KR20210124010A - Methods and compositions for treatment of coronavirus infection - Google Patents

Abstract

A method of treating a viral infection, such as a viral infection caused by a virus of the coronaviridae family, is provided. Compositions having at least oleandrine are used to treat viral infections.

Description

Methods and compositions for treatment of coronavirus infection

Integration by reference

Pursuant to 37 CFR 1.52(e)(5), this application was filed in electronic format through EFS, which includes a list of sequences incorporated herein by reference. The sequence information contained in the electronic file named PBI22PCT9_SEQ_ST25.txt, size 1 KB, created on July 10, 2020 using Patents 3.5.1 and Checker 4.4.6, is incorporated by reference in its entirety.

The present invention relates to arenaviridae infection, buniaviridae infection, flaviviridae infection, togaviridae infection, paramyxoviridae infection, retroviridae infection, coronaviridae infection, or filobiridae infection in a mammal It relates to an antiviral composition and its use for the treatment of Some embodiments relate to the treatment of hemorrhagic viral infections.

Yes Solarium (Nerium) a member of four meander up Leeum (Nerium oleander) is a species of ornamental plant widely distributed in subtropical Asia, the southwestern United States and the Mediterranean. Its medicinal and toxicological properties have long been recognized. For example, it has been proposed for use in the treatment of hemorrhoids, ulcers, leprosy, snake bites, cancer, tumors, neurological disorders, warts, and cell proliferative diseases. Zibbu et al. (J. Chem. Pharm. Res. (2010), 2(6), 351-358) provides a brief review of the chemical and pharmacological activities of Nerium oleander.

Extraction of components from plants of the Nerium species has traditionally been performed using boiling water, cold water, supercritical fluid or organic solvents.

ANVIRZEL™ (US Pat. No. 5,135,745 to Ozel) contains a concentrated or powdered form of a hot-water extract of Nerium oleander. Muller et al. ( Pharmazie . (1991) Sept. 46(9), 657-663) disclose results on the analysis of water extracts of Nerium oleander. They report that the polysaccharide present is mainly galacturonic acid. Other sugars include rhamnose, arabinose and galactose. Polysaccharide content and individual sugar composition of polysaccharides in boiling water extract of Nerium oleander have also been reported by Newman et al. (J. Herbal Pharmacotherapy , (2001) vol 1, pp. 1-16). The compositional analysis of ANVIRZEL™, a hot water extract, is described in Newman et al. (Anal. Chem. (2000), 72(15), 3547-3552). U.S. Pat. No. 5,869,060 to Selvaraj et al. relates to an extract of Nerium sp. and a method of production. To prepare the extract, the plant material is put in water and brought to a boil. The crude extract was then separated from the plant material and sterilized by filtration. Then, the resulting extract may be freeze-dried to prepare a powder. U.S. Pat. No. 6,565,897 (Pre-Patent Publication No. 20020114852 to Selvaraj et al. and PCT International Publication No. WO 2000/016793) 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) disclosed a hydrothermal extract of Nerium oleander and its fractionation by liquid chromatography using a mixture of chloroform, methanol and water. do. They also report that N. oleander leaf extract was used to treat type 2 diabetes. US20060188585 published on August 24, 2006 by Panyosan discloses a hydrothermal extract of Nerium oleander. US 10323055, issued Jun. 18, 2019, by Smothers discloses a method for extracting plant material with aloe and water to provide an extract comprising aloe and cardiac glycosides. US20070154573 published July 5, 2007 by Rashan et al. discloses a cold water extract of Nerium oleander and uses thereof.

Erdemoglu et al. ( J. Ethnopharmacol. (2003) Nov. 89(1), 123-129) based on anti-nociceptive and anti-inflammatory activity, aqueous and Comparative results of ethanol extracts are disclosed. Fartyal et al. (J. Sci. Innov. Res. (2014), 3(4), 426-432) compared methanol, aqueous and petroleum ether extracts of Nerium oleander based on their antibacterial activity. start

Organic solvent extracts of Nerium oleander were also obtained from Adome et al. (Afr. Health Sci. (2003) Aug. 3(2), 77-86; ethanol extract), el-Shazly et al. (J. Egypt Soc. Parasitol. (1996), Aug. 26(2), 461-473; ethanol extract), Begum et al. (Phytochemistry (1999) Feb. 50(3), 435-438; methanol extract), Zia et al. ( J. Ethnolpharmacol. (1995) Nov. 49(1), 33-39; methanol extract) and Vlasenko et al. (Farmatsiia. (1972) Sept.-Oct. 21(5), 46-47; alcoholic extract) is initiated by Turkmen et al. (J. Planar Chroma. (2013), 26(3), 279-283) disclose aqueous ethanol extracts of Nerium oleander leaves and stems. US 3833472, issued September 3, 1974 by Yamauchi, discloses extraction of Nerium odorum SOL (Nerium oleander Linn) leaves with water, an organic solvent or an aqueous organic solvent, wherein the leaves are It is heated to 60°C-170°C and then extracted, and the organic solvent is methanol, ethanol, propyl ether or chloroform.

Supercritical fluid extracts of Nerium species are known (US 8394434, US 8187644, US 7402325), disorders of the nervous system (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, WO2019055119A1).

Triterpenes are known to have a wide variety of therapeutic activities. Some of the known triterpenes include oleanolic acid, ursolic acid, betulinic acid, bardoxolone, maslinic acid, and the like. The therapeutic activity of triterpenes was primarily evaluated individually rather than in combination with triterpenes.

Oleanolic acids belong to a class of triterpenoids represented by compounds such as bardoxolone that have been shown to be potent activators of the innate cellular phase 2 detoxification pathway, where activation of the transcription factor Nrf2 contains an antioxidant transcriptional response element (ARE). It results in a transcriptional increase in the program of downstream antioxidant genes. Although bardoxolone itself has been extensively investigated in clinical trials in inflammatory diseases, phase 3 clinical trials in chronic kidney disease suggest that it may be associated with the known cytotoxicity of certain triterpenoids, including bardoxolone, at elevated concentrations. It was terminated due to side effects.

Compositions containing triterpenes along with other therapeutic ingredients are found as plant extracts. Fumiko et al. (Biol. Pharm. Bull (2002), 25(11), 1485-1487) disclose the evaluation of a methanol extract of Rosmarimus officinalis L. for the treatment of trypanosomiasis. Addington et al. (US 8481086, US 9220778, US 9358293, US 20160243143 A1) described a supercritical fluid extract of Nerium oleander containing oleandrine and triterpenes (SCF; PBI-05204) for the treatment of neurological diseases. to start Addington et al. (US 9011937, US 20150283191 A1) discloses a triterpene-containing fraction (PBI-04711) of an SCF extract of Nerium oleander containing oleandrine and triterpenes for the treatment of neurological diseases. 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) discloses an extract of Betula utilis bark containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other ingredients. Wang et al. (Molecules (2016 ), 21, 139) discloses the Alstonia scholaris extract containing a mixture of olreahnolsan, ursolic acid, betulinic acid and the 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 extracts 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.

Oleanolic acid (O or OA), ursolic acid (U or UA) and betulinic acid (B or BA) are PBI-05204 (PBI-23; supercritical fluid extract of Nerium oleander) and PBI 04711 (PBI-05204 of the tree There are three major triterpene components found in terpene-containing fractions 0-4). We previously reported (Van Kanegan et al., Nature Scientific Reports (May 2016), 6:25626. doi:10.1038/srep25626), their neuroprotection in brain slice oxygenated glucose deprivation (OGD) model analysis at similar concentrations. The contribution of triterpenes to efficacy was reported by comparing the activities. We found that PBI 05204 (PBI) and PBI-04711 (fractions 0-4) confer neuroprotective activity.

Extracts of Nerium species are known to contain many different classes of compounds: cardiac glycosides, glycosides, steroids, triterpenes, polysaccharides and other compounds. Certain compounds include oleandrine; neritaloside; odoroside; oleanolic acid; ursolic acid; betulinic acid; oleandrigenin; Oliaside A; betulin (urs-12-ene-3β,28-diol); 28-norurs-12-en-3β-ol; urs-12-en-3β-ol; 3β,3β-hydroxy-12-oleanene-28-oic acid; 3β,20α-dihydroxyurs-21-en-28-oic acid; 3β,27-dihydroxy-12-ursen-28-oic acid; 3β,13β-dihydroxyurs-11-en-28-oic acid; 3β,12α-dihydroxyoleanane-28,13β-olide; 3β,27-dihydroxy-12-oleanane-28-oic acid; and other ingredients.

Viral hemorrhagic fever (VHF) can be caused by five distinct viral families: arenaviridae, buniaviridae, filobiridae, flaviviridae and paramyxoviridae. Filoviruses, such as Ebola virus (EBOV) and Marburg virus (MARV), are the most pathogenic viruses known to humans and are among the viruses responsible for viral hemorrhagic fever with mortality rates of up to 90%. Each virion contains a single-stranded negative-sense RNA molecule. Other than supportive management or symptomatic therapy, there are no commercially effective and prophylactic drugs that can treat EBOV (Ebolavirus) and MARV (Marburgvirus) infections, ie filovirus infections. Five Ebola viruses have been identified: Tai Forest (formerly Ivory Coast), Sudan, Zaire, Reston and Bundibugyo.

Negative-sense single-stranded enveloped RNA viruses ((-)-(ss)-envRNAV) are of the Arenaviridae, Bunyavirales order, Philoviridae, Orthomyxoviridae, paramic viruses of the family Sobiridae and Rhabdoviridae. Negative viral RNA is complementary to mRNA and must be converted to positive RNA by RNA polymerase prior to translation. Thus, the purified RNA of the negative sense virus is not infective by itself as it must be converted to positive sense RNA for replication. Exemplary viruses and infections from the family Arenaviridae include Lassa virus, aseptic meningitis, Guanarito virus, Junin virus, Lujo virus, Machupo virus, Sa Sabia virus and Whitewater Arroyo virus. Exemplary viruses and infections from the family Buniaviridae include Hantavirus, Crimean-Congo hemorrhagic fever orthonairovirus. Exemplary viruses and infections of the family Paramyxoviridae include mumps virus, nipa virus, Hendra virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV), and NDV. Exemplary viruses and infections of the family Orthomyxoviridae include influenza virus (A-C), isa virus, togotovirus, quaranzavirus, H1N1, H2N2, H3N2, H1N2, Spanish flu, Asian flu, Hong Kong flu, Russian flu includes Exemplary viruses and infections from the family Rhabdoviridae include rabies virus, vesiculovirus, lyssavirus, and cytorhabdovirus.

Flaviviruses are positive single-stranded enveloped RNA viruses ((+)-(ss)-envRNAV). They are mainly found in arthropods, ticks and mosquitoes, and cause widespread morbidity and mortality worldwide. Some of the mosquito-transmitted viruses include yellow fever, dengue fever, Japanese encephalitis, West Nile virus and Zika virus. Some of the tick-borne viral infections include tick encephalitis, kasanur forest disease, alkhurma disease, and Omsk hemorrhagic fever. Although not a hemorrhagic infection, Poisan virus is a flavivirus. (+)-(ss)-envRNAV is of the family Coronaviridae (human and animal pathogens), the family Flaviviridae (human and animal pathogens), and Togaviridae. subfamily (human and animal pathogens) and the Arterviridae family (animal pathogens).

Coronavirus (CoV) is the common name for the coronavirus. In humans, CoV causes respiratory infections that are usually mild but can be fatal in rare forms such as SARS (Severe Acute Respiratory Syndrome)-CoV, MERS (Middle East Respiratory Syndrome)-CoV, and COVID-19. CoVs have nucleocapsids of helical symmetry, and genome sizes range from about 26 to about 32 kilobases. Other exemplary human CoVs include CoV 229E, CoV NL63, CoV OC43, CoV HKU1 and CoV HKU20. The envelope of CoV carries three glycoproteins: S-spike protein: receptor binding, cell fusion, major antigen; E-envelope protein: a small envelope-related protein; and M-membrane protein: transmembrane-budding and envelope formation. Several types of CoVs have a fourth glycoprotein, HE-hemagglutinin-esterase. The genome has a 5' methylated cap and 3' poly-A and functions directly as mRNA. CoV entry into human cells occurs through endocytosis and membrane fusion. Replication occurs in the cytoplasm of a cell. CoV is delivered by respiratory secretion aerosol, fecal-oral route, and mechanical delivery. Most viral growth occurs in epithelial cells. Sometimes the liver, kidney, heart or eye, as well as other cell types such as macrophages, can become infected. In cold-type respiratory infections, growth appears to be confined to the epithelium of the upper respiratory tract. Coronavirus infections are very common and occur worldwide. The incidence of infection is seasonal, and it occurs most often in children in winter. Adult infections are uncommon. The number of coronavirus serotypes and the extent of antigenic variation are unknown. Since reinfection appears to occur throughout life and implies multiple serotypes (at least 4 known) and/or antigenic variants, the chances of immunization against all serotypes with a single vaccine are very low. SARS is a type of viral pneumonia and has symptoms such as fever, dry cough, shortness of breath (shortness of breath), headache, and hypoxemia (low blood oxygen concentration). Common laboratory findings include lymphopenia (decreased lymphocyte count) and mild aminotransferase levels (indicative of liver damage). Death can result from progressive respiratory failure due to alveolar damage. The typical clinical course of SARS is improvement in symptoms during the first week of infection and then worsening in the second week. There remains a significant need for effective antiviral treatments (compositions and methods) against human CoV.

Oleandrin and extracts of Nerium oleander have been shown to prevent incorporation of the gp120 envelope glycoprotein of HIV-1 into mature viral particles and inhibit in vitro viral infection (Singh et al., "Nerium oleander"). Derived cardiac glycoside oleandrine is a novel HIV infection inhibitor" Fitoterapia (2013) 84, 32-39).

Oleandrin exhibited 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 different levels of antiviral activity, but have not been evaluated against many viruses. Betulinic acid showed some antiviral activity against HSV-1 strain 1C, influenza A H7N1, ECHO 6 and HIV-1. Oleanolic acid showed antiviral activity against HIV-1, HEP C and HCV H strain NS5B. Ursolic acid exhibited antiviral activity against HIV-1, HEP C, HCV H strains NS5B, HSV-1, HSV-2, ADV-3, ADV-8, ADV-11, HEP B, ENTV CVB1 and ENTV EV71. The antiviral activity of oleandrine, oleanolic acid, ursolic acid and betulinic acid cannot be predicted even from their efficacy against specific viruses. There are viruses in which oleandrine, oleanolic acid, ursolic acid and/or betulinic acid have little or no antiviral activity, which means that oleandrine, oleanolic acid, ursolic acid and/or betulinic acid are antiviral against viruses of certain genera. It means that it is not possible to predict a priori whether or not it will show activity.

Barrows et al. (“Screen of FDA Approved Drugs for Inhibitors of Zika Virus Infections” in Cell Host Microbe (2016), 20, 259-270) show that digoxin exhibits antiviral activity against Zika virus, but at too high a dose Report possible toxicity. Cheung et al. (“Antiviral activity of lanatoside C against dengue virus infection” in Antiviral Res. (2014) 111, 93-99) reported that lanatoside C exhibited antiviral activity against dengue virus. do.

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 cellular DNA. Once integrated, HTLV-1 continues to exist only as a provirus that can spread from cell to cell via viral synapses. Free virions are rarely produced, and the virus is usually present in genital secretions, but no virus is detectable in plasma. HTLV-1 primarily infects CD4 + T-lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL). It is a rare but aggressive hematologic malignancy that is highly resistant to treatment and generally has poor clinical outcomes, in addition to several autoimmune/inflammatory diseases including infectious dermatitis, rheumatoid arthritis, uveitis, keratoconjunctivitis, Sica syndrome, Sjogren's syndrome, HAM/TSP, etc. not. HAM/TSP is clinically characterized by chronic progressive convulsive paralysis, urinary incontinence and mild sensory impairment. Although ATLL is etiologically associated with viral latency, oncogenic transformation, and clonal expansion of HTLV-1-infected cells, inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paralysis (HAM/TSP) are autologous. It is caused by immune and/or proviral replication and immune disease responses to the expression of viral antigens. HAM/TSP is a progressive neuroinflammatory disease that results in deterioration and demyelination of the lower spinal cord. Circulating T cells infected with HTLV-1 invade the central nervous system (CNS), resulting in an immunopathogenic response to the virus and possibly components of the CNS. Nerve damage and subsequent degeneration can cause serious disability in HAM/TSP patients. Persistence of proviral replication and proliferation of HTLV-1 infected cells in the CNS triggers a targeted cytotoxic T-cell response to viral antigens, which may be responsible for autoimmune destruction of neural tissue.

Although cardiac glycosides have been demonstrated to exhibit some antiviral activity against some viruses, certain compounds exhibit very different levels of antiviral activity against different viruses, which, when evaluated against the same virus, some exhibit very poor antiviral activity. and some have better antiviral activity.

There remains a need for improved pharmaceutical compositions containing oleandrine, oleanolic acid, ursolic acid, betulinic acid or any combination thereof that are therapeutically active against certain viral infections.

The present invention provides pharmaceutical compositions and methods for treating and preventing viral infections in mammalian subjects. The present invention also provides pharmaceutical compositions and methods for treating viral infections, eg, viral hemorrhagic fever (VHF) infections in a mammalian subject. The invention also provides a method of treating a viral infection in a mammal by administration of a pharmaceutical composition. The present inventors have succeeded in preparing antiviral compositions that exhibit sufficient antiviral activity for use in treating viral infections in humans and animals. The present inventors have developed corresponding treatment methods using specific dosing regimens. The present invention also provides a prophylactic method of treating a subject at risk of contracting a viral infection, said method comprising administering to the subject one or more doses of the antiviral composition repeatedly over an extended treatment period prior to the subject becoming virally infected. including chronic administration. thereby preventing the subject from contracting a viral infection, wherein the antiviral composition comprises oleandrine.

In some embodiments, the antiviral composition is administered to a subject having virally infected cells, wherein the cells display an elevated ratio of Na, alpha-3 to alpha-1 isoforms of K-ATPase.

