-2), CoV 229E, CoV NL63, CoV 0C43, CoV HKU1, and CoV HKU20.
Method A. Antiviral Composition therapy
[00324] A subject presenting with CoV infection is prescribed antiviral
composition, and
therapeutically relevant doses are administered to the subject according to a
prescribed dosing
regimen for a period of time. The subject's level of therapeutic response is
determined
periodically. The level of therapeutic response can be determined by
determining the subject's
CoV virus titer in blood or plasma. If the level of therapeutic response is
too low at one dose,
then the dose is escalated according to a predetermined dose escalation
schedule until the
desired level of therapeutic response in the subject is achieved. Treatment of
the subject with
antiviral composition is continued as needed and the dose or dosing regimen
can be adjusted as
needed until the patient reaches the desired clinical endpoint.
Method B. Combination therapy: antiviral composition with another agent
[00325] Method A, above, is followed except that the subject is prescribed and
administered
one or more other therapeutic agents for the treatment of CoV infection or
symptoms thereof.
Then one or more other therapeutic agents can be administered before, after or
with the antiviral
composition. Dose escalation (or de-escalation) of the one or more other
therapeutic agents can
also be done. Exemplary other therapeutic agents are described herein.
Example 27
Treatment of COVID-19 infection in a subject using ANVIRZELTM
[00326] A child (infant) presenting with COVID-19 was administered ANVIRZELTM
as
follows to treat symptoms associated with COVID-19. The subject's viral
infection was
worsening prior to administration of ANVIRZELTM. The subject was prescribed
and
administered ANVIRZELTM according to the following dosing regimen: initial
dose- 0.25 mL
of reconstituted ANVIRZELTM, then 0.5 mL of reconstituted ANVIRZELTM every
twelve hours
for a period of two to three days. The subject's COVID-19 infection resolved,
and no drug-
related toxicity was observed.
Date Recue/Date Received 2021-04-13
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Example 28
In vitro evaluation of oleandrin against COVID-19 virus
[00327] The purpose of this study was to determine the impact of oleandrin on
infectivity of
progeny virions.
[00328] A stock solution of oleandrin in methanol (10 mg oleandrin/mL) was
prepared. The
stock solution was used to prepare incubation media containing DMSO (0.1% or
0.01% v/v in
aqueous culture medium RPMI 1640 and oleandrin (20 microg/mL, 10 microg/mL,1.0
microg/mL, or 0.1 microg/mL). The incubation solutions are as follows.
Incubation medium ID
Oleandrin cone (microg/mL) 0.1% aq. DMSO 0.01% aq. DMSO
20 20A 20B
10A 10B
1.0 1.0A 1.0B
0.1 0.1A 0.1B
0 (control media) ¨> OA OB
[00329] Uninfected Vero cells (target initial cell count 1x106) in culture
were incubated in
each of the indicated incubation media in vials for 30 min at 37 C. A viral
inoculate of SARS-
CoV-2 was then added to each vial to achieve a target initial viral titer
(about PFU/mL 1x104).
The target MOI (multiplicity of infection) of about 0.1. The solutions were
incubated for an
additional 2 h at 37 C to achieve infection of the Vero cells. The infected
Vero cells were then
washed with control vehicle to remove extracellular virus and oleandrin. New
aliquots of each
incubation medium were added to each respective vial of infected Vero cells.
Those receiving
oleandrin in the second aliquot were denoted as "+ treatment Post-infection",
and those not
receiving oleandrin in the second aliquot were denoted as "- treatment Post-
infection" (FIGS.
23A-23D). The viral titer for each vial was determined at about 24 h and about
48 h after
infection.
[00330] As a means of determining the potential toxicity of oleandrin against
Vero cells,
parallel cultures, based upon the ones above, were prepared for uninfected
Vero cells.
Date Recue/Date Received 2021-04-13
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[00331] The data acquired included quantity of virus produced, infectivity of
progeny virus,
and relative safety (nontoxicity) of oleandrin in infected and uninfected
cells.
Example 29
In vitro evaluation of oleandrin against COVID-19 virus
[00332] The purpose of this assay was to determine the direct antiviral
activity of oleandrin
against SARS-CoV-2.
[00333] Growth media was removed from confluent monolayers of approximately
106 Vero
CCL81 cells in 6-well plates Oleandrin was serially diluted in culture media
and added to Vero-
E6 cells seeded in 96 well plates. The growth media was replaced with 200111
of maintenance
media containing either 1.0 g/ml, 0.5 g/ml, 10Ong/ml, 50ng/ml, lOng/ml, or
5ng/mloleandrin,
or matched DMSO-only controls. The plates were incubated at 37 C for about 30
minutes prior
to addition of virus.
[00334] SARS-CoV-2 virus was added to Oleandrin treated cells and untreated
cells at a MOI
(multiplicity of infection) of 0.4 (entry assay) or 0.02 (replication assay).