In some embodiments, the viral infection is arenaviridae, arterviridae, buniaviridae, filobiridae, flaviviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, retroviridae (particularly , genus Deltaretroviridae), Coronaviridae, or Togaviridae. In some embodiments, the viral infection is caused by (+)-ss-envRNAV or (-)-ss-envRNAV.

Some embodiments of the present invention relate to compositions and methods for treating filovirus infection, flavivirus infection, henipavirus infection, alphavirus infection or togavirus infection. Treatable viral infections include, at least, Ebolavirus, Marburg virus, alphavirus, flavivirus, yellow fever, dengue fever, Japanese encephalitis, West Nile virus, Zika virus, Venezuelan equine encephalomyelitis (VEE) virus, chikunguya. Virus, Western Equine Encephalomyelitis (Encephalitis) (WEE) Virus, Eastern Equine Encephalomyelitis (Encephalitis) (EEE) Virus, Tick Encephalitis, Kyasanur Forest Disease, Alkhurma Disease, Omsk Hemorrhagic Fever, Hendra Virus , Nipah virus deltaretrovirus genus, HTLV-1 virus and species thereof.

Some embodiments of the present invention are arenaviridae, Atterviridae, Buniaviridae, Philobiridae, Flaviviridae (Flaviviridae), Orthomyxoviridae, Paramyxoviridae Viral infection from viruses of the family Rhabdoviridae, Retrovaridae high (genus Deltaretrovirus), Coronaviridae, (+)-ss-envRNAV, (-)-ss-envRNAV or Togaviridae family It relates to compositions and methods for the treatment of

Some embodiments of the present invention are for treating a viral infection from a virus of the genus Henipavirus, genus Ebolavirus, genus Flavivirus, genus Marburgvirus, genus Deltaretrovirus, coronavirus (CoV), or genus Alphavirus. compositions and methods.

In some embodiments, the (+)-ss-envRNAV is a virus selected from the group consisting of Coronaviridae, Flaviviridae, Togaviridae, and Arterviridae.

In some embodiments, the (+)-ss-envRNAV is a coronavirus that is pathogenic to humans. In some embodiments, the coronavirus spike protein binds to the ACE2 (angiotensin converting enzyme 2) receptor 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 OC43, CoV HKU1 and CoV HKU20.

In some embodiments, (+)-ss-envRNAV is flavivirus, yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, tick-borne encephalitis virus, Kyasanur Forest Disease virus , Alkhurma disease virus, Omsk hemorrhagic fever virus and Powassan virus.

In some embodiments, (+)-ss-envRNAV is an arbovirus, 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 It is a virus of the family Togaviridae selected from the group consisting of.

In some embodiments, (-)-(ss)-envRNAV is from the family Arenaviridae, the Bunyavirales order, the Philovidira family, the Orthomyxoviridae family, the Paramyxoviridae family A, or the Rhabdo family. It is a virus selected from the group consisting of the family Viridae.

In some embodiments, the arenaviridae family virus is Lassa virus, aseptic meningitis, Guanarito virus, Junin virus, Lujo virus, Machupo virus, Sabia (Sabia) virus and Whitewater Arroyo virus.

In some embodiments, the buniavidae virus is selected from the group consisting of Hantavirus, Crimean-Congo hemorrhagic fever orthonairovirus.

In some embodiments, the Paramyxoviridae virus is selected from the group consisting of mumps virus, nipa virus, Hendra virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) and NDV.

In some embodiments, the Orthomyxoviridae family virus is influenza virus (A-C), isa virus, togotovirus, quaranzavirus, H1N1, H2N2, H3N2, H1N2, Spanish flu, Asian flu, Hong Kong flu, Russian flu is selected from the group consisting of

In some embodiments, the Rhabdoviridae virus is selected from the group consisting of rabies virus, vesiculovirus, lyssavirus, and cytorhabdovirus.

The present invention also provides an embodiment for the treatment of a HTLV-1-associated disease or a neuro-inflammatory disease. In some embodiments, the HTLV-1-associated disease or neuroinflammatory disease is myelopathy/tropical convulsive palsy (HAM/TSP), adult T-cell leukemia/lymphoma (ATLL), autoimmune disease, inflammatory disease, infectious dermatitis, rheumatoid arthritis , uveitis, keratoconjunctivitis, Sica syndrome, Sjogren's syndrome, and Strongyloides stercoralis.

The present invention also provides a method for reducing intercellular propagation of HTLV-1 by inhibiting the infectivity of HTLV-1 particles released into the culture supernatant of the treated cells and also inhibiting the Env-dependent formation of viral synapses, said method comprises administering to a subject in need thereof an effective amount of an antiviral composition.

In some embodiments, the present invention provides (a) specific cardiac glycoside(s); b) a plurality of triterpenes; or c) providing an antiviral composition comprising (consisting essentially of) a combination of the specific cardiac glycoside(s) and a plurality of triterpenes.

One aspect of the invention provides a method of treating a viral infection in a subject by chronic administration of an antiviral composition to the subject. A subject is treated by chronically administering to the subject a therapeutically effective amount (a therapeutically appropriate dosage) of the composition, thereby providing relief of symptoms associated with a viral infection or amelioration of the viral infection. Administration of the composition to a subject may begin immediately after infection or within 1 to 5 days after infection, or at the earliest point after definitive diagnosis of viral infection. The virus may be any virus described herein.

Accordingly, the present invention provides a method of treating a viral infection in a mammal comprising administering to the mammal a therapeutically effective amount of one or more antiviral compositions. One or more doses are administered on a daily, weekly or monthly basis. One or more doses per day may be administered. The virus may be any virus described herein.

The invention also provides a method of treating a viral infection in a subject in need thereof, said method comprising:

determining whether the subject has a viral infection;

directing administration of an antiviral composition;

administering an initial dose of the antiviral composition to the subject according to the prescribed initial dosing regimen for a period of time;

periodically determining the adequacy of the subject's clinical response and/or therapeutic response to treatment with the antiviral composition; and

If the subject's clinical response and/or therapeutic response is appropriate, continue treatment with the antiviral composition until the desired clinical endpoint is reached; or

if the subject's clinical response and/or therapeutic response is insufficient at the initial dose and initial dosing regimen, the dose is escalated or decreased until the desired clinical response and/or therapeutic response is achieved in the subject.

Treatment of the subject with the antiviral composition continues as needed. The dosage or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint(s), such as, for example, reduction or alleviation of certain symptoms associated with viral infection. Determining the adequacy of a clinical response and/or therapeutic response can be performed by a clinician familiar with viral infections.

The individual steps of the method of the invention may be performed in separate facilities or in the same facility.

The present invention provides alternatives to all of the embodiments described herein, wherein oleandrine is substituted for digoxin or used in combination with digoxin. The method of the present invention may use oleandrine, digoxin, or a combination of oleandrine and digoxin. Thus, oleandrine, digoxin, oleandrine-containing composition, digoxin-containing composition, or oleandrine-containing composition and digoxin-containing composition can be used in the method of the present invention. Cardiac glycosides may be considered to mean oleandrine, digoxin or a combination thereof. The cardiac glycoside-containing composition comprises oleandrine, digoxin, or a combination thereof.

The present invention also provides a method of treating a coronavirus infection, in particular a coronavirus infection pathogenic to humans, e.g., SARS-CoV-2 infection, said method to a subject having said infection. and chronic administration of a therapeutically effective amount of a cardiac glycoside (a composition containing a cardiac glycoside).

The present invention also provides a dual route method for treating a coronavirus infection, in particular a coronavirus infection pathogenic to humans, e.g., SARS-CoV-2 infection, said method having said infection chronically administering to a subject a therapeutically effective amount of a cardiac glycoside (a composition containing a cardiac glycoside) to inhibit viral replication of the coronavirus and reduce the infectivity of a progeny virus of the coronavirus.

The present invention also provides a method of repeatedly administering (via any of the modes of administration discussed herein) a plurality of therapeutically effective amounts of a cardiac glycoside (composition containing a cardiac glycoside) to a subject having said infection, thereby causing a coronavirus infection, particularly SARS-CoV. -2 provides a method for treating an infection. The one or more doses may be administered daily, one or more days per week, optionally one or more weeks per month, optionally one or more months per year.

The present invention also provides a method of treating a coronavirus infection in a human, comprising administering to a subject a cardiac glycoside (composition containing a cardiac glycoside) at a dose of 1 to 10 daily for a treatment period of 2 days to about 2 months. administering. During the treatment period, it may be administered 2 to 8 times, 2 to 6 times or 4 times daily. The dosage may be administered for from 2 days to about 60 days, from 2 days to about 45 days, from 2 days to about 30 days, from 2 days to about 21 days, or from 2 days to about 14 days. Such administration may be via any of the modes of administration discussed herein. Systemic administration that provides therapeutically effective plasma levels of oleandrine and/or digoxin in said subject is preferred.

In some embodiments, one or more doses of oleandrine are administered daily for multiple days until the viral infection is resolved. In some embodiments, one or more doses of cardiac glycoside (composition containing cardiac glycosides) are administered daily for days and weeks until the viral infection is cured. One or more doses may be administered daily. It may be administered once, twice, three times, four times, five times, six or more times daily.

In some embodiments, for example, the concentration of oleandrine and/or digoxin in the plasma of an infected subject treated for a coronavirus infection is about 10 μg/mL or less, about 5 μg/mL or less, about 2.5 μg/mL or less , about 2 μg/mL or less, or about 1 μg/mL or less. In some embodiments, the concentration of oleandrine and/or digoxin in the plasma of a subject treated for coronavirus infection is at least about 0.0001 μg/mL, at least about 0.0005 μg/mL, at least about 0.001 μg/mL, or at least about 0.0015 μg ./mL or greater, about 0.01 µg/mL or greater, about 0.015 µg/mL or greater, about 0.1 µg/mL or greater, about 0.15 µg/mL or greater, about 0.05 µg/mL or greater, or about 0.075 µg/mL or greater. In some embodiments, the concentration of oleandrine and/or digoxin in the plasma of the treated infected subject is about 10 μg/mL to about 0.0001 μg/mL, about 5 μg/mL to about 0.0005 μg/mL, about 1 μg/mL mL to about 0.001 μg/mL, about 0.5 μg/mL to about 0.001 μg/mL, about 0.1 μg/mL to about 0.001 μg/mL, about 0.05 μg/mL to about 0.001 μg/mL, about 0.01 μg/mL to about 0.001 μg/mL, about 0.005 μg/mL to about 0.001 μg/mL. The present invention includes all combinations and selections of the plasma concentration ranges described herein.

The antiviral composition is administered chronically, i.e. daily, every other day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every week, every other week, every 2 weeks, every 3 weeks, every month, every other month. , semi-monthly, bi-monthly, bi-monthly, quarterly, bi-quarterly, every three months, seasonally, semi-annually, and/or annually. The duration of treatment may be one or more weeks, one or more months, one or more quarters, and/or one or more years. An effective amount of cardiac glycoside (cardial glycoside-containing composition) is administered at least once per day.

In some embodiments, the subject is administered between 140 micrograms and 315 micrograms of cardiac glycosides per day. In some embodiments, the dosage comprises from 20 micrograms to 750 micrograms, from 12 micrograms to 300 micrograms, or from 12 micrograms to 120 micrograms of cardiac glycosides. The daily dosage of cardiac glycoside may range from 20 micrograms to 750 micrograms, 0.01 micrograms to 100 mg, or 0.01 micrograms to 100 micrograms of cardiac glycosides. The recommended daily dosage of oleandrine present in the SCF extract is generally from about 0.25 to about 50 micrograms twice daily, or from about 0.9 to 5 micrograms twice daily or about every 12 hours. A dosage may be from about 0.5 to about 100 micrograms/day, from about 1 to about 80 micrograms/day, from about 1.5 to about 60 micrograms/day, from about 1.8 to about 60 micrograms/day, from about 1.8 to about 40 micrograms /may be a day. The maximum tolerated dose is about 100 micrograms/day, about 80 micrograms/day, about 60 micrograms/day, about 40 micrograms/day, about 38.4 micrograms/day, or about 30 micrograms/day, wherein the minimum effective dosage is about 0.5 micrograms/day, about 1 micrograms/day, about 1.5 micrograms/day, about 1.8 micrograms/day, about 2 micrograms/day, or about 5 micrograms/day. Suitable dosages comprising cardiac glycoside and triterpene are 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 microgram/kg/day, about 0.05-0.35 microgram/kg/day, about 0.05-0.22 microgram/kg/day, about 0.05-0.4 microgram /kg/day or about 0.05-0.3 micrograms/kg/day. In some embodiments, the dosage of oleandrine 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 present invention includes all combinations of doses presented herein.

In some embodiments, the cardiac glycoside is administered in at least two administration phases: a loading phase and a maintenance phase. The loading phase continues until steady-state plasma levels of cardiac glycosides are achieved. The maintenance phase begins at the start of treatment or after completion of the loading phase. Dosage titration may occur during the loading phase and/or maintenance phase.

All dosing regimens, dosing schedules and dosages described herein are considered suitable; However, some dosing regimens, dosing schedules, and dosages may be more suitable for some subjects than others. Target clinical endpoints are used to guide the dosage.

The viral composition may be administered systemically. Systemic administration modes include parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, peroral, pulmonary, and oral administration. The composition may also be administered via injection or intravenously. The composition may also be administered to the same subject by two or more routes. In some embodiments, the composition is administered by a combination of any two or more administration modes selected from the group consisting of parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, oral, pulmonary, and oral administration. do.

The present invention also provides a sublingual formulation comprising oleandrine and a liquid carrier. The present invention also provides a method of treating a viral infection, particularly a coronavirus infection. The method comprises sublingually administering to a subject having said viral infection a plurality of doses of an oleandrine containing (digoxin containing) composition, as defined herein. The one or more doses may be administered two or more days per week and one or more weeks per month, optionally more than one month per year.

In some embodiments, the antiviral composition comprises oleandrine (or digoxin or a combination of oleandrine and digoxin) and an oil. The oil may contain medium chain triglycerides. The antiviral composition may comprise one, two or more oleandrine-containing extracts and one or more pharmaceutical excipients.

When present in the antiviral composition, additional cardiac glycosides may additionally be included: odoroside, neritaloside or oleandrigenin. In some embodiments, the composition comprises a) one or more triterpenes; b) one or more steroids; c) at least one triterpene derivative; d) one or more steroid derivatives; or e) a combination thereof. In some embodiments, the composition comprises a cardiac glycoside and a) two or three triterpenes; b) two or three triterpene derivatives; c) two or three triterpene salts; or d) combinations thereof. In some embodiments, the triterpene is selected from the group consisting of oleanolic acid, ursolic acid, betulinic acid and salts or derivatives thereof.

Some embodiments of the present invention include the pharmaceutical composition comprising at least one pharmaceutical excipient and an 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) comprises at least two triterpenes and excludes cardiac glycosides; e) comprises at least three triterpenes and excludes cardiac glycosides; or f) at least one cardiac glycoside, for example oleandrine, digoxin. As used herein, the terms triterpenes and cardiac glycosides also include salts and derivatives thereof, unless otherwise specified.

The cardiac glycosides may be present in the pharmaceutical composition in pure form or as an extract fraction containing one or more cardiac glycosides. The triterpene(s) may be present in the pharmaceutical composition in pure form or as part of an extract containing the triterpene(s). In some embodiments, the cardiac glycoside is present as a primary therapeutic component, meaning a component mainly responsible for antiviral activity in the pharmaceutical composition. In some embodiments, the triterpene(s) is present as the primary therapeutic component(s), meaning the component(s) primarily responsible for antiviral activity in the pharmaceutical composition.

In some embodiments, the oleandrine-containing extract is obtained by extraction of plant material. The extract may include a boiling water extract of plant material, a cold water extract, a supercritical fluid (SCF) extract, a subcritical liquid extract, an organic solvent extract, or a combination thereof. In some embodiments, the extract is prepared by subcritical liquid extraction (biomass) of Nerium (oleander) plant mass, optionally using an extraction fluid comprising alcohol, subcritical liquid carbon dioxide (biomass). In some embodiments, the oleandrine-containing composition comprises two or more different types of oleandrine-containing extracts.

An embodiment of the present invention is oleandrine-containing biomass (plant material) is Nerium species, Nerium oleander or Nerium oleander L (oleander family), Nerium odourum (Nerium odourum), white oleander, Pink oleander, Thevetia sp., Thevetia peruviana, yellow oleander, Thevetia nerifolia, Agrobacterium tumefaciens, any of the above species of cell culture (cell mass), or a combination thereof. In some embodiments, biomass comprises leaves, stems, flowers, bark, fruit, seeds, sap and/or pods.

In some embodiments, the extract comprises at least one other pharmacologically active agent that is obtained with the cardiac glycoside during extraction when the extract is administered to a subject, thereby contributing to the therapeutic efficacy of the cardiac glycoside. In some embodiments, the composition further comprises one or more other non-cardiac glycoside therapeutically effective agents, ie, one or more agents that are not cardiac glycosides. In some embodiments, the composition further comprises one or more antiviral compound(s). In some embodiments, the antiviral composition excludes pharmacologically active polysaccharides.

In some embodiments, the extract is such as one or more cardiac glycosides and one or more cardiac glycoside precursors (eg, cardenolide, cardadienolide and cardatrienolide), all of which are agglutinins of cardiac glycosides. Licon components, for example digitoxin, acetyl digitoxin, digitoxygenin, digoxin, acetyl digoxin, digoxigenin, medigoxin, strophanthine, cymarin, huavine or strophantidine. The extract may further include one or more glycol components of cardiac glycosides (eg, glucoside, fructoside and/or glucuronide) as cardiac glycoside precursors. Accordingly, the antiviral composition may comprise at least two cardiac glycoside precursors selected from the group consisting of at least one aglycone component and at least one glycoside component and at least one cardiac glycoside. The extract may also contain one or more other non-cardiac glycoside therapeutically effective agents obtained from Nerium spp. or Thevetia spp. plant material.

In some embodiments, a composition containing oleandrine (OL), oleanolic acid (OA), ursolic acid (UA), and betulinic acid (BA) is pure oleadrine when equivalent doses based on oleandrine content are compared. more effective than

In some embodiments, the molar ratio of total triterpene content (OA + UA + BA) to oleandrine is from about 15:1 to about 5:1, or from about 12:1 to about 8:1, or from 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 It is in the range of about 10:1.

In some embodiments, the molar ratio of individual triterpenes to oleandrine ranges 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).

In some embodiments, the other therapeutic agent, such as that obtained by extraction of Nerium sp. or Thebetia sp. plant material, is not a polysaccharide obtained during the preparation of the extract, which means that it is an acidic homopolygalacturonan or arabinogalacturonan. Meaning I'm not In some embodiments, the extract excludes other therapeutic agents and/or excludes acidic homopolygalacturonan or arabinogalacturonan obtained during preparation of the extract.

In some embodiments, the other therapeutic agent, eg, Nerium spp. or Thebetia spp. plant material, is a polysaccharide obtained during the preparation of the extract, eg, acidic homopolygalacturonan or arabinogalacturonan. In some embodiments, the extract comprises another therapeutic agent and/or comprises acidic homopolygalacturonan or arabinogalacturonan obtained during the preparation of the extract from the plant material.

In some embodiments, the extract is oleandrine, and cardiac glycosides, sugars, non-saccharides, steroids, triterpenes, polysaccharides, saccharides, alkaloids, fats, proteins, neritaloside, odoroside, oleanolic acid, ursolic acid, betul Linic acid, oleandrigenin, oleaside A, betulin (urs-12-ene-3β,28-diol), 28-norurs-12-en-3β-ol, urs-12-en-3β-ol, 3β,3β-hydroxyl-12-oleanene-28-oic acid, 3β,20α-dihydroxyurs-21-en-28-oic acid, 3β,27-dihydroxyl-12-ursen-28-oic acid, 3β,13β-dihydroxyurus-11-en-28-oic acid, 3β,12α-dihydroxyoleanane-28,13β-olide, 3β,27-dihydroxy-12-oleanane-28 -oic acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumaroyl-CoA, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid , cardenolide B-1, cardenolide B-2, oleagenin, neridizinoside, nerizoside, orodoside-H, 3-beta-O-(D-digitinosyl)-5 -beta, 14 beta consisting of galacturonic acid, rhamnose, arabinose, xylose and galactose, polysaccharides having a MW in the range of 17000-120000 D or about 35000 D, about 3000 D, about 5500 D or about 12000 D -dihydroxy-card-20(22)-enolide pectic polysaccharide, cardenolide monoglycoside, cardenolide N-1, cardenolide N-2, cardenolide N-3, Cardenolide N-4, pregnan, 4,6-diene-3,12,20-trione, 20R-hydroxypregna-4,6-diene-3,12-dione, 16beta,17beta -epoxy-12beta-hydroxypregna-4,6-diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A ), 20S,21-dihydroxypregna-4,6-diene-3,12-dione (neridione B), neriucoumaric acid (neriucou maric acid), isoneriucoumaric acid, oleanderoic acid, oleanderen, 8-alpha-methoxyrapdan-18-oic acid, 12-ursen, cannero side, neriumoside, 3β- O- (D-digitinosyl)-2α-hydroxy-8,14β-epoxy-5β-carda-16:17, 20:22-dienolide, 3β- O- ( D-digitinosyl)-2α,14β-dihydroxy-5β-carda-16:17,20:22-dienolide, 3β,27-dihydroxy-urs-18-en-13,28-olide , 3β,22α,28-trihydroxy-25-nor-lup-1(10),20(29)-dien-2-one, cis -karenine (3β-hydroxy-28-Zp-coumaroyloxy -urs-12-en-27-oic acid), trans -karenine (3-β-hydroxy-28-Ep-coumaroyloxy-urs-12-en-27-oic acid), 3beta-hydroxy -5alpha-carda-14(15),20(22)-dienolide (beta-anhydroepidigitoxigenin, 3beta-O-(D-digitalosyl)-21-hydro Roxy-5beta-carda-8,14,16,20(22)-tetraenolide (nerium mogenin-A-3beta-D-digitaloside), proseragenin, neridienone A, 3beta,27-dihydroxyl-12-ursene-28-oic acid, 3beta,13beta-dihydroxyurus-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-oleanene-28-oic acid, (20S, 24R)-epoxydammaran-3beta,25-diol, 20beta,28-epoxy-28alpha-methoxytaraxasteran-3beta-ol, 20beta , 28-epoxytaraxaster-21-en-3beta-ol, 28-nor-urs-12-ene-3beta,17beta-diol, 3beta-hydroxyurs-12-en-28-aldehyde, alpha-neriursate (neri) ursate), beta-neriursate, 3 alpha-acetophenoxy-urs-12-en-28-oic acid, 3 beta-acetophenoxy-urs-12-en-28-oic acid, oleanderoic acid, Kanerodione, 3β- p -hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursen-28-oic acid, 28-hydroxy-20(29)-lupen-3,7- Dion, kanerocin, 3 alpha-hydroxy-urs-18,20-dien-28-oic acid, D-sarmentose, D-digitinose, neridizinoside, nerizoside, isoricinore isoricinoleic acid, gentiobiosylnerigoside, gentiobiosylbeaumontoside, gentiobiosyloleandrin, polynerin, 12β-hydroxy- 5β-carda-8,14,16,20(22)-tetraenolide, 8β-hydroxy-digitoxigenin, Δ16-8β-hydroxy-digitoxygenin, Δ16- neriagenin, uvaol, ursolic aldehyde, 27(p-coumaroyloxy)ursolic acid, oleanderol, 16-anhydride-deactyl-nerigoside , 9-D-hydroxy-cis-12-octadecanoic acid, adigoside, adinerine, alpha-amirin, beta-sitosterol, campestrol, natural rubber (caoutchouc) , capric acid, caprylic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl-nerigoside, polyandrin ), pseudocuramine, quercetin, quercetin-3-hamnoglucoside (rhamnoglu) coside), quercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospeside, urehitoxin and uzarigenin (uzarigenin) includes one or more compounds selected from the group consisting of. Additional components that may be present in the extract are disclosed in Gupta et al. (IJPSR, 2010(, 1(3), 21-27, the entire disclosure of which is incorporated herein by reference).

Oleandrin can also be obtained from an extract of a suspension culture derived from Agrobacterium tumefaciens-transformed calli. Hot water, organic solvents, aqueous organic solvents or supercritical fluid extracts of Agrobacterium may be used according to the invention.

Oleandrin can also be obtained from an extract of an in vitro Nerium oleander microculture, whereby the shoot culture is a Nerium oleander variety, such as, for example, Splendens Giganteum, It may be initiated from seedlings and/or shoot apex of Revanche or Alsace or other varieties. Hot water, organic solvent, aqueous organic solvent or supercritical fluid extract of micro-cultured Nerium oleander according to the present invention may be used.

Extracts can also be obtained by extracting a mass of cells (eg, present in cell culture) of any of the above plant species.

The invention also provides for the use of cardiac glycosides in the manufacture of a medicament for the treatment of a viral infection in a subject. In some embodiments, the preparation of such a medicament comprises providing one or more antiviral compounds of the present invention; incorporating a single dose of the antiviral compound(s) into the pharmaceutical formulation; and packaging the pharmaceutical formulation. In some embodiments, manufacturing may be performed as described in PCT International Application No. PCT/US06/29061. Manufacturing may also include delivering the packaged formulation to a vendor (retailer, wholesaler and/or distributor); selling or providing the packaged formulation to a subject having a viral infection; It may include one or more additional steps, such as including a label and package insert providing instructions regarding the use of the formulation, dosing regimen, administration, dosage and toxicity profile with the medicament. In some embodiments, treatment of a viral infection comprises determining whether the subject has a viral infection; instructing the subject to administer the pharmaceutical formulation according to the dosing regimen; administering to the subject one or more pharmaceutical formulations, wherein the one or more pharmaceutical formulations are administered according to a dosing regimen.

The pharmaceutical composition may further comprise a combination of at least one substance selected from the group consisting of water-soluble (miscible) co-solvents, water-insoluble (immiscible) co-solvents, surfactants, antioxidants, chelating agents and absorption enhancers. can

The solubilizing agent is at least a single surfactant, but it also comprises a) a surfactant and a water miscible solvent; b) surfactants and water immiscible solvents; c) surfactants, antioxidants; d) surfactants, antioxidants and water miscible solvents; e) surfactants, antioxidants and water immiscible solvents; f) surfactants, water miscible solvents and water immiscible solvents; or g) a combination of materials such as a surfactant, an antioxidant, a combination of a water miscible solvent and a water immiscible solvent.

The pharmaceutical composition comprises a) at least one liquid carrier; b) at least one emulsifier; c) at least one solubilizing agent; d) at least one dispersant; e) at least one other excipient; or f) optionally a combination thereof.

In some embodiments, the water miscible solvent is a 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.

The present invention includes all combinations of examples, embodiments and sub-embodiments of the invention disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the description herein and describe exemplary embodiments of the claimed invention. Those skilled in the art will be able to practice the present invention without undue experimentation in light of these drawings and the description herein.
1 to 2 depict charts summarizing the in vitro dose response antiviral activity of various compositions against Ebolavirus.
3 to 4 depict charts summarizing the in vitro dose response antiviral activity of various compositions against Marburgvirus.
Figure 5 depicts a chart summarizing the in vitro dose response antiviral activity of oleandrine against Zika virus (SIKV strain PRVABC59) in Vero E6 cells.
6 depicts a chart summarizing the in vitro dose response antiviral activity of digoxin against Zika virus (SIKV strain PRVABC59) in Vero E6 cells.
7 depicts a chart summarizing the in vitro dose response antiviral activity of various compositions (oleandrine, digoxin and PBI-05204) against Ebolavirus in Vero E6 cells.
8 depicts a chart summarizing the in vitro dose response antiviral activity of various compositions (oleandrine, digoxin and PBI-05204) against Marburgvirus in Vero E6 cells.
9 depicts a chart summarizing the in vitro cell viability of Vero E6 cells in the presence of various compositions (oleandrine, digoxin and PBI-05204).
10A and 10B depict charts summarizing the ability of compositions (oleandrine and PBI-05204) to inhibit Ebolavirus in Vero E6 cells immediately after exposure to virus: FIG. 10A 2 h post infection; Figure 10b 24 hours post infection.
11A and 11B depict charts summarizing the ability of compositions (oleandrine and PBI-05204) to inhibit Marburgvirus in Vero E6 cells immediately after exposure to virus: FIG. 11A is 2 hours post infection; 11B 24 hours post infection.
12A and 12B depict charts summarizing the ability of compositions (oleandrine and PBI-05204) to inhibit the product of infectious progeny by virus infected Vero E6 cells exposed to oleandrine. 12a-Ebolavirus; 12b-Marburg virus.
13A and 13B show the in vitro dose response antiviral activity of various compositions (oleandrine, digoxin and PBI-05204) against Venezuelan equine encephalomyelitis virus ( FIG. 13A ) and Western equine encephalomyelitis virus ( FIG. 13B ) in Vero E6 cells. A summary chart is shown.
14 is a diagram summarizing the effect of vehicle control, oleandrine or extracts of N. oleander on HTLV-1 replication or release of newly synthesized viral particles, as determined by quantification of HTLV-1 p19 ^Gag. shown (see Examples 19 and 20). Untreated (UT) cells are shown for comparison. All data are presented from at least three independent experiments. Data represent mean ± standard deviation (error bars) of the experiment.
15 shows a chart summarizing the relative cytotoxicity of vehicle control, oleandrine and N. oleander extracts against HTLV-1 + SLB1 lymphoma T-cell lines. All data are presented from at least three independent experiments. Data represent mean ± standard deviation (error bars) of the experiment.
16A-16F show representative micrographs of annexin V-FITC (green) and PI (red) staining results with DIC phase difference in the merged images. Individual annexin V-FITC and PI fluorescence channel images are also provided. Scale bar, 20 μm.
17 is a chart summarizing the effect of vehicle control, oleandrine or N. oleander extract on HTLV-1 replication and release of newly synthesized viral particles from oleandrine-treated HTLV-1 + lymphoma T-cells. indicates.
18 presents a chart summarizing the relative cytotoxicity of vehicle control, oleandrine or N. oleander extracts against treated huPBMCs.
19 presents a chart summarizing the relative inhibition of HTLV-1 transduction in vehicle control, co-culture assay huPBMCs containing oleandrine or N. oleander extracts.
20 depicts representative photomicrographs: fluorescence microscopy (upper panel) and immunoblotting (lower panel) of a GFP-expressing HTLV-1 + SLB1 T-cell line.
21 depicts representative micrographs of the virological synapses between huPBMCs and mitomycin C-treated HTLV-1 + SLB1/pLenti-GFP lymphoblasts (green cells).
22 shows a plot of the mean data with standard deviations (error bars) from the quantification of the micrographs of FIG. 21 .
23A-23D show VERO E6 cells infected with SARS-CoV-2 virus treated with oleandrine (red bars) or control vehicle (culture medium) (black bars) at 24 and 48 hours after “treatment”. A log plot of SARS-CoV-2 virus titer (PFU/mL) versus time (h) is shown (Example 28). Cells were pretreated with oleandrine prior to infection. After the initial incubation for 2 hours after infection, the infected cells were washed to remove extracellular viruses and oleandrin. Thereafter, the recovered infected cells were treated as follows. Infected cells were treated with oleandrine (FIG. 23A: 1 μg/mL in 0.1% aqueous DMSO with RPMI 1640 culture medium as aqueous component; FIG. 23C: 0.1 μg/mL in 0.01% aqueous DMSO with RPMI 1640) or Just treated with control vehicle ( FIG. 23B: 0.1% aqueous DMSO with RPMI 1640; FIG. 23D: 0.01% aqueous DMSO with RPMI 1640) and virus titers were determined.
Figure 24a shows the viral replication inhibition rate (Y1, left axis) and Vero-E6 cell number (Y2, right axis: olean for the cells) versus the concentration of oleandrine (μg/mL) in the culture medium 24 hours after infection. A double y-axis plot of the potential cytotoxic expression of Drin) is shown (Example 29). Figure 24b is for the culture of Figure 24, but was taken 48 hours after infection.
25 shows a plot of the percentage of Vero-E6 cells (cell titer) versus the concentration of oleandrine (μg/mL) in the culture medium at 24 h after continuous exposure of the cells to the indicated concentrations of oleandrine. (Example 30).
26A-26B are oleandrine (blue circles) or control vehicle (culture medium) (red circles) at 24 hours ( FIG. 26A ) and 48 hours ( FIG. 26B ) after "treatment" following infection with SARS-CoV-2 virus. ) of oleandrine in culture medium for VERO CCL-81 cells (Seropithecus aetiops renal normal cells; https://www.atcc.org/products/all/CCL-81.aspx) treated with A log chart of SARS-CoV-2 virus titer (PFU/mL) versus concentration is shown (Example 31).
In Figures 26a and 26b, fold reductions in viral titers were determined at 24 hours (Figure 26c) and 48 hours (Figure 26d).
27A-27D show SARS-CoV-2 on VERO E6 cells infected with SARS-CoV-2 virus treated with oleandrine (blue circles) or control vehicle (culture medium) at 24 and 48 hours after “treatment”. A log chart of virus titer (PFU/mL) versus time (h) is shown (Example 28). Cells were pretreated with oleandrine prior to infection. After the initial incubation for 2 hours after infection, the infected cells were washed to remove extracellular viruses and oleandrin. The recovered infected cells were then treated as follows. Infected cells were treated with oleandrine ( FIG. 27A : 0.005 μg/mL in aqueous DMSO (0.005%) with RPMI 1640 culture medium as aqueous component; FIG. 27B : 0.01 μg/mL in aqueous DMSO (0.01%) with RPMI 1640) /mL) or; 27C: 0.05 μg/mL in aqueous DMSO (0.05%) with RPMI 1640; 27B: Treatment with 0.1 μg/mL in aqueous DMSO (0.1%) with RPMI 1640) and determination of viral titers.
Figures 28a and 28b show oleandrine (dark blue circles (Experiment 2) and light blue circles (Experiment 3) after infection with SARS-CoV-2 virus at 24 hours (Figure 28A) and 48 hours (Figure 28B) after “treatment”. )) or control vehicle (culture medium) (dark red squares (Experiment 2) and light red squares (Experiment 3)) versus SARS-CoV-2 virus titers (PFU/mL) in culture medium for VERO 81 cells treated A log chart of oleandrine concentrations is shown. Experiment 2 and Experiment 3 are merely duplicate runs of the assay.
29A and 29B depict bar graphs of viral titer versus oleandrine concentration in culture medium, where viral titers were measured at 24 hours ( FIG. 29A ) and 48 hours ( FIG. 29B ) post infection. For some samples, cells were treated with oleandrine (blue bars) or DMSO control vehicle (red bars) before and after infection (2 h). For other samples, cells were treated with oleandrine (hashed blue bars: 12 hours post infection, hollow blue bars: 24 hours post infection) or DMSO control vehicle (hashed red bars: 12 hours post infection, open red bars). : 24 hours).
30A and 30B depict charts for evaluation of anti-COVID-19 activity of dual extract combination compositions (PBI-A). In FIG. 30B , the μg/ml (oleandrine concentration) indication assumes that PBI-A was supplied as a 1 mg/ml (oleandrine concentration) solution. Viral titer (Log ₁₀ (PFU/mL)) versus Log ₁₀ dilution factor ( FIG. 30A ) or viral titer versus Log ₁₀ concentration of oleandrine ( FIG. 30B ) was determined. 30A is for data processing analysis before infection of Example 31. 30b is for the post-infection treatment analysis of Example 34.

The present invention provides a method of treating a viral infection in a subject by chronically or acutely administering to the subject an effective amount of one or more antiviral compositions (or pharmaceutical compositions comprising an antiviral composition and one or more pharmaceutical excipients). The composition is administered according to a dosing regimen most suitable for the subject, and the dosage and suitability of the dosing regimen can be clinically determined according to conventional clinical practice and clinical treatment endpoints for viral infections.

As used herein, the term “subject” is understood to mean a warm-blooded animal such as a mammal, eg, a cat, a dog, a mouse, a guinea pig, a horse, a cow, a sheep and a human.