Oleandrin remained
in the wells during a lhr incubation at 37 C.
[00335] After lhr absorption, inoculation media was removed and washed 1 time
with PBS
(standard phosphate buffer saline).
[00336] Media alone (no oleandrin) was added back to oleandrin-treated wells
designated as
"Pretreatment" on data slides. Media with indicated concentrations of
oleandrin was added back
to wells designated as "Duration" on data slides.
[00337] Plates were fixed at either 24 (entry assay) or 48 (replication assay)
hours post-
infection and immunostained with virus-specific antibody and fluorescently
labeled secondary
antibody.
[00338] Cells were imaged using an Operetta and data was analyzed using custom
algorithms
in Harmonia software to determine the percent of infected cells in each well.
[00339] Results are depicted in FIGS. 24A and 24B.
Example 30
In vitro evaluation of oleandrin toxicity against Vero-E6 cells
[00340] The purpose of this assay was to determine the relative potential
toxicity of oleandrin
against Vero-E6 cells.
Date Recue/Date Received 2021-04-13
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[00341] Oleandrin was serially diluted in culture media and added to Vero-E6
cells seeded in
96 well plates and incubated at 37 C for about 24 h. Cell count was obtained
using the CellTiter
Glo assay.
[00342] The results are depicted in FIG. 25.
Example 31
In vitro evaluation of oleandrin against COVID-19 virus
[00343] The purpose of this study was to determine a dose response of COVID-19
virus
toward treatment with oleandrin.
[00344] The procedure of Example 28 was repeated except that lower
concentrations of
oleandrin were used: 1 microg/mL, 0.5 microg/mL, 0.1 microg/mL, 0.05
microg/mL, 0.01
microg/mL, and 0.005 microg/mL. In addition, VERO CCL-81 cells were used
instead of
VERO E6 cells.
[00345] The viral titer was determined according to Example 28, and the fold
reduction in
viral titer was calculated by comparison to control samples. The results are
depicted in FIGS.
26A-26D, 27A-27D, and 28A and 28B.
Example 32
Sublingual liquid dosage form
[00346] A sublingual dosage form comprising oleandrin was made by mixing
oleandrin or
oleandrin-containing composition (e.g. oleandrin-containing extract; 2 wt %)
with medium
chain triglyceride (95 wt%) and flavoring agent (3 wt%). The oleandrin content
in the dosage
form was about 25 microg/mL.
Example 33
Preparation of Subcritical fluid extract of Nerium oleander
[00347] An improved process for the preparation of an oleandrin-containing
extract was
developed by employing subcritical liquid extraction rather than supercritical
fluid extraction
of Nerium oleander biomass.
[00348] Dried and powdered biomass was placed in an extraction chamber, which
was then
sealed. Carbon dioxide (about 95% wt) and alcohol (about 5% wt; methanol or
ethanol) were
injected into the chamber. The interior temperature and pressure of the
chamber were such that
Date Recue/Date Received 2021-04-13
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the extraction medium was maintained in the subcritical liquid phase, rather
than the
supercritical fluid phase, for a majority or substantially all of the
extraction time period:
temperature in the range of about 2 C to about 16 C (about 7 C to about 8 C),
and pressure in
the range of about 115 to about 135 bar (about 124 bar). The extraction period
was about 4 h
to about 12 h (about 6 to about 10 h). The extraction milieu was then filtered
and the supernatant
collected. The carbon dioxide was vented from the supernatant, and the
resulting crude extract
was diluted into ethanol (about 9 parts ethanol : about 1 part extract) and
frozen at about -50 C
for at least 12 h. The solution was thawed and filtered (100 micron pore size
filter). The filtrate
was concentrated to about 10% of its original volume and then sterile filtered
(0.2 micron pore
size filter). The concentrated extract was then diluted with 50% aqueous
ethanol to a
concentration of about 1.5 mg of extract per mL of solution.
[00349] The resulting subcritical liquid (SbCL) extract comprised oleandrin
and one or more
other compounds extractable from Nerium oleander, said one or more other
compounds being
as defined herein.
Example 34
In vitro evaluation of oleandrin against COVID-19 virus
[00350] The purpose of this study was to determine the impact of oleandrin on
infectivity of
progeny virions without oleandrin pretreatment (as per Example 28).
[00351] The procedure of Example 28 was repeated except that cells were not
pre-treated
with oleandrin prior to infection. Instead, the infected cells were treated
with oleandrin or
control vehicle at 12 h and 24 h post-infection. Moreover, VERO CCL-81 cells
were used
instead of VERO E6 cells, and lower concentrations of oleandrin were used: 1
microg/mL, 0.5
microg/mL, 0.1 microg/mL, and 0.05 microg/mL. The data are depicted in FIGS.
29A and 29B.
Example 35
In vivo evaluation of oleandrin against COVID-19 virus
[00352] The purpose of this study was to determine the efficacy of oleandrin-
containing
extract (OCE) in treating subjects already infected with COVID-19 virus.