As used herein, a subject at risk for viral infection includes: a) a subject residing in a geographic area inhabited by mosquitoes, particularly Aedes egypti (Aedes albopictus) mosquitoes; b) subjects living near or with people infected with the virus; c) a subject having sexual intercourse with a person with a viral infection; d) a subject residing in a geographic area inhabited by mites, particularly Ixodes marx, Ixodes scapularis or Ixodes cooke species mites; e) a subject living and residing in a geographic area inhabited by fruit bats; f) subjects living in tropical areas; g) persons living in Africa; h) a subject in contact with the bodily fluids of another subject 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 fertile female, or a pregnant female.

Subjects treated in accordance with the present invention will exhibit a therapeutic response. By "therapeutic response" is meant that a subject suffering from a viral infection enjoys at least one of the following clinical benefits as a result of treatment with cardiac glycosides: a reduction in the concentration of active virus in the subject's blood or plasma, the subject's blood or Eradication of active virus from plasma, amelioration of infection, reduction of incidence of symptoms associated with infection, partial or complete remission of infection or increased time to progression of infection and/or reduction of infectivity of the virus causing said viral infection. The therapeutic response may be a total or partial therapeutic response.

As used herein, “time to progression” is the period, length, or duration from when a viral infection is diagnosed (or treated) until the infection begins to worsen. The period during which the level of infection is maintained without further infection progression, and that period ends when the infection begins to progress again. The progression of the disease is determined by “staging” a subject suffering from an infection prior to or at the beginning of treatment. For example, a subject's health is determined prior to or at the start of treatment. The subject is then treated with an antiviral composition and the virus concentration is monitored periodically. At some point later, the symptoms of infection may worsen, thus marking the progression of the infection and the end of the "time to progression". "Time to progression" is the period during which the infection has not progressed or the level or severity of the infection has not worsened.

Dosage regimens include therapeutically relevant doses (or effective doses) of one or more cardiac glycosides and/or triterpenes administered according to a dosing schedule. Thus, a therapeutically relevant dosage is a therapeutic dosage in which a therapeutic response of a viral infection to treatment with the antiviral composition is observed and the antiviral composition can be administered to a subject without undue unwanted or deleterious side effects. Therapeutically relevant dosages may cause some side effects in the patient, but are not fatal to the subject. A dose at which the level of clinical benefit for the subject to which the antiviral composition is administered exceeds the level of adverse side effects experienced by the subject as a result of administration of the antiviral composition or these component(s). Therapeutically relevant dosages will vary from subject to subject according to various established pharmacological, pharmacodynamic and pharmacokinetic principles. However, a therapeutically relevant dosage (eg, a dosage relative to oleandrine) is typically about 25 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms, or about 750 micrograms. cardiac glycoside per day, or the amount of cardiac glycoside per dose is in the range of about 25-750 micrograms, or about 25 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms or about 750 micrograms per day may not exceed grams of cardiac glycosides. Another example of a therapeutically relevant dose (eg, individually or in combination with a triterpene) is generally about 0.1 mg/kg to 100 mg/kg, about 0.1 mg/kg to about 500 per kg body weight. mg/kg, about 100 to about 1000 mg/kg, 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 range. It is known in the art that the actual amount of an antiviral composition needed to provide a targeted therapeutic result in a subject may vary from subject to subject depending upon the underlying principles of pharmacy.

Digoxin treatment can be carried out using two or more administration phases: a loading phase and a maintenance phase. The loading phase may use the next dosing regimen until steady state plasma levels of digoxin are achieved, and the maintenance phase may use the next dosing regimen after completion of the loading phase.

Therapeutically relevant dosages may be administered according to any dosing regimen typically used for the treatment of viral infections. A therapeutically relevant dosage may be administered once, twice, three or more times daily. Every other day, every 3 days, every 4 days, every 5 days, every half week, every week, every other week, every 3 weeks, every 4 weeks, monthly, every other month, every half month, every 3 months, every 4 months, every half year , annually, or any combination of the above to arrive at a suitable dosing schedule. For example, a therapeutically relevant dose may be administered one or more times daily (up to 10 times a day for highest doses) for at least one week.

^{Example 15 is oleandrine (active alone), Anvirzel™} for the treatment of Ebolavirus ( FIGS. 1 and 2 ) and Marburg virus ( FIGS. 3 and 4 ) infections, both of which are filoviruses. ) (hydrothermal extraction of Nerium oleander) and PBI 05204 (supercritical fluid (SCF) extract of Nerium oleander) in vitro assays used to evaluate the efficacy of compositions are provided.

The hot water extract can be administered orally, sublingually, subcutaneously and intramuscularly. One embodiment is a liquid formulation available under the trade designations ANVIRZEL™ (Nerium Biotechnology, Inc., San Antonio, TX; Salud Integral Medical Clinic, Tegucigalpa, Honduras; www.saludintegral.com; www.anvirzel.com). For sublingual administration, the usual dosing regimen is 1.5 ml per day or 0.5 ml 3 times per day. For injection administration, a typical dosing regimen is from about 1 to about 2 ml/day, or from about 0.1 to about 0.4 ml/m ² /day for at least about 1 week to about 6 months or more, or from about 1 week to about 6 months or more. 0.4 to about 0.8 ml/m ² /day, or about 0.8 to about 1.2 ml/m ² /day for at least about 1 week to about 6 months or more. Because the maximum tolerated dose of ANVIRZEL™ is much higher, higher doses can be used. ANVIRZEL™ contains oleandrine, oleandrigenin and polysaccharides extracted from Nerium oleander (hot water extraction). Commercially available vials are lyophilized powders (before reconstitution with water prior to administration) comprising about 200 to about 900 μg of oleandrine, about 500 to about 700 μg of oleandrigenin and a polysaccharide extracted from Nerium oleander. , about 150 mg of oleander extract. Said vial also contains one or more osmotic agents, for example mannitol, sodium chloride, one or more buffers, for example sodium ascorbate with ascorbic acid, one or more preservatives, for example propylparaben, a pharmaceutical excipient such as methylparaben. can do.

The experiment was set up by adding the composition to cells at 40 micrograms/mL, then adding the virus and incubating for 1 hour. When the virus is added to the cells, the final concentration of the composition is 20 micrograms/mL. Compositions containing different amounts of oleandrine can be adjusted according to the concentration of oleandrine they contain, which can be converted to a molar concentration. 1 to 4 show the efficacy based on the oleandrine content of the extract. OL itself is effective. PBI-05204, an SCF extract of Nerium oleander containing OL, OA, UA and BA, is substantially more effective than OL itself. ^{Anvirgel TM} , a boiling water extract of Nerium Oleander, is more effective than OL itself. Both extracts clearly show efficacy in the nanomolar range. The percentage of oleandrine in the PBI-05204 extract (1.74%) ^{is higher than in Anvirgel™} (0.459%, 4.59 micrograms/mg). At the highest dose of PBI-05204, it completely inhibited EBOV and MARV infection, whereas Anvirgel ^TM achieved complete inhibition, as toxicity is observed at doses greater than 20 micrograms/mL in combination with ^{Anvirgel TM.} did not show The data demonstrate the best antiviral activity against Ebolavirus and Marburg virus against PBI-05204. Combination of triterpenes in PBI-05204 increased the antiviral activity of oleandrine.

Example 6 provides a detailed description of an in vitro assay used to evaluate the efficacy of cardiac glycosides for the treatment of Zika virus (flavivirus) infection. Vero E6 cells were infected with Zika virus (ZIKV strain PRVABC59) at an MOI of 0.2 in the presence of oleandrine ( FIG. 5 ) or digoxin ( FIG. 6 ). Cells were incubated with virus and cardiac glycosides for 1 h before inoculum and non-absorbable cardiac glycosides (if present) were removed. Cells were immersed in fresh medium and cultured for 48 hours, fixed with formalin, and stained for ZIKV infection. The data demonstrate antiviral activity against Zika virus against both cardiac glycosides; Oleandrine exhibited higher (nearly 8-fold greater) antiviral activity than digoxin.

Example 14 provides a detailed description of the assays used to evaluate the antiviral activity of test compositions against Zika virus and Dengue virus. The data indicate that oleandrine exhibits efficacy against Zika virus and Dengue virus.

7 depicts a chart summarizing the in vitro dose response antiviral activity of various compositions (oleandrine, digoxin and PBI-05204) against Ebolavirus (EBOV) in Vero E6 cells. 8 depicts a chart summarizing the in vitro dose response antiviral activity of various compositions (oleandrine, digoxin and PBI-05204) against Marburgvirus (MARV) in Vero E6 cells. 9 depicts a chart summarizing the in vitro cell viability of Vero E6 cells in the presence of various compositions (oleandrine, digoxin and PBI-05204). 7-8 , host cells were exposed to the composition prior to viral infection. Vero E6 cells were infected with EBOV/Kik (Fig. 7, MOI = 1) or MARV/Ci67 (Fig. 8, MOI = 1) in the presence of oleandrine-containing plant extracts oleandrine, digoxin or PBI-05204. . After 1 hour, the inoculum and compounds were removed and fresh medium was added to the cells. After 48 hours, cells were fixed and immunostained to detect cells infected with EBOV or MARV. Infected cells were enumerated using Operetta.

Cell viability was tested in the presence of the composition to ensure that no false positives were observed with respect to antiviral activity. For the data of Figure 9, Vero E6 cells were treated with the compound as above. ATP levels were measured by CellTiter-Glo as a measure of cell viability. It was determined that oleandrine, digoxin and PBI-05204 did not reduce cell viability, meaning that the antiviral activity detailed in other figures herein is not due to false positives caused by the cytotoxicity of individual compounds. .

Accordingly, the present invention provides a method of treating a viral infection in a mammal or host cell, the method comprising administering an antiviral composition to the mammal or host cell prior to contracting the viral infection; or upon viral infection of the host cell, the antiviral composition reduces the viral concentration and ameliorates, reduces or eliminates the viral infection.

The antiviral compositions and methods of the present invention are also useful for treating a viral infection that occurs prior to administration of the antiviral composition. Vero E6 cells were infected with EBOV ( FIGS. 10A , 10B ) or MARV ( FIGS. 11A , 11B ). At 2 hours post infection ( FIGS. 10A, 11A ) or 24 hours post infection ( FIGS. 10B , 11B ), oleandrine or PBI-05204 was added to the cells for 1 hour, then discarded and the cells returned to the culture medium.

10A and 10B depict charts summarizing the ability of compositions (oleandrine and PBI-05204) to inhibit Ebolavirus in Vero E6 cells immediately after exposure to virus: FIG. 10A - 2 hours post infection; Figure 10b - 24 hours post infection. When the antiviral composition is administered within 2 hours (or up to 12 hours) after viral infection, the viral concentration antiviral composition provides an effective treatment and reduces the EBOV virus concentration. Although the viral composition is effective even after 24 hours, its efficacy decreases as time passes after the initial viral infection. The same evaluation was performed for MARV. 11A and 11B depict charts summarizing the ability of compositions (oleandrine and PBI-05204) to inhibit Marburgvirus in Vero E6 cells immediately after exposure to virus: 11a- 2 hours post infection; Figure 11b - 24 hours post infection. When the antiviral composition is administered within 2 hours (or up to 12 hours) after viral infection, the viral concentration antiviral composition provides an effective treatment and reduces the MARV virus concentration. Although the viral composition is effective even after 24 hours, its efficacy decreases as time passes after the initial viral infection.

Given that the antiviral activity of the composition herein is reduced against a single generation of virus-infected cells, for example within 24 hours after infection, whether the antiviral composition can inhibit viral proliferation, i.e., inhibit the production of infectious progeny. evaluated whether it could be done. Vero E6 cells were infected with EBOV or MARV in the presence of oleandrine or PBI-05204 and cultured for 48 hours. The supernatant from the infected cell culture was passed through fresh Vero E6 cells, incubated for 1 hour and then discarded. Cells containing the passed supernatant were incubated for 48 hours. Cells infected with EBOV (B) or MARV (C) were assessed as described herein. Control infection rates were 66% for EBOV and 67% for MARV. The antiviral composition of the present invention inhibited the production of infectious progeny.

Accordingly, the antiviral composition of the present invention may be administered prophylactically prior to viral infection, in order to inhibit viral infection after exposure to the virus: a); b) to inhibit or reduce viral replication and production of infectious progeny, after viral infection; or c) a combination of a) and b).

The antiviral activity of the antiviral composition against Togaviridae alphavirus was evaluated using VEE virus and WEE virus in Vero E6 cells. 13A and 13B show in vitro dose response antiviral activity of various compositions (oleandrine, digoxin and PBI-05204) against Venezuelan equine encephalomyelitis virus ( FIG. 13A ) and Western equine encephalomyelitis virus ( FIG. 13B ) in Vero E6 cells. A chart summarizing the 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 18 h in the presence or absence of the indicated compounds. Infected cells were detected as before and enumerated in operetta. The antiviral composition of the present invention has been found to be effective.

Therefore, the present invention relates to the arenaviridae family virus, the philoviridae family virus, the flaviviridae family virus (the flavivirus genus), the retroviridae family virus, the deltaretrovirus genus virus, the coronaviridae family virus, paramic A method of treating a viral infection caused by a Sovidae virus or a Togaviridae virus is provided, the method comprising administering an effective amount of an antiviral composition, thereby exposing the virus to the antiviral composition, thereby causing the viral infection including treating the

We evaluated the use of oleandrine and its extracts described herein for the treatment of HTLV-1 (human T-cell leukemia virus type 1; enveloped retrovirus; deltaretrovirus genus) infection. To determine whether purified oleandrine compounds or N. oleander extracts can inhibit HTLV-1 provirus replication and/or ^{the production and release of p19 Gag-} containing viral particles, virus-producing HTLV-1-transformed SLB1 Lymphoma T-cell lines were treated with increasing concentrations of oleandrine or N. oleander extract or sterile vehicle control (MilliQ-treated _{20% DMSO in ddH 2} O), then _{under 10% CO 2 at} 37° C. for 72 hours. cultured. Cells were later pelleted by centrifugation, and ^{the relative levels of extracellular p19 Gag-} containing virus particles released into the culture supernatant were quantified by performing Anti-HTLV-1 p19 ^Gag ELISA (Zeptometrix).

14 shows vehicle control (1.5 μl, 7.5 μl, or 15 μl) or increasing concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml) of oleandrine compound or N. oleander extract for 72 hours. ^{Quantification data of HTLV-1 p19 Gag} expressed by treated HTLV-1 + SLB1 lymphoma T-cell lines are shown (Examples 19 and 20). Virus replication and extracellular particle release into culture supernatants were ^{quantified by performing Anti-HTLV-1 p19 Gag} ELISA (Zeptometrix). Oleandrine did not significantly inhibit HTLV-1 replication or release of newly synthesized viral particles. We confirmed that neither the extract nor oleandrin alone ^{significantly inhibited viral replication or release of p19 Gag-} containing particles into the culture supernatant. Therefore, we did not expect any further antiviral activity. However, we unexpectedly found that viral particles collected from treated cells exhibited reduced infectivity in primary human peripheral blood mononuclear cells (huPBMCs). In contrast to HIV-1, extracellular HTLV-1 particles are less infective, and viral transmission usually occurs through direct cell-to-cell interactions via viral synapses.

Accordingly, the present invention provides a method for producing HTLV-1 viral particles with reduced infectivity, wherein the method provides the HTLV-1 viral particles with reduced infectivity, using the antiviral composition of the present invention to produce HTLV -1 involves processing viral particles.

To confirm that the observed antiviral activity is not an artifact due to the potential cytotoxicity of the antiviral composition to HTLV-1 + SLB1 lymphoblasts, we investigated purified oleagin from treated HTLV-1 + SLB1 lymphoblast cultures. The cytotoxicity of various dilutions of andrine compounds and N. oleander extracts was evaluated (Example 21). SLB1 T-cells were treated with increasing concentrations (10, 50 and 100 μg/ml) of oleandrine or N. oleander extracts for 72 hours as described herein. As a negative control, cells were treated with increasing amounts of vehicle solution (1.5, 7.5 and 15 μl) corresponding to the volumes used for drug-treated cultures. Cyclophosphamide (50 μM; Sigma-Aldrich) treated cells were included as positive controls for apoptosis. The samples were then 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 counting triplicate fields of view using a fluorescence microscope and a 20x objective.

The results ( FIGS. 15 and 16A-16F ) indicate that the lowest concentration (10 μg/ml) of oleandrine and N. oleander extract did not induce significant cytotoxicity/apoptosis. However, higher concentrations of crude plant extracts (about 50 and about 100 μg/ml) induced significantly more cell death than oleandrine compounds. This is consistent with the fact that oleandrine accounts for about 1.23% of the extract of N. oleander. Cytotoxicity induced by oleandrine was not significantly higher in treated HTLV-1 + SLB1 cells than in vehicle control.

We then investigated whether oleandrine or N. oleander extract could inhibit viral propagation from green fluorescent protein (GFP)-expressing HTLV-1 + lymphoma T-cell lines into huPBMCs in a co-culture assay (Example 20). . For this study, HTLV-1 + SLB1 lymphoma T-cells were treated with increasing concentrations of oleandrine compound or N. oleander extract or vehicle control in 96-well microtiter plates for 72 h, followed by virus-containing supernatants. were collected and used to directly infect primary cultured human peripheral blood mononuclear cells (huPBMC) in vitro. ^{After 72 h, the relative levels of extracellular p19 Gag-} containing viral particles released into the culture supernatant as a result of direct infection were quantified by performing Anti-HTLV-1 p19 ^{Gag ELISA.}

HTLV-1 + SLB1 lymphoma T-cell lines were treated with vehicle control for 72 hours or with increasing concentrations of N. oleander or oleandrine compounds (10 μg/ml, 50 μg/ml and 100 μg/ml). Next, virus-containing supernatants were collected and used to directly infect primary huPBMCs. Vehicle control, N. oleander extract or oleandrine was also included in the culture medium of huPBMC. After 72 h, culture supernatants were collected and the relative amounts of extracellular viral particles produced were quantified by performing ^{Anti-HTLV-1 p19 Gag ELISA.}

The data (FIG. 17) shows that both oleandrine and N. oleander extracts were released into the culture supernatant of treated cells, even at the lowest concentration (10 μg/ml), as compared to the corresponding amount of the vehicle control. This ^{indicates that the infectivity of p19 Gag-} containing virus particles was inhibited. Both oleandrine and crude extract inhibited the formation of viral synapses and the propagation of HTLV-1 in vitro. Extracellular viral particles produced by oleandrine-treated HTLV-1 + lymphoma T cells show reduced infectivity in primary huPBMCs. Importantly, oleandrine exhibits antiviral activity against enveloped viruses by reducing the incorporation of envelope glycoproteins into mature particles, representing a unique step in the retroviral infection cycle.