[00353] Subjects representing a broad demographic distribution and presenting
with COVID-
19 infection were evaluated to determine clinical status prior to sublingual,
buccal or peroral
administration of OCE, prepared according to the dosage form of Example 32.
The composition
Date Recue/Date Received 2021-04-13
- 97 -
was safely administered to subject by placing drops of liquid in the subject's
mouth. The dosing
regimen was approximately 0.5 mL per dose and four doses per day (one dose
about every six
hours), which approximates about 50 microg of oleandrin per day.
Alternatively, half the total
daily dose was administered. All subjects experienced a complete recovery.
Example 36
Preparation of ethanolic extract of Nerium oleander
[00354] The purpose of this was to prepare an ethanolic extract by extraction
of Nerium
oleander biomass with aqueous ethanol.
[00355] Ground dried leaves were repeatedly treated with aqueous ethanol (90%
v/v ethanol;
10% v/v water). The combined ethanolic supernatants were combined and filtered
and then
concentrated by evaporation in vacuo to reduce the amount of ethanol and water
therein and
provide crude ethanolic extract comprising about 25 mg of oleandrin/mL of
extract (which has
about 50% v/v ethanol content).
Example 37
Preparation of dosage form comprising a combination of extracts of Nerium
oleander
[00356] The purpose of this was to prepare a dosage form according to Example
32 except
that a portion (1 wt %) of the ethanolic extract of Example 36 is combined
with a portion (1 wt
%) of the SbCL extract of Example 33, medium chain triglyceride (95 wt %), and
flavoring
agent (3 wt %).
Example 38
In vivo evaluation of digoxin against COVID-19 virus
[00357] The purpose of this study is to determine the efficacy of digoxin-
containing
composition (DCC) in treating subjects already infected with COVID-19 virus.
Commercially
available dosage form containing digoxin is purchased.
[00358] Subjects presenting with COVID-19 infection are evaluated to determine
clinical
status prior to peroral or systemic administration of DCC. Commercially
available
compositions are described herein. The safe dosing regimen for each is
described in the
respective NDA and package inserts. The composition is safely administered to
each subject
Date Recue/Date Received 2021-04-13
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according to the intended route of administration. Clinical monitoring is
conducted to
determine therapeutic response and the dose is titrated accordingly.
Example 39
Determination of Genome to infectious particle ratio in SARS-CoV-2 infection
treated with
oleandrin
[00359] The purpose of this study is to determine whether the inhibition of
SARS-CoV-2 by
oleandrin was at the level of total or infectious particle production.
[00360] To quantify genome copies for the samples, 2001.11 of sample was
extracted with a
5:1 volume ratio of TRIzol LS (Ambion, Carlsbad, CA), utilizing standard
manufacturers
protocols and resuspending in 110 water. Extracted RNA were tested for SARS-
CoV-
2 by qRT-PCR following a previously published assay (26). Briefly, the N gene
was
amplified using the following primers and probe:
forward primer [5'-
TAATCAGACAAGGAACTGATTA-3'] (SEQ ID NO. 1); reverse primer [5'-
CGAAGGTGTGACTTCCATG-3'] (SEQ ID NO. 2); and probe [5' -FAM-
GCAAATTGTGCAATTTGCGG-TAMRA-3'; (SEQ ID NO. 3)]. A 20111 reaction
mixture was prepared using the iTaq Universal probes One-Step kit (BioRad,
Hercules, CA),
according to manufacturer instructions: A reaction mix (2x: 101.1L), iScript
reverse transcriptase
(0.5 OA primers (10 M: 1.0 pL), probe (10 M: 0.5 OA extracted RNA (4 L) and
water
(3 L). The qRT-PCR reactions were conducted using the thermocycler
StepOnePlusTM Real-
Time PCR Systems (Applied Biosystems). Reactions were incubated at 50 C for
5min and
95 C for 20sec followed by 40 cycles at 95 C for 5sec and 60 C for 30sec. The
positive control
RNA sequence (nucleotides 26,044 - 29,883 of COVID-2019 genome) was used to
estimate the
RNA copy numbers of N gene in the samples under evaluation.
[00361] As used herein, the term "about" or "approximately" are taken to mean
10%, 5%,
+2.5% or +1% of a specified valued. As used herein, the term "substantially"
is taken to mean
"to a large degree" or "at least a majority of" or "more than 50% of'
[00362] The above is a detailed description of particular embodiments of the
invention. It will
be appreciated that, although specific embodiments of the invention have been
described herein
for purposes of illustration, various modifications may be made without
departing from the
Date Recue/Date Received 2021-04-13
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spirit and scope of the invention. Accordingly, the invention is not limited
except as by the
appended claims. All of the embodiments disclosed and claimed herein can be
made and executed
without undue experimentation in light of the present disclosure.
Date Recue/Date Received 2021-04-13