To confirm that the observed antiviral activity was not an artifact due to the potential cytotoxicity of the antiviral composition to the treated huPBMCs, in the treated huPBMCs, compared to the vehicle negative control, purified oleandrine and N. oleander The cytotoxicity of the extract was investigated (Example 21). Primary buffy-coat huPBMCs were isolated, stimulated with plant phytohemagglutinin (PHA) and cultured in the presence of recombinant human interleukin-2 (hIL-2). Cells were then treated with increasing concentrations of oleandrine or N. oleander extract or increasing volumes of vehicle for 72 hours. Samples were then stained with annexin V-FITC and PI, and the relative percentage of apoptotic (i.e., annexin V-FITC and/or PI-positive) cells per field was measured by confocal fluorescence microscopy and triplicate counting ( counting in-triplicate).

The cytotoxic effects of vehicle control, N. oleander extract and oleandrine compound were evaluated by treating primary huPBMCs for 72 hours, and then the cultures were stained with annexin V-FITC and PI. Relative percentages of apoptotic (ie, annexin V-FITC and/or PI-positive) cells were quantified by fluorescence microscopy and counting triplicate fields using a 20x objective. Total cell number was determined using DIC phase contrast microscopy. Cyclophosphamide (50 μM) treated cells were included as a positive control for apoptosis. NA indicates that the number of cells in this sample is too low for an accurate assessment due to higher toxicity.

The data ( FIG. 18 ) show that oleandrine exhibited moderate cytotoxicity (eg, 35-37% at the lowest concentration) in huPBMCs compared to the vehicle control. In contrast, the N. oleander extract was significantly cytotoxic and induced high levels of programmed cell death even at the lowest concentrations. huPBMCs were somewhat more sensitive to purified oleandrine than HTLV-1 + SLB1 lymphoblasts. However, huPBMCs were much more sensitive to the crude N. oleander extract, which also contained other cytotoxic compounds such as triterpenes described herein.

In addition, we investigated whether oleandrine or N. oleander extract could interfere with the delivery of HTLV-1 particles targeting huPBMC in co-culture experiments (Example 22). For this study, virus-producing HTLV-1 + SLB1 T-cell lines were treated with mitomycin C followed by increasing amounts of oleandrine, N. oleander extract or vehicle control for 15 min or 3 h. . SLB1 cells were washed twice with serum-free medium, an equal number of huPBMCs were added to each well, and samples were co-cultured for 72 hours in complete medium at 37° C. under _{10% CO 2 in a humidified incubator.} The relative intercellular transmission of HTLV-1 was assessed by performing an ^{Anti-HTLV-1 p19 Gag} ELISA to measure the level of extracellular virus released into the culture supernatant.

Primary huPBMCs were co-cultured with mitomycin C-treated HTLV-1 + SLB1 lymphoma T-cells, either as vehicle control, or at increasing concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml) of N Pre-treated with oleander extract or oleandrine compound for 15 min or 3 h. Vehicle controls, extracts and compounds were also present in the co-culture medium. After 72 h, the supernatant was collected and Anti-HTLV-1 p19 ^Gag ELISAs were performed to quantify the amount of extracellular virus particle release.

The results shown in Figure 19 demonstrate that oleandrine and N. oleander extracts inhibited the propagation of HTLV-1 compared to vehicle control; However, no difference was observed between 15 min and 3 h of pretreatment of HTLV-1 + SLB1 cells.

We also investigated whether oleandrine inhibits virological synapse-forming and HTLV-1 propagation in a co-culture assay (Example 22). GFP-expressing HTLV-1 + SLB1 T-cell line transfected SLB1 lymphoma T-cells with pLenti-6.2/V5-DEST-GFP vector after 2 weeks of selection against blasticidin (5 μg/ml; Life Technologies) was created by introducing GFP-positive clones were screened, expanded and passed repeatedly by fluorescence microscopy ( FIG. 20 top panel) and immunoblotting ( FIG. 20 bottom panel). DIC phase contrast images are provided for comparison.

huPBMC and mitomycin C-treated HTLV-1 pretreated with vehicle control or increasing amounts (10 μg/ml, 50 μg/ml and 100 μg/ml) of N. oleander extract or oleandrine compound for 3 hours + Viral synapse formation between SLB1/pLenti-GFP lymphoblasts (green cells) was visualized by fluorescence microscopy ( FIG. 21 ). Quantify the relative percentage of infected (i.e., HTLV-1 gp21-positive, red) huPBMCs (GFP-negative) in 20 fields of view (n = 20) by fluorescence microscopy using a 20x objective (see arrows in vehicle control panel). to evaluate the spread of the virus. Fluorescence microscopy data were quantified ( FIG. 22 ). The data confirm that the antiviral composition inhibits viral synapse formation and propagation of HTLV-1 in a co-culture assay.

Therefore, the present invention also inhibits (reduces) the infectivity of HTLV-1 particles released into the culture supernatant of treated cells, and also reduces intercellular propagation of HTLV-1 by inhibiting Env-dependent formation of viral synapses. provide a way The method comprises treating virus-infected cells (in vitro or in vivo) with an effective amount of an antiviral composition.

The antiviral activity of the compositions herein was evaluated against Rhinovirus infection. Rhinoviruses belong to the family Picornaviridae and the genus Enterovirus. It is an unenveloped, (+) polarity ss-RNA virus. Oleandrine was found to be inactive against rhinovirus at the concentrations and assays used herein, as it does not inhibit viral replication.

CoV infection can be treated in vivo, as detailed in Example 26, wherein the antiviral composition is administered to a subject as a monotherapy or a combination therapy. The efficacy of oleandrine on CoV was established in vivo according to Example 27. In a small portion of orange juice, 0.25 ml of reconstituted ANVIRZEL™ was administered to the child. Then, 0.5 ml of reconstituted ANVIRZEL™ was administered every 12 hours for about 2-3 days. The infant has recovered from COVID-19 infection.

Further evidence for the efficacy of oleandrine (composition containing oleandrine) against coronavirus, e.g., SARS-CoV-2 (COVID-19), was obtained through an in vitro evaluation according to Example 28 and Vero cells were pretreated with oleandrine and then infected with SARS-CoV-2. After cell infection, extracellular virus and oleandrine were washed away, and infected cells were washed with oleandrine ( FIG. 23A: 1 μg/mL in 0.1 % v/v aqueous DMSO; FIG. 23C: 0.1 in 0.01 % v/v aqueous DMSO; FIG. μg/mL) or aqueous DMSO as control vehicle ( FIG. 23B : 0.1 % v/v aqueous DMSO; FIG. 23D : 0.01 % v/v aqueous DMSO). Results were a) oleandrine pretreatment reduced viral load by 1368-fold at 24 h and 369-fold at 48 h; b) oleandrine is effective over the entire concentration range from about 0.1 to about 1.0 μg/mL, high doses are slightly better than low doses, and oleandrine is even at much lower concentrations, e.g. 0.01 to 0.1 very likely to be effective at μg/mL; c) repeated administration of oleandrine is required, as a single dose cannot completely halt viral replication; d) 30 min pre-incubation of Vero cells with oleandrine is slightly effective in reducing initial viral infection, indicating that it does not affect the infectivity of progeny virions. The results also indicate that oleandrine at concentrations of 0.1 and 1.0 μg/mL are not excessively toxic to Vero cells. Results showed that oleandrine was reduced by a) by about 1 log ₁₀ without continuous drug treatment; and b) inhibits the infectivity of the progeny virus by about >3 log _{10 (no toxicity) with continuous drug treatment.}

To determine whether oleandrine directly inhibits viral replication, Vero-E6 cells were infected with SARS-CoV-2 virus and treated with oleandrine at various concentrations according to Example 29. The results are shown in Figures 24A and 24B. At the 24 h time point ( FIG. 24A ), antiviral activity was observed in wells treated with oleandrine only during the uptake phase (pretreatment data), with an estimated IC _{50 of 0.625 μg/mL.} In wells treated with oleandrine during the assay period (period data), oleandrine significantly limited viral entry and/or viral replication even in the presence of large amounts of inoculated virus. At the 48 h time point ( FIG. 24B ), minimal antiviral activity was observed until the end of the period in wells treated with oleandrine only during the uptake phase (pretreatment data). In wells treated with oleandrine during the assay period (period data), oleandrine significantly limited viral infection. Potential methods of action include inhibition of viral replication, assembly and/or shedding.

To confirm that the observed antiviral activity of oleandrine against SARS-CoV-2 was not due to the cytotoxicity of oleandrine on Vero-E6 cells, at 24 h (Fig. 24a) and 48 h (Fig. 24b). ), the cell titers were measured. At concentrations of oleandrine above 1.0 μg/mL, cytotoxicity appeared and potentially interfered with the assay. However, at the oleandrine concentration of 0.625 μg/mL or less, the interference of cytotoxicity was significantly reduced, confirming the strong antiviral activity of oleandrine even at a very low concentration. Further evidence of the degree of toxicity of oleandrine to Vero-E6 cells was observed in the analysis of Example 30 ( FIG. 25 ). At an oleandrine concentration of 0.625 μg/mL, about 80% of Vero cells were viable at the 24 h time point, and less toxicity was observed at the lower concentration. It should be understood that the toxicity of oleandrine on Vero-E6 cells does not suggest that oleandrine is toxic to humans. This measure of toxicity is used to determine the potential effect of background cell death when simply measuring antiviral activity.

Thus, oleandrine has at least a dual mechanism (pathway) for treating viral infections, particularly coronavirus infections, eg SARS-CoV-2 infection: i.e., a) direct inhibition of viral replication; and b) reduced infectivity of the progeny virus.

Moreover, oleandrine has antiviral activity even at very low doses, and oleandrine has a significant prophylactic effect. This was demonstrated according to Example 31 in which VERO CCL-81 cells were infected with SARS-CoV-2. Cells were pretreated with oleandrine prior to infection. After the initial incubation for 2 hours after infection, the infected cells were washed to remove extracellular viruses and oleandrin. The recovered infected cells were then treated as follows. Infected cells were treated with oleandrine (various concentrations in aqueous DMSO with RPMI 1640 culture medium as aqueous component) or only control vehicle (aqueous DMSO with RPMI 164), followed by “treatment” for 24 hours ( Viral titers were measured at Figure 26a) and 48 hours (Figure 26b). In the absence of oleandrine, SARS-CoV-2 reached high titers (approximately 6 log ₁₀ plaque forming units (pfu)/ml) by the 24 h time point and maintained titers at later time points: limit of detection for analysis or consistently below it. Oleandrin concentrations between 1 μg/mL and 0.05 μg/mL significantly reduced virus titers in just 24 hours. The two higher doses reduced virus titers essentially below the limit of detection and no cytotoxicity was observed at any of the oleandrin concentrations tested. A fold reduction in virus titer was calculated for these samples. Fold reductions in viral titers ( FIGS. 26C and 26D ) ranged from about 1,000-fold to about 40,000-fold at the 48-hour time point, and were observed from about 1,000-fold to about 20,000-fold at the 24 hour time point. Although the 10 ng/ml dose had no significant effect compared to the DMSO control at 24 h post-infection, the titer was significantly reduced at 48 h post-infection. Importantly, the decrease due to oleandrine increased at the highest concentration measured at 48 h compared to 24 h. The increased prophylactic efficacy of oleandrine over time (24 hours versus 48 hours) was _{reflected in its EC 50} values, which were calculated to be 11.98 ng/ml at 24 hours post infection and 7.07 ng at 48 hours post infection. Calculated as /ml.

The Vero 81 cells described above underwent genomic analysis to determine whether inhibition of SARS-CoV-2 was at global or infectious particle production levels. RNA was extracted from cell culture supernatants of prophylactic studies and genomic equivalents were quantified via qRT-PCR (Example 39). The prophylactic effect of oleandrine, initially observed through infectivity assays, was confirmed at the genomic equivalent level. At 24 h post infection, oleandrine significantly reduced the SARS-CoV-2 genome in the supernatant at the 4 highest doses. The prophylactic effect of oleandrine, initially observed through infectivity assays, was confirmed at the genomic equivalent level. At 24 h post infection, oleandrine significantly reduced the SARS-CoV-2 genome in the supernatant at the 4 highest doses.

At 24 and 48 hours post infection, additional studies were performed to determine the dose response of COVID-19 infection to oleandrine ( FIGS. 27A-27B ). A dose response was observed, and increasing the concentration of oleandrine in the culture medium resulted in a greater decrease in virus titer; However, even at the lowest concentration tested (0.05 μg/mL), the titers decreased at 24 h, and the titers decreased even more at 48 h post-infection. The highest dose reduced infectious SARS-CoV-2 titers by more than 1,000-fold, the 0.5 μg/ml and 100 ng/ml doses caused a more than 100-fold reduction, and the 50 ng/ml dose caused a 78-fold decrease.

28A and 28B show the results of a duplicate study performed in triplicate each to determine the dose-response of COVID-19 to treatment with oleandrine at various concentrations (0.005 to 1 μg/mL) in culture medium. . Significant antiviral activity was also observed at 24 and 48 hours post infection in Vero 81 cells for concentrations of 0.01 μg/mL or higher. A large decrease in virus titer was observed even at very low concentrations of 0.05 μg/mL.

A study according to Example 34 was conducted to determine the antiviral efficacy of oleandrine after infection. Vero 81 cells were not pretreated with oleandrine prior to infection. Instead, cells were infected with the COVID-19 virus and then treated with oleandrine (at the indicated concentrations) at 12 and 24 hours post infection. Viral titers were then measured at 24 hours ( FIG. 29A ) and 48 hours ( FIG. 29B ) post infection. The data show that even with a single treatment, oleandrine can exert antiviral activity for at least 12 hours, at least 24 hours, or at least 36 hours after infection. It is important to note that this assay is a time-compressed assay compared to human viral infection. The 24 hour time point corresponds to about 5 to 7 days post infection in humans, and the 48 hour time point corresponds to about 10 to 14 days post infection in humans.

The analysis of Examples 31 and 34 was repeated using a dual extract combination composition (PBI-A, PBI-A containing 1% wt of the ethanolic extract 1% wt of Example 36 dissolved in DMSO (98% wt)). did. Figure 30a describes in detail the evaluation result of the double extract combination composition according to the analysis of Example 31. 30B details the results of evaluation of the double extract (1 % wt) according to the analysis of Example 34. 30A demonstrates the relative antiviral (anti-COVID-19) efficacy of PBI-A based on relative dilutions of the original stock solution. The data in FIG. 30B is based on the relative concentrations (μg/mL) of oleandrine in the assay solution. The dotted line in each graph represents the lowest concentration of virus detectable using CFU (Viral Colony Forming Unit) assay.

Based on the results of Figures 30a and 30b. The dual extract combination composition is effective as an antiviral agent against COVID-19 at concentrations of 0.05 to 1.0 μg/ml, in the same range as that observed for pure oleandrine.

It is also important to observe that the concentrations of oleandrine evaluated in the assay are clinically relevant in terms of dose and plasma concentrations.

Evidence for the safety of oleandrine-containing compositions was further provided by exposing the cells to solutions containing different concentrations of oleandrine, followed by an in vitro cellular assay to determine the release of lactate dehydrogenase. . Up to a concentration of 1 μg/mL, no additional toxicity was found compared to the control vehicle.

Efficacy of oleandrine (oleandrine-containing composition, oleandrine-containing extract) in the treatment of COVID-19 viral infection was demonstrated by using oleandrine-containing sublingual formulations (Examples 32 or 37) under the FDA's Extended Access Program. 35 was further established by administering to a subject. Subjects between the ages of 18 and 78 years were administered a 15 μg dose of oleandrine (as a dual extract composition) four times daily, approximately 6 hours apart, or three 15 mg doses daily, approximately 8 hours apart. Prior to initiation of treatment, the subject's clinical status and/or viral titers were observed. Some subjects received palliative care or hospice treatment. Clinical status and/or viral titers were determined periodically during treatment periods of 1-2 weeks, 10-14 days. After initiation of treatment, the following results were observed.

Additional in vivo studies under the FDA's Extended Access Program were conducted in a second group of human subjects who displayed different levels of COVID-19-related symptoms. Prior to initiating treatment, the subject's clinical status and/or viral titer was determined to confirm COVID-19 infection. Some subjects had moderate to severe symptoms. Age subjects were administered oleandrine (as a dual extract composition) at a dose of 15 μg 4 times a day at about 6 hour intervals. Clinical status and/or viral titers were determined periodically during treatment periods of 1-2 weeks, 10-14 days. All subjects fully recovered from COVID-19 infection within 5 to 12 days after initiation of treatment.

Oleandrine has also been shown to produce a strong anti-inflammatory response, which may help prevent a hyper-inflammatory response to SARS-CoV-2 infection.

Accordingly, the present invention provides a method of treating a COVID-19 virus infection, comprising administering to a subject suffering from said infection multiple doses of cardiac glycoside (composition containing cardiac glycoside or extract containing cardiac glycoside). The multiple doses may be divided into one or more doses per day for at least two days per week, optionally for at least one week per month, further optionally for at least one month per year. A preferred cardiac glycoside is oleandrine.

Accordingly, the present invention provides a method of treating a coronavirus infection, in particular a coronavirus that is pathogenic to humans, eg SARS-CoV-2 infection. The method comprises chronically administering to a subject having the infection a therapeutically effective amount of a cardiac glycoside (a composition containing a cardiac glycoside). Chronic administration can be achieved by repeatedly administering one or more (plurality) therapeutically effective doses of cardiac glycosides (compositions containing cardiac glycosides). One or more doses may be administered daily, for at least one day per week, optionally for at least one week per month, optionally for at least one month per year.

Accordingly, the present invention provides a method of treating a virus, eg, a CoV infection, in a subject in need thereof (especially a human subject), comprising: a) an antiviral composition comprising at least one dose of an antiviral composition comprising oleandrine; or b) administering to the subject one or more doses of an antiviral composition comprising oleandrine and one or more other compounds extracted from Nerium species. Oleandrin may be present as part of an extract of Nerium species, the extract comprising: a) a supercritical fluid extract; b) hot water extract; c) organic solvent extract; d) an aqueous organic solvent extract; e) an extract using a supercritical fluid, optionally with one or more organic solvents (extraction modifiers); f) optionally an extract using a subcritical liquid with at least one organic solvent (extraction modifier) or g) any combination of any two or more of said extracts.

PBI-05204 (this specification and in US 8187644 B2 to Addington published on May 29, 2012; in US 7402325 B2 to Addington, published July 22, 2008; 8394434 B2, the entire disclosure of which is incorporated herein by reference) as the major pharmacologically active ingredients cardiac glycosides (oleandrine, OL) and triterpenes (oleanolic acid (OA), ursolic acid (UA) and betulinic acid) (BA)). The molar ratio of OL to total triterpenes 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 oleandrine when compared on an OL equimolar basis. PBI-04711 is part of PBI-05204, but does not contain cardiac glycosides (OL). The molar ratio of OA:UA:BA in PBI-04711 is about 3:2.2:1. PBI-04711 also has antiviral activity. Thus, an antiviral composition comprising OL, OA, UA and BA is more effective than a composition comprising OL as the sole active ingredient based on the equimolar content of the OL. In some embodiments, the molar ratio of individual triterpenes to oleandrine ranges 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).

Antiviral compositions comprising oleandrine as the only antiviral agent are within the scope of the present invention. Antiviral compositions comprising digoxin as the only antiviral agent are within the scope of the present invention.

Antiviral compositions comprising oleandrine and a plurality of triterpenes as antiviral agents are within the scope of the present invention. In some embodiments, the antiviral composition comprises oleandrine, oleanolic acid (free acid, salt, derivative or prodrug thereof), ursolic acid (free acid, salt, derivative or prodrug thereof) and betulinic acid (free acid, salt, derivatives or prodrugs). The molar ratios of the compounds are as described herein.

Antiviral compositions comprising a plurality of triterpenes as main active ingredients (which means excluding steroids, cardiac glycosides and pharmacologically active ingredients) are also within the scope of the present invention. As described above, PBI 04711 contains OA, UA and BA as main active ingredients, and exhibits antiviral activity. In some embodiments, the triterpene-based antiviral composition comprises OA, UA and BA, each independently selected at each occurrence from the free acid form, salt form, deuterated form and derivative form.

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.

In some embodiments, the antiviral composition comprises oleanolic acid (free acid, salt, derivative or prodrug thereof) and ursolic acid (free acid, salt, derivative or prodrug thereof) present in a molar ratio of OA to UA as described herein. drug) at least. OA is present in a higher molar amount than UA.

In some embodiments, the antiviral composition comprises oleanolic acid (free acid, salt, derivative or prodrug thereof) and betulinic acid (free acid, salt, derivative or prodrug thereof) present in a molar ratio of OA to BA as described herein. drug) at least. OA is present in a higher molar amount than BA.

In some embodiments, the antiviral composition comprises oleanolic acid (free acid, salt, derivative or prodrug thereof), ursolic acid (free acid, salt, derivative thereof) present in a molar ratio of OA to UA to BA as described herein. or a prodrug) and betulinic acid (a free acid, salt, derivative or prodrug thereof). OA is present in a higher molar amount than both UA and BA.

In some embodiments, the triterpene-based antiviral composition excludes cardiac glycosides.

Generally, arenaviridae infection, arterviridae infection, filobiridae infection, flaviviridae infection (flavivirus genus), deltaretrovirus genus, coronaviridae, paramyxoviridae, orthomyxoviridae, or a subject with a Togaviridae infection is treated as follows. A subject is evaluated to determine if the subject is infected with the virus. Administration of antiviral compositions is indicated. An initial dose of the antiviral composition is administered to a subject according to a prescribed dosing regimen for a period of time (treatment period). The subject's clinical response and therapeutic response level are determined periodically. If the level of therapeutic response at one dose is too low, the dose is increased according to a predetermined dose escalation schedule until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition continues as needed. The dosage or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint(s), such as cessation of infection itself, reduction of infection-related symptoms, and/or reduction of progression of infection.

When a clinician intends to treat a virally infected subject with a combination of an antiviral composition and one or more other therapeutic agents, it is known that the viral infection the subject has is at least partially therapeutically responsive to said one or more other therapeutic agents. The methods of the present invention then comprise administering to a subject in need thereof a therapeutically relevant dosage of an antiviral composition and a therapeutically relevant dosage of said one or more other therapeutic agents, wherein the antiviral composition is administered in a first administration. and the one or more other therapeutic agents are administered according to the 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.

The antiviral composition(s) of the present invention may be administered as primary antiviral therapy, adjuvant antiviral therapy or co-antiviral therapy. The methods of the present invention comprise individually or concurrently administering an antiviral composition with one or more other known antiviral compositions, wherein the antiviral composition of the present invention is associated with a known antiviral composition (compound) or viral infection. It means that the composition may be administered before, during or after administration of the composition for treating a condition. For example, a drug used to treat inflammation, vomiting, nausea, headache, fever, diarrhea, nausea, urticaria, conjunctivitis, malaise, myalgia, arthralgia, seizures or paralysis may be administered with or separately from the antiviral composition of the present invention. can .

The one or more other therapeutic agents may be administered at a dosage or dosing regimen recognized by the clinician to be therapeutically effective, or at a dosage recognized by the clinician to be sub-therapeutically effective. The clinical benefit and/or therapeutic effect provided by the administration of the combination of the antiviral composition and one or more other therapeutic agents may be additive or synergistic, and this level of benefit or effect may differ from the administration of the combination and the individual antiviral compositions and the one or more other therapeutic agents. It is determined by comparison of administration of different therapeutic agents. One or more other therapeutic agents are the United States Food and Drug Administration (FDA), World Health Organization, European Medicines Agency (EMEA), Therapeutic Goods Administration (TGA, Australia), Pan American Health Organization (PAHO), Drug and Medical Device Safety Agency (Medsafe, New Zealand). ) or according to dosages and dosing regimens suggested or described by various health departments worldwide.

Exemplary other therapeutic agents that may be included in the antiviral composition of the present invention for the treatment of viral infections include antiretroviral agents, interferon alpha (IFN-a), zidovudine, lamivudine, cyclosporine A, CHOP containing arsenic trioxide, sodium valpro ate, methotrexate, azathioprine, one or more symptom-relieving drugs, steroid sparing drugs, corticosteroids, cyclophosphamide, immunosuppressants, anti-inflammatory drugs, Janus kinase inhibitors, tofacitinib, calcineurin inhibitors, tacrolimus, mTOR inhibitors , sirolimus, everolimus, IMDH inhibitor, azathioprine, leflunomide, mycophenolate, biologics, abatacept, adalimumab, Anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab (rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, monoclonal antibody, basiliximab, daclizumab, Polyclonal antibody, nucleoside analogue, reverse transcriptase inhibitor, emtricitabine, telbivudine, abacavir, adefovir, didanosine, emtricitabine, entecavir, stavudine, tenofovir, azithromycin, macrolide -type antibiotics, protease inhibitors, interferons, immune response modulators, mRNA synthesis inhibitors, protein synthesis inhibitors, thiazolides, CYP3A4 inhibitors, heterocyclic biguanidines, CCR5 receptor inhibitors and combinations thereof. Plasma separation and/or radiation are also included.Antibodies to certain viruses may also be administered to a subject treated with the antiviral composition of the present invention. can Plasma obtained from the blood of a survivor of a first viral infection may be administered to another subject having the same type of viral infection, who may also be administered an antiviral composition of the present invention. For example, plasma from a COVID-19 infection survivor may be administered to another subject suffering from a COVID-19 infection, who may also be administered an antiviral composition of the present invention.

Example 5 provides an exemplary procedure for the treatment of a Zika virus infection in a mammal. Example 12 provides exemplary procedures for the treatment of filovirus infections (Ebolavirus, Marburgvirus) in a mammal. Example 13 is a flavivirus infection (yellow fever, dengue fever, Japanese encephalitis, West Nile virus, Zika virus, tick encephalitis, Kasanur forest disease, alkhurma disease, Omsk hemorrhagic fever, Powasan virus infection) in mammals. Exemplary procedures for treatment are provided. Example 25 provides an exemplary procedure for the treatment of a deltaretroviral genus (HTLV-1) infection.

The antiviral compound (triterpene(s), cardiac glycoside(s), etc.) present in the pharmaceutical composition may be present in unmodified form, salt form, derivative form, or a combination thereof. As used herein, the term “derivative” refers to: a) a chemical substance structurally related to and theoretically derived from a first chemical substance; b) a compound formed from a compound that is imaginable to arise from a similar first compound or another first compound when one atom of the first compound is replaced by another atom or group of atoms; c) compounds derived from the parent compound and containing the essential elements of the parent compound; or d) can be prepared from a first compound of similar structure in one or more steps. For example, derivatives include deuterated forms, oxidized forms, dehydrated, unsaturated, polymer conjugated or glycosylated forms, or include esters, amides, lactones, homologues, ethers, thioethers, cyano, amino, alkylamino, sulfonyl phydryl, heterocyclic, heterocyclic ring-fused, polymerized, pegylated, benzylidenyl, triazolyl, piperazinyl or deuterated forms thereof.

As used herein, the term “oleandrine” is intended to mean all known forms of oleandrine, unless otherwise specified. Oleandrin may exist in racemic, optically pure or optically enriched form. Nerium oleander plant material can be obtained from commercial plant suppliers such as, for example, Aldridge Nursery of Atasco, Texas.

Supercritical fluid (SCF) extracts may be prepared as detailed in US 7,402,325, US 8394434, US 8187644 or PCT International Publication No. WP 2007/016176 A2, the entire disclosures of which are incorporated herein by reference. Extraction can be performed with supercritical carbon dioxide in the presence or absence of a modifier (organic solvent) such as ethanol.

Other extracts containing cardiac glycosides, particularly oleandrine, can be prepared by a variety of different processes. The extract can be prepared according to a process developed by Dr. Huseyin Ziya Ozel (US Pat. No. 5,135,745), which describes a procedure for preparing a boiling water extract. It is reported that the aqueous extract contains several polysaccharides with molecular weights of 2KD to 30KD, oleandrine and oleandrigenin, orodoside and neritaloside. Polysaccharides are known to include acidic homopolygalacturonan or arabinogalacturonan. U.S. Patent No. 5,869,060 to Selvaraj et al discloses a boiling water extract of Nerium species and a method for preparing the same, for example Example 2. Then, the resulting extract may be freeze-dried to prepare a powder. U.S. Patent No. 6,565,897 (U.S. Patent Publication No. 20020114852 to Selvaraj et al. and PCT International Publication No. WO 2000/016793) discloses a hot water extraction process for the preparation of substantially sterile extracts. Erdemoglu et al. (J. Ethnopharmacol. (2003) Nov. 89(1), 123-129) compared aqueous and ethanolic extracts of plants including Nerium oleander based on anti-nociceptive and anti-inflammatory activity. is starting. The organic solvent extract of Nerium oleander was prepared by Adome et al. (Afr. Health Sci. (2003) Aug. 3(2), 77-86; ethanol extract), el-Shazly et al. (J. Egypt Soc. Parasitol). (1996), Aug. 26(2), 461-473; ethanol extract), Begum et al. (Phytochemistry (1999) Feb. 50(3), 435-438; methanol extract), Zia et al. (J) Ethnolpharmacol. (1995) Nov. 49(1), 33-39; methanol extract) and Vlasenko et al. (Farmatsiia. (1972) Sept.-Oct. 21(5), 46-47; alcoholic extract). is initiated by US Patent Application Publication No. 20040247660 to Singh et al. discloses the preparation of protein stabilized liposomal formulations of oleandrine for use in the treatment of cancer. US Patent Application Publication No. 20050026849 to Singh et al. discloses water-soluble formulations of oleandrin containing cyclodextrins. US Patent Application Publication No. 20040082521 to Singh et al. discloses the preparation of protein-stabilized nanoparticle formulations of oleandrin from boiling water extracts.

Oleandrin is also found in Agrobacterium tumefaciens-transformed calli (Ibrahim et al., "Stimulation of oleandrin production by combination Agrobacterium tumefaciens mediated transformation and fungal elicitation in Nerium oleander cell cultures" in Enz. Microbial Techno (2007), 41(3), 331-336, the entire disclosure of which is incorporated herein by reference). Hot water, organic solvents, aqueous organic solvents, or supercritical fluid extracts of Agrobacterium may be used in accordance with the present invention.

Oleandrin can also be obtained from extracts of Nerium olinger microcultures in vitro, whereby the shoot cultures are grown on seedlings and/or Nerium olingander varieties Splendens Giganteum, Revanche or Alsace. (Alsace), or other cultivars (Vila et al., "Micropropagation of Oleander (Nerium oleander L.)" in HortScience (2010), 45(1), 98-102, the entire contents of which are incorporated herein by reference). Hot water, organic solvents, aqueous organic solvents, or supercritical fluid extracts of microcultured Nerium oleander can be used according to the present invention.

The extracts also differ in their polysaccharide and carbohydrate content. The boiling water extract contained 407.3 glucose equivalents of carbohydrate compared to the standard curve prepared with glucose, while _{analysis of the SCF CO 2} extract found carbohydrate levels found at very low levels below the limit of quantitation. However, the amount of carbohydrates in the boiling water extract of Nerium oleander was at least 100 times greater than that in the _{SCF CO 2 extract.} The polysaccharide content of the SCF extract may be 0%, <0.5%, <0.1%, <0.05% or <0.01% by weight. In some embodiments, the SCF extract excludes polysaccharides obtained during extraction of the plant mass.

The partial composition of the SCF CO ₂ extract and the boiling water extract was determined by DART TOF-MS (time-of-flight mass spectrometer real-time direct analysis) on a JEOL AccuTOF-DART mass spectrometer (JEOL USA, Peabody, MA, USA).

SCF extracts of Nerium species or Thevetia species are mixtures of pharmacologically active compounds such as oleandrin and triterpenes. The extract obtained by the SCF process is a viscous semi-solid (after solvent removal) that is substantially water-insoluble at ambient temperature. The SCF extract contains many different components with various different ranges of water solubility. Extracts from the supercritical fluid process theoretically contain 0.9 to 2.5 wt % oleandrine or 1.7 to 2.1 wt % oleandrine or 1.7 to 2.0 wt % oleandrine. SCF extracts containing varying amounts of oleandrine were obtained. In one embodiment, the SCF extract comprises about 2% oleandrine by weight. SCF extract contains 3-10 times higher concentration of oleandrin than boiling water extract. This was confirmed by HPLC and LC/MS/MS (tandem mass spectrometry) analysis.

The SCF extract contains oleandrine and triterpene oleanolic acid, betulinic acid and ursolic acid and other ingredients described herein. The content of oleandrine and triterpene may vary from batch to batch; 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.

The content of the individual components may vary by ± 25 %, ± 20 %, ± 15 %, ± 10 % or ± 5 % relative to the indicated values. Thus, the content of oleandrine in the SCF extract will be in the range of 20 mg ± 5 mg per mg of SCF extract (which is ± 25% of 20 mg).

Oleandrine, oleanolic acid, ursolic acid, betulinic acid and their derivatives can also be purchased from Sigma-Aldrich (www.sigmaaldrich.com; St. Louis, MO, USA). Digoxin is available from HIKMA Pharmaceuticals International LTD (NDA N012648, elixir, 0.05 mg/mL; tablets, 0.125 mg, 0.25 mg), VistaPharm Inc. (NDA A213000, elixir, 0.05 mg/mL), Sandoz Inc. (NDA A040481), injection Yes, 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).

As used herein, the individually named triterpenes may in each case be independently selected from their native (unmodified, free acid) form, salt form, derivative form, prodrug form, or combinations thereof. . Compositions and methods containing triterpenes in deuterated form are also within the scope of the present invention.

Oleanolic acid derivatives, prodrugs and salts are disclosed in US 20150011627 A1 by Gribble et al., published Jan. 8, 2015, US 20140343108 A1 by Rong et al., published Nov. 20, 2014, in US 20140343108 A1 in Nov. 20, 2014. US 20140343064 A1 by Xu et al., published on Jun. 26, 2014, US 20140179928 A1 by Anderson et al., published on Jun. 26, 2014, US20140100227 A1 by Bender et al., published Apr. 10, 2014, 3, 2014 US 20140088188 A1 by Jiang et al. published March 27, 2014 US 20140088163 A1 by Jiang et al. published March 27, 2014, US 20140066408 A1 by Jiang et al. published March 6, 2014 , US 20130317007 A1 by Anderson et al., published Nov. 28, 2013, US201303303607 A1 by Gribble et al., published Nov. 14, 2013, and US201303303607 A1 by Anderson et al., published Sep. 27, 2012 US 20120245374, US 20120238767 A1 by Jiang et al. published September 20, 2012, US 20120237629 A1 by Shode et al. published September 20, 2012, Anderson et al. US 20120214814 A1 to al., US 20120165279 A1 to Lee et al. published Jun. 28, 2012, US 20110294752 A1 to Arntzen et al. published Dec. 1, 2011, US 20110294752 A1 to Apr. 21, 2011 by Lee et al. US 20110091398 A1 by Majeed et al., published 29 July 2010, US 201000189824 A1, 201 by Arntzen et al. published on July 29, 2010 US 20100048911 A1 to Jiang et al., published Feb. 25, 0, and US 20060073222 A1 to Arntzen et al., published Apr. 6, 2006, the entire contents of which are incorporated herein by reference. .

Ursolic acid derivatives, prodrugs and salts are disclosed in US 20150011627 A by Gribble et al., published Jan. 8, 2015, US 20130303607 A1 by Gribble et al., published Nov. 14, 2013, Aug. 6, 2015 US 20150218206 A1 by Yoon et al. published on Nov. 30, 2004, US 6824811 by Fritsche et al., published on 30 November 2004, US 7718635 by Ochiai et al. published on 8 May 2010, 5, 2014 US 8729055 to Lin et al., issued Sept. 20, and US 9120839, Yoon et al., Sep. 1, 2015, issued Sept. 1, 2015, the entire contents of which are incorporated herein by reference.

Betulinic acid derivatives, prodrugs and salts are disclosed in US 20150011627 A by Gribble et al., published Jan. 8, 2015, US 20130303607 by Gribble et al., published Nov. 14, 2013, in US 20130303607, Sep. 20, 2012 Shode et al. published US 20120237629 A1, Regueiro-Ren et al. US 20170204133 A1 published July 20, 2017, US 20170096446 A1 published April 6, 2017 by Nitz et al. US 20150337004 A1 by Parthasaradhi Reddy et al, published on Nov. 26, 2015, US 20150119373 A1 by Parthasaradhi Reddy et al., published on Apr. 30, 2015, Yan et al., published on Oct. 2, 2014. US 20140296546 A1 by Swidorski et al. published Aug. 28, 2014, US 20140243298 A1 by Swidorski et al. published Aug. 7, 2014, US 20140221328 A1 by Parthasaradhi Reddy et al., published Aug. 7, 2014. US 20140066416 A1 by Leunis et al., published Mar. 6, 2014, US 20130065868 A1, by Durst et al., published Mar. 14, 2013, Regueiro-Ren et al, Jan. 31, 2013 US 20130029954 A1 by ., US 20120302530 A1 by Zhang et al. published on Nov. 29, 2012, US 20120214775 A1 by Power et al. published on Aug. 23, 2012, published on Apr. 26, 2012 US 20120101149 A1 by Honda et al. published September 15, 2011, US 20110224182 A1 by Bullock et al. published September 15, 2011, US 20110313191 A1 by Hemp et al. published December 22, 2011, September 2011 US 20110224159 A1 by Pichette et al. published on 15th September, US 20110218204 by Parthasaradhi Reddy et al. published on 8 September 2011, US 20090203661 A1 by Safe et al. published on 13 August 2009, US 20090131714 A1 by Krasutsky et al. published May 21, 2009, US 20090076290 by Krasutsky et al. published March 19, 2009, US 20090076290 by Leunis et al., published March 12, 2009 20090068257 A1, US 20080293682 by Mukherjee et al. published on November 27, 2008, US 20070072835 A1 by Pezzuto et al. published on March 29, 2007, Jansen et al published on November 9, 2006 US 20060252733 to ., US 2006025274 A1 to O'Neill et al. published on Nov. 9, 2006 , the entire contents of which are incorporated herein by reference.

The antiviral composition may be formulated in any suitable pharmaceutically acceptable formulation. Parenteral, otic, ocular, nasal, inhaled, buccal, sublingual, enteral, topical, buccal, oral and injectable formulations are particularly useful. Certain formulations include solid or liquid formulations. Exemplary suitable dosage forms are tablets, capsules, pills, caplets, troches, sachets, solutions, suspensions, dispersions, vials, bags, bottles, injections, iv (intravenous), im (intramuscular). or ip (intraperitoneal) administrable liquids and other formulations known to those skilled in the art of pharmaceutical science.

Because viral infections can 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, cardiac glycoside-containing compositions include inhalable compositions and oral compositions, sublingual compositions and oral compositions, inhalable compositions and sublingual compositions, inhalable compositions and parenteral compositions, sublingual compositions and parenteral compositions, oral compositions and parenteral compositions, or Other such combinations may advantageously be administered

Suitable formulations containing an antiviral composition can be prepared by mixing the antiviral composition with a pharmaceutically acceptable excipient described herein, which is also described in Pi et al. (Cur. Drug Deliv. (Mar. 2016), 13(8), "Ursolic acid nanocrystals for improving dissolution and bioavailability: the effect of different particle sizes" in 13(8), 1358-1366), Yang et al. (Int. J. Nanomed. (2013), 8) (1), "Self-microemulsifying drug delivery system for improving oral bioavailability of oleanolic acid: design and evaluation" in 2917-2926), Li et al. (Biol. Pharm. Bull. (2014), 37(6)) , "Development and evaluation of optimized sucrose ester stabilized oleanolic acid nanosuspensions prepared by wet ball milling with experimental design" in 926-937), Zhang et al. (J. Pharm. Sci. (June 2014), 103) (6), 1711-1719), “Enhancing the oral bioavailability of triterpenes via lipid nanospheres: preparation, characterization and absorption evaluation”), Godugu et al. (PLoS One (Mar 2014), 9(3):e89919). “Method for improving oral bioavailability and effectiveness of novel anticancer drugs berberine and betulinic acid” in “Oral bioavailability and effectiveness” in Zhao et al. Preparation and Characterization of Betulin Nanoparticles for Hypoglycemic Drugs"), Yang et al. (Food Chem. (May 2012), 132(1), 319-325 using a supercritical anti-solvent (SAS) process) Physicochemical properties and oral bioavailability of ursolic acid nanoparticles), Cao et al. (Mol. Pharm. (Aug. 2012), 9(8), 2127-2135), “Ethylene of oleanolic acid for PEPT1-mediated transport. Glycol-Linked Amino Acid Diester Prodrugs: Synthesis, Intestinal Permeability and Pharmacokinetics"), Li et al .(Parm. Res. (Aug 2011), 28(8), 2020-2033), "Preparation, biological and pharmacokinetic studies of sucrose ester-stabilized nanosuspensions of oleanolic acid"), Tong et al. (Int. J. Pharm. (Feb 2011), 404(1-2), "Spray Freeze Drying with Polyvinylpyrrolidone and Sodium Caprate to Improve Dissolution and Oral Bioavailability of Oleanolic Acid, BCS Class IV Compounds" in 404(1-2), 148-158); Xi et al. (“Formulation Development and Bioavailability Evaluation of Self-Nanoemulsifying Drug Delivery Systems of Oleanolic Acid” in AAPS PharmSciTech (2009), 10(1), 172-182), Chen et al. (J. Pharm. Pharmacol. (Feb 2005), 57(2), 259-264, "Oleanolic Acid Nano Suspension: Preparation, In Vitro Characterization and Improved Hepatoprotective Effect"), the entire disclosure of which is incorporated herein by reference.

Suitable formulations are also disclosed in US 8187644 B2 to Addingington, issued 29 May 2012, US 7402325 B2 to Addington, 22 July 2008, and US 8394434 B2 to Addington et al. may be prepared according to the present invention, the entire disclosure of which is incorporated herein by reference. Suitable formulations can also be prepared as described in Examples 13-15.

An effective or therapeutically relevant amount of an antiviral compound (cardiac glycoside, triterpene, or a combination thereof) is specifically contemplated. It is understood that the term “effective amount” contemplates a pharmaceutically effective amount. A pharmaceutically effective amount is an amount of active ingredient sufficient for the desired or desired therapeutic response, ie, an amount sufficient to elicit a significant biological response when administered to a patient. A significant biological response may occur as a result of administration of single or multiple doses of the active agent. A dosage may include one or more formulations. The specific dosage level for any patient will depend on a variety of factors including the indication being treated, the severity of the indication, the patient's health, age, sex, weight, diet, pharmacological response, the particular formulation used, and other such factors. will understand

A preferred dosage for oral administration is a maximum of 5 formulations, although 1 to 10 formulations may be administered in a single dose. Exemplary formulations may contain 0.01 to 100 mg or 0.01 to 100 micrograms of antiviral composition per formulation, for a total of 0.1 to 500 mg per dose (1 to 10 dose levels). Dosages will be administered according to a dosing regimen that may be predetermined and/or adjusted to achieve a particular therapeutic response or clinical benefit in the subject.

The cardiac glycoside may be present in the formulation in an amount sufficient to provide the subject with an initial dose of oleandrine from about 20 to about 100 micrograms, from about 12 micrograms to about 300 micrograms, or from about 12 micrograms to about 120 micrograms. may exist. The dosage form is a nerium containing about 20 to about 100 micrograms, about 0.01 micrograms to about 100 mg or about 0.01 micrograms to about 100 micrograms of oleandrine, oleandrine extract or oleandrine of oleandrine. Oleander extract may be included.

The antiviral agent may be included in an oral formulation. Some embodiments of the formulation are not enteric coated and release a charge of the antiviral composition within a period of 0.5 to 1 hour or less. Some embodiments of the dosage form are enteric coated and release a dose of the antiviral composition downstream of the stomach, eg, from the jejunum, ileum, small intestine and/or large intestine (colon). The enteric coated formulation will release the antiviral composition into the systemic circulation within 1-10 hours after oral administration.

The antiviral composition may 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 a dosage form exhibiting two or more of these release types. have. The release profile of the antiviral composition from the formulation may be a zero order, pseudo-0 order, first order, pseudo-first order or sigmoidal release profile. The plasma concentration profile for triterpene in a subject administered the antiviral composition may exhibit one or more maxima.

Based on human clinical data, it is expected that 50% to 75% of the dose of oleandrine will be orally bioavailable, such that about 10 to about 20 micrograms, about 20 to about 40 micrograms of oleandrine per dosage form; from about 30 to about 50 micrograms, from about 40 to about 60 micrograms, from about 50 to about 75 micrograms, from about 75 to about 100 micrograms. Given an average blood volume of 5 liters in an adult human, expected oleandrine plasma concentrations are from about 0.05 to about 2 ng/ml, from about 0.005 to about 10 ng/mL, from about 0.005 to about 8 ng/mL, from about 0.01 to about 0.01 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 dosage of oleandrine present in SCF extracts is generally from about 0.2 to about 4.5 micrograms per kg body weight, twice daily. The dosage of oleandrine is from about 0.2 to about 1 micrograms/kg body weight/day, from about 0.5 to about 1.0 micrograms/kg body weight/day, from about 0.75 to about 1.5 micrograms/kg body weight/day, from about 1.5 to about 2.52 micrograms/kg body weight/day, about 2.5 to about 3.0 micrograms/kg body weight/day, about 3.0 to 4.0 micrograms/kg body weight/day, or about 3.5 to 4.5 micrograms oleandrine/kg body weight/day . The maximum tolerated dose of oleandrine may be from about 3.5 micrograms/kg body weight/day to about 4.0 micrograms/kg body weight/day. The minimum effective dosage may be about 0.5 micrograms/day, about 1 micrograms/day, about 1.5 micrograms/day, about 1.8 micrograms/day, about 2 micrograms/day, or about 5 micrograms/day.

Antiviral compositions can be administered in low to high doses due to the combination of triterpenes present and the molar ratios in which they are present. A therapeutically effective dosage for humans is about 100-1000 mg or 100-1000 micrograms of antiviral composition per kg body weight. These doses may be administered up to 10 times in 24 hours. Other suitable dosage ranges are specified below.

It should be noted that a compound herein may have more than one function in a composition or formulation of the present invention. For example, the compound may act as both a surfactant and a water miscible solvent, or a surfactant and a water immiscible solvent.

Liquid compositions may include one or more pharmaceutically acceptable liquid carriers. Liquid carriers may be aqueous, non-aqueous, polar, non-polar and/or organic carriers. Liquid carriers include, for example, but are not limited to, water miscible solvents, water immiscible solvents, water, buffers, and mixtures thereof.

As used herein, the terms "aqueous solvent" or "water miscible solvent" are used interchangeably and contain at least 5% solvent that does not form a two-phase mixture with water or is sufficiently soluble in water without separation of the liquid phase. refers to an organic liquid that provides an aqueous solvent mixture that The solvent is suitable for administration to humans or animals. Exemplary water-soluble solvents include, but are not limited to, PEG (poly(ethylene glycol)), PEG 400 (poly(ethylene glycol, which has an approximate molecular weight of about 400), ethanol, acetone, alkanols, alcohols, ethers , propylene glycol, glycerin, triacetin, poly(propylene glycol), PVP (poly(vinyl pyrrolidone)), dimethylsulfoxide, N,N-dimethylformamide, formamide, N,N-dimethylacetamide, pyridine , propanol, N-methylacetamide, butanol, solution (2-pyrrolidone), and Pharmasolve (N-methyl-2-pyrrolidone).

As used herein, the terms "water insoluble solvent" or "water immiscible solvent" are used interchangeably and form a two-phase mixture with water, or cause phase separation when the concentration of solvent in water exceeds 5%. It refers to an organic liquid that provides. The solvent is suitable for administration to humans or animals. Exemplary water insoluble solvents include, but are not limited to, medium chain/long chain triglycerides, oil, castor oil, corn oil, vitamin E, vitamin E derivatives, oleic acid, fatty acids, olive oil, softisan 645 (diglycerides) Lyl Caprylate/Caprate/Stearate/Hydroxy Stearate Adipate), Miglyol, Captex (Captex 350: Glyceryl Tricaprylate/Caprate/Lorate Triglyceride; Captex 355: Glyceryl Trika) Prylate/Caprate Triglycerides; Captex 355 EP/NF: Glyceryl Tricaprylate/Caprate Medium Chain Triglycerides).

Suitable solvents are listed in "Guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Drugs for Human Use (ICH) for Industrial Q3C Impurities: Residual Solvents" (1997), which states that what amounts of residual solvents is considered safe in Exemplary solvents are listed as class 2 or class 3 solvents. Class 3 solvents include, for example, acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butylmethyl ether, cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid, heptane, iso Butyl 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 includes

Other materials that can be used as the water-immiscible solvent in the present invention include the following materials: 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-ethitexanoate); 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- and diglycerides; Capmul MCM C8: glyceryl monocaprylate; Capmul MCM C10: glyceryl monocaprate; Capmul PG-8: propylene glycol monocaprylate; Capmul PG-12: propylene glycol monolaurate; Caprol 10G10O: decaglycerol decaoleate; Caprol 3GO: triglycerol mono oleate; Caprol ET: polyglycerol esters of mixed fatty acids; Caprol MPGO: hexaglycerol dioleate; Caprol PGE 860: contains decaglycerol mono-, dioleate.

As used herein, "surfactant" refers to a compound comprising a polar or charged hydrophilic moiety as well as a non-polar hydrophobic (lipophilic) moiety; That is, surfactants are amphiphilic. The term surfactant may refer to a mixture of one or more compounds. Surfactants may be solubilizers, emulsifiers or dispersants. Surfactants may be hydrophilic or hydrophobic.

The hydrophilic surfactant may be any hydrophilic surfactant suitable for use in pharmaceutical compositions. Such surfactants may 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 present invention.

Similarly, the hydrophobic surfactant may be any hydrophobic surfactant suitable for use in pharmaceutical compositions. Generally, suitable hydrophobic surfactants will have an HLB value of less than about 10. Mixtures of hydrophobic surfactants are also within the scope of the present invention.

Examples of further suitable solubilizers include alcohols and polyols such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediol and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide , polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrin and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of from about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, sold under the trade name Tetraglycol from BASF) or methoxy PEG (Union Carbide); Amides such as 2-pyrrolidone, 2-piperidone, caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide and poly vinylpyrrolidone; 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 solubilizing agents known in the art, such as dimethyl acetamide, dimethyl isosorbide (Arlasolve DMI (ICI)), N-methyl pyrrolidone (Pharmasolve (ISP)), monooctanoin, diethylene glycol nonoethyl ether (which is available from Gattefosse under the trade name Transcutol), and water. Mixtures of solubilizers are also within the scope of the present invention.

Except where indicated, the compounds referred to herein are readily available from standard commercial sources.

Although not required, the composition or formulation may 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 dyeing agents, one or more It may further comprise one or more electrolytes, one or more salts, one or more stabilizers, one or more tonicity modifiers, one or more diluents, or combinations thereof.

The composition of the present invention may also contain oils such as fixed oil, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil; fatty acids such as oleic acid, stearic acid and isostearic acid; fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. The composition may also contain alcohols such as ethanol, isopropanol, hexadecyl alcohol, glycerol and propylene glycol; glycerol ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol; ethers such as poly(ethylene glycol) 450; petroleum hydrocarbons such as mineral oil and petrolatum; water; pharmaceutically suitable surfactants, suspending or emulsifying agents; or mixtures thereof.

It should be understood that compounds used in the field of pharmaceutical formulation generally serve a variety of functions or purposes. Thus, when a named compound is mentioned only once or used to define one or more terms herein, that purpose or function should not be construed as limited to the only named purpose or function(s).

One or more components of the formulation may be present in the form of their free base, free acid, or pharmaceutically or analytically acceptable salt thereof. "Pharmaceutically or analytically acceptable salt," as used herein, 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 inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, sulfamic acid, phosphoric acid, nitric acid and others known to those skilled in the art. Salts include organic acids such as amino acids, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, prepared from sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethane disulfonic acid, oxalic acid, isethionic acid and others known to those skilled in the art. On the other hand, when the pharmacologically active ingredient has an acid functional group, a pharmaceutically acceptable base is added to form a pharmaceutically acceptable salt. A list of other suitable salts can be found in Remington's Pharmaceutical Sciences, 17th (ed., Mack Publishing Company, PA, Easton, 1985, p. 1418), the disclosure of which is incorporated herein by reference.

The phrase "pharmaceutically acceptable" is used for contact with tissues of humans and animals, within the scope of sound medical judgment, and proportionate to a reasonable benefit/risk rate without undue toxicity, irritation, allergic reaction, or other problems or complications. Used to refer to compounds, materials, compositions and/or formulations suitable for the following.

The formulations may be prepared by any conventional means known in the pharmaceutical industry. Liquid formulations can be prepared by providing in a container one or more liquid carriers and an antiviral composition. One or more other excipients may be included in the liquid formulation. Solid dosage forms can be prepared by providing one or more solid carriers and an antiviral composition. One or more other excipients may be included in the solid dosage form.

The formulation may be packaged using conventional packaging equipment and materials. Formulations may be contained in packs, bottles, vias, bags, syringes, envelopes, packets, blister packs, boxes, ampoules or other containers.

The composition of the present invention may be included in any formulation. Certain formulations include solid or liquid formulations. Exemplary suitable dosage forms include tablets, capsules, pills, caplets, troches, chassis and other dosage forms known to those skilled in the art of pharmaceutical science.

In light of the above description and the following examples, those skilled 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 which describe in detail specific procedures for practicing the embodiments of the present invention. All references to these examples are for the purpose of illustration. The following examples are not to be considered exhaustive, but merely exemplify some of the many examples contemplated by the present invention.

Vero CCL81 cells were used for prophylactic and therapeutic assays (ATCC, Manassas, VA). Plaque assays were performed on Vero E6 cells kindly provided by Vineet Menachery (UTMB, Galveston, TX). Cells were maintained in a 37° C. incubator with 5% CO _{2 .} Delbecco's Modified Eagle medium (Gibco) supplemented with 5% Fetal Bovine Serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) and 1% Penicillium/Streptomycin (Gibco, Grand Island, NY) , Grand Island, NY) were used to propagate cells. Maintenance medium reduced FBS to 2%, but was otherwise the same. SARS-CoV-2, strain USA_WA1/2020 (Genbank accession MT020880) was provided by the World Reference Center for Emerging Viruses and Arboviruses. All studies used the NextGen sequence Vero passage 4 stock of SARS-CoV-2.

Example 1

Supercritical fluid extraction of powdered oleander leaves

Method A. With carbon dioxide

Powdered oleander leaves are prepared by harvesting, washing and drying the oleander leaf material, and then milling and dewatering the oliander leaf material in a grinding and dewatering apparatus such as those described in US Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. It was prepared by passing it through. The weight of the starting material used was 3.94 kg.

The starting material was mixed with _{pure CO 2} in an extractor apparatus at a pressure of 300 bar (30 MPa, 4351 psi) and a temperature of 50 °C (122ºF). A total of 197 kg of CO ₂ was used so that the ratio of solvent to raw material was 50:1. Then, _{the mixture of CO 2} and the raw material was passed through a separator device to change the pressure and temperature of the mixture, and the extract was separated from carbon dioxide.

The extract (65 g) was obtained as a fragrant brown sticky viscous substance. Chlorophyll and other residual chromogenic compounds may have caused the color. For accurate yield determination, the tube and separator were rinsed with acetone and the acetone evaporated to give an additional 9 g of extract. The total extraction 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 yield 560.1 mg, or 0.76%.

Method B. With a mixture of carbon dioxide and ethanol

Powdered oleander leaves are prepared by harvesting, washing and drying the oleander leaf material, and then milling and dewatering the oliander leaf material in a grinding and dewatering apparatus such as those described in US Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. It was prepared by passing it through. The weight of the starting material used was 3.85 kg.

_{The starting material was mixed with pure CO 2} and 5% ethanol as modifier at a pressure of 280 bar (28 MPa, 4061 psi) and a temperature of 50° C. (122° F) in an extractor apparatus. A total of 160 kg of CO ₂ and 8 kg of ethanol were used so that the ratio of solvent to raw material was 43.6 to 1. Then, _{a mixture of CO 2} , ethanol and raw material was passed through a separator device to change the pressure and temperature of the mixture, and the extract was separated from carbon dioxide.

The extract (207 g) was obtained as a dark green sticky viscous mass, apparently containing some chlorophyll, after removal of the ethanol. Based on the weight of the starting material, the yield of the extract was 5.38%. The content of oleandrine in the extract was calculated using high pressure liquid chromatography and mass spectrometry in a yield of 1.89 g, or 0.91 %.

Example 2

Hot-water extraction of powdered oleander leaves.

(comparative example)

Hot water extraction is typically used to extract oleandrin and other active ingredients from oleander leaves. Examples of hot water extraction processes can be found in US Pat. Nos. 5,135,745 and 5,869,060.

Hot water extraction was performed using 5 g of powdered oleander leaves. To the powdered oliander leaves (based on the weight of the oliander starting material) 10 volumes of boiling water were added and the mixture was stirred constantly for 6 hours. The mixture was then filtered, 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 weight of the dried extract material was about 1.44 g. 34.21 mg of the extract was dissolved in water, and analysis of the oleandrine content was performed using high pressure liquid chromatography and mass spectrometry. The amount of oleandrine was measured to be 3.68 mg. The yield of oleandrine based on the amount of extract was calculated to be 0.26%.

Example 3

Preparation of a pharmaceutical composition.

Method A. Cremophor-Based Drug Delivery System

The following ingredients were provided in the amounts indicated.

The excipients were dispensed into bottles and shaken in a New Brunswick Scientific C24KC refrigerated incubator shaker at 60° C. for 24 hours to ensure homogeneity. The samples were then followed and visually inspected for dissolution. Both excipients and antiviral composition were completely dissolved for all formulations after 24 hours.

Method B. GMO/Cremophor-Based Drug Delivery System

The following ingredients were provided in the amounts indicated.

The procedure of Method A was followed.

Method C. Labrasol-Based Drug Delivery System

The following ingredients were provided in the amounts indicated.

The procedure of Method A was followed.

Method D. Vitamin E-TPGS-based micellar formation system

The following ingredients were provided in the amounts indicated.

The procedure of Method A was followed.

Method E. Multi-Component Drug Delivery System

The following ingredients were provided in the amounts indicated.

The procedure of Method A was followed.

Method F. Multi-Component Drug Delivery System

The following ingredients were provided in the amounts indicated as included in the capsules.

The procedure of Method A was followed.

Example 4

Preparation of enteric coated capsules

Step I: Preparation of Liquid-Filled Capsules

Hard gelatin capsules (50 counts, size 00) were filled with the liquid composition of Example 3. These capsules were manually filled with 800 mg of formulation and then sealed by hand with 50% ethanol/50% aqueous solution. The capsules were then hand banded with a 22% gelatin solution containing the following ingredients in the amounts indicated below.

The gelatin solution was thoroughly mixed and allowed to swell for 1-2 hours. After the expansion period, the solution was tightly covered and placed in a 55 °C oven to liquefy. Banding was performed when the entire gelatin solution became liquid.

Using a pointed, round 3/0 artist brush, the gelatin solution was painted onto the capsules. A banding kit provided by Shionogi was used. After banding, the capsules were held at ambient conditions for 12 hours to cure the bands.

Step II: Coating of Liquid-Filled Capsules

Coating dispersions were prepared from the ingredients listed in the table below.

When using banding capsules according to step I, the dispersion was applied to the capsules at a coating level of 20.0 mg/cm 2 . The following conditions were used to coat the capsules.

* The spray nozzle is set so that the nozzle and spray path are under the inlet air flow path.

Example 5

Treatment of a Zika Virus Infection in a Subject

Method A. Antiviral Composition Therapy

A subject exhibiting a Zika virus infection is prescribed an antiviral composition, and a therapeutically relevant dose is administered to the subject according to a predetermined dosing regimen over a period of time. The subject's level of response to treatment is determined periodically. A therapeutic response level can be determined by determining a subject's Zikavirus titre in blood or plasma. If the level of therapeutic response in one dose is too low, the dose is increased according to a predetermined schedule of dose escalation until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition may continue as needed, and the dosage or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Other Agents

Method A above is followed except that one or more other therapeutic agents have been prescribed and administered to the subject for treatment of a Zika virus infection or symptoms thereof. One or more other therapeutic agents may then be administered with, or before or after administration of the antiviral composition. Dosage escalation (or reduction) of one or more other therapeutic agents may also be performed.

Example 6

In vitro evaluation of antiviral activity against Zika virus infection

Method A. Pure Compounds

Vero E6 cells (also known as Vero C1008 cells, ATTC No. CRL-1586; https://www.atcc.org/Products/All/CRL-1586.aspx) were subjected to multiple infection with an MOI of 0.2 in the presence of cardiac glycosides. ) was infected with ZIKV (Zika virus strain PRVABC59, ATCC VR-1843; https://www.atcc.org/Products/All/VR-1843.aspx). After cells were incubated with virus and compound for 1 hour, the inoculum and compound were discarded. After providing fresh medium to the cells and incubating for 48 hours, they were fixed with formalin and stained for ZIKV infection. Representative infection rates of oleandrine ( FIG. 1A ) and digoxin ( FIG. 1B ) as determined by scintigraphy are shown. Other compounds were evaluated under the same conditions and exhibited very variable levels of antiviral activity against Zika virus.

Method B. Compound in extract form

Extracts containing the target compound being tested are 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% by weight of oleandrine contains 20 micrograms of oleandrine per mg of extract. Thus, if the intended amount of oleandrine for evaluation is 20 micrograms, then 1 mg of extract will be used in the analysis.

Example 7

Preparation of Tablets Containing Antiviral Compositions

An initial purification mixture of 3% Syloid 244FP and 97% microcrystalline cellulose (MCC) was mixed. Then, the conventional batch composition prepared according to Example 3 was incorporated into the Syloid/MCC mixture through wet granulation. This mixture is labeled "initial tabletting mixture" in the table below. Additional MMC was added as extra-granules to increase the compressibility. This addition to the initial purification mixture is marked as "extragranular addition". The resulting mixture from the add-granule addition was of the same composition as the "final purification mixture".

Syloid 244FP is colloidal silicon dioxide manufactured by Grace Davison. Colloidal silicon dioxide is commonly used to serve several functions such as adsorbent, glidant and tablet disintegrant. Syloid 244FP was chosen because of its ability to adsorb three times its weight in oil and its 5.5 micron particle size.

Example 8

HPLC analysis of solutions containing oleandrine

Using the following conditions (symmetric C18 column (5.0 μm, 150 x 4.6 mm ID; water); mobile phase of MeOH:water = 54:46 (v/v) and flow rate at 1.0 ml/min), the sample (olean Drin standards, SCF extract and hot water extract) were analyzed on HPLC (water).

The detection wavelength was set to 217 nm. Samples were prepared by dissolving the compound or extract in a fixed amount of HPLC solvent to achieve an approximate target concentration of oleandrine. The retention time of oleandrine can be determined using an internal standard. The concentration of oleandrine can be determined/calibrated by developing/developing a signal response curve using an internal standard.

Example 9

Preparation of pharmaceutical compositions

The pharmaceutical composition of the present invention can be prepared by any of the following methods. Mixing can be carried out under wet or dry conditions. The pharmaceutical composition may be compressed, dried, or both during manufacture. The pharmaceutical composition may be divided into dosage forms.

Method A.

At least one pharmaceutical excipient is admixed with at least one antiviral compound disclosed herein.

Method B.

At least one pharmaceutical excipient is admixed with at least two antiviral compounds disclosed herein.

Method C.

At least one pharmaceutical excipient is admixed with at least one cardiac glycoside disclosed herein.

Method D.

At least one pharmaceutical excipient is admixed with at least two triterpenes disclosed herein.

Method E.

At least one pharmaceutical excipient is admixed with at least one cardiac glycoside disclosed herein and at least two triterpenes disclosed herein.

Method D.

At least one pharmaceutical excipient is admixed with at least three triterpenes disclosed herein.

Example 10

Preparation of triterpene mixtures

The following compositions were prepared by mixing the indicated triterpenes in the approximate molar ratios indicated.

For each composition, three different respective solutions were prepared, so the total concentration of triterpenes in each solution was approximately 9 μM, 18 μM or 36 μM.

Example 11

Preparation of antiviral compositions

Antiviral compositions can be prepared by mixing the individual triterpene components to form a mixture. The triterpene mixture prepared above, which provided acceptable antiviral activity, was formulated into an antiviral composition.

Antiviral composition with oleanolic acid and ursolic acid

Known amounts of oleanolic acid and ursolic acid are mixed according to a predetermined molar ratio of the components as defined herein. The ingredients can be mixed in solid form or mixed with a solvent such as methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone ( NMP), water or mixtures thereof. The resulting mixture contained the components in relative molar ratios as described herein.

For pharmaceutically acceptable antiviral compositions, at least one pharmaceutically acceptable excipient is admixed with the pharmacologically active agent. The antiviral composition is formulated for administration to a mammal.

Antiviral composition with oleanolic acid and betulinic acid

A known amount of oleanolic acid and betulinic acid was mixed according to a predetermined molar ratio of the components as defined herein. The ingredients can be mixed in solid form or mixed with a solvent such as methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone ( NMP), water or mixtures thereof. The resulting mixture contained the components in relative molar ratios as described herein.

For pharmaceutically acceptable antiviral compositions, at least one pharmaceutically acceptable excipient is admixed with the pharmacologically active agent. The antiviral composition is formulated for administration to a mammal.

Antiviral composition with oleanolic acid, ursolic acid and betulinic acid

Known amounts of oleanolic acid, ursolic acid and betulinic acid are mixed according to a predetermined molar ratio of the components as defined herein. The components are mixed in solid form or mixed in a solvent, for example a solvent. The ingredients can be mixed in solid form or mixed with a solvent such as methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone ( NMP), water or mixtures thereof. The resulting mixture contained the components in relative molar ratios as described herein.

For pharmaceutically acceptable antiviral compositions, one or more pharmaceutically acceptable excipients are admixed with the pharmacologically active agent. The antiviral composition is formulated for administration to a mammal.

Antiviral composition with oleadrine, oleanolic acid, ursolic acid and betulinic acid

Known amounts of oleadrine, oleanolic acid, ursolic acid and betulinic acid are mixed according to a predetermined molar ratio of the components as defined herein. The ingredients can be mixed in solid form or mixed with a solvent such as methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone ( NMP), water or mixtures thereof. The resulting mixture contained the components in relative molar ratios as described herein.

For pharmaceutically acceptable antiviral compositions, one or more pharmaceutically acceptable excipients are admixed with the pharmacologically active agent. The antiviral composition is formulated for administration to a mammal.

Example 12

treatment of filovirus infection in a subject

Exemplary filovirus infections include Ebolavirus and Marburg virus.

Method A. Antiviral Composition Therapy

A subject exhibiting a filovirus infection is prescribed an antiviral composition, and a therapeutically relevant dose is administered to the subject according to the prescribed dosing regimen for a period of time. The subject's level of response to treatment is determined periodically. A therapeutic response level can be determined by determining a subject's filovirus titer in blood or plasma. If the level of therapeutic response in one dose is too low, the dose is increased according to a pre-determined schedule of dose escalation until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition may continue as needed, and the dosage or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Other Agents

Method A is followed except that the subject has prescribed and administered one or more other therapeutic agents for the treatment of filovirus infection or symptoms thereof. One or more other therapeutic agents may then be administered with, or before or after administration of the antiviral composition. Dosage escalation (or reduction) of one or more other therapeutic agents may also be performed.

Example 13

Treatment of Flavivirus Infection in a Subject

Exemplary flavivirus infections include yellow fever, dengue fever, Japanese encephalitis, West Nile virus, Zika virus, tick encephalitis, kasanur forest disease, Alkhurma disease, chikungunya virus, Omsk hemorrhagic fever, Powassan ), including viral infections.

Method A. Antiviral Composition Therapy

A subject exhibiting a flavivirus infection is prescribed an antiviral composition, and a therapeutically relevant dose is administered to the subject according to the prescribed dosing regimen for a period of time. The subject's level of response to treatment is determined periodically. A therapeutic response level can be determined by determining a subject's flavivirus titer in blood or plasma. If the level of therapeutic response in one dose is too low, the dose is increased according to a pre-determined schedule of dose escalation until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition may continue as needed, and the dosage or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Other Agents

Method A above is followed except that one or more other therapeutic agents are prescribed and administered to the subject for treatment of a flavivirus infection or symptoms thereof. One or more other therapeutic agents may then be administered with, or before or after administration of the antiviral composition. Dosage escalation (or reduction) of one or more other therapeutic agents may also be performed.

Example 14

Evaluation of antiviral activity against Zika virus and dengue virus

CPE-based antiviral assays were performed by infecting target cells in the presence or absence of test compositions at a range of concentrations. Infection of target cells results in cellular lesion effects and cell death. In this type of assay, a decrease in CPE and a corresponding increase in cell viability in the presence of the test composition is used as an indicator of antiviral activity. For CPE-based assays, cell viability was determined with a neutral red readout. Viable cells contain a neutral red color within their lysosomes. Absorption of neutral red depends on the ability of living cells to maintain a lower pH in the lysosome than in the cytoplasm, and this activation process requires ATP. Once inside the lysosome, a neutral red dye is charged and retained within the cell. After 3 h incubation with neutral red (0.033%), the extracellular dye was removed, the cells were washed with PBS, and the intracellular neutral red was lysed with 50% ethanol + 1% acetic acid solution. The amount of neutral red in solution was quantified by reading the absorbance (optical density) of each well at 490 nm.

Adherent cell lines were used to evaluate the antiviral activity of the compositions against a panel of viruses. Prior to adding the virus to the cells, the composition was pre-incubated with the target cells for 30 minutes. The composition was present in the cell culture medium during the infection culture period. For each infection assay, viability assays were established in parallel using the same concentration of the composition (replicas) to determine the cytotoxic effect of the composition in the absence of virus.

The antiviral activity of the test composition was determined by comparing the level of infection (immunostaining-based assay) or viability (CPE-based assay) of cells under the test conditions with the level of infection or viability of uninfected cells. Cytotoxic effects were assessed in uninfected cells by comparing viability in the presence of inhibitor to viability of mock-treated cells. Cytotoxicity was determined by the XTT viability assay, which was performed at the same time points as reads for the corresponding infection assay.

The test composition was dissolved in 100% methanol. By performing an 8-fold dilution, starting with 50 μM as the highest concentration tested, 8 concentrations of the composition were generated (in duplicate). The highest tested concentration of the composition (50 μM) 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 reflecting the final concentration of methanol in each composition test condition. Where possible, the EC50 and CC50 of the composition were determined for each assay using GraphPad Prism software.

Antiviral activity was assessed by the degree of protection against virus-induced cellular lesion effects (CPE). Cells were challenged with virus in the presence of different concentrations of control or composition. The degree of protection against CPE was measured by quantifying cell viability at different test conditions, 6 days post-infection (ZIKV, Zika virus) or 7 days post-infection (DENV, dengue virus), untreated cells and cells treated with vehicle alone. (infection medium) was monitored by comparing the values.

Quality control for neutral analysis was performed on all plates: i) signal versus background (S/B) values; ii) inhibition by known inhibitors, and iii) the variance of the assay as measured by the coefficient of variation (C.V.) of all data points. The overall variation in the infection assay ranged from 3.4% to 9.5%, and the overall variation in the viability assay ranged from 1.4% to 3.2%, calculated as the average of all C.V. values. The signal versus background (S/B) range for the infection assay ranged from 2.9 to 11.0, and the signal versus background (S/B) range for the viability assay ranged from 6.5 to 29.9.

Protection of DENV2-induced cytopathic effect (CPE) with neutral red readout: For the DENV2 antiviral assay, the 08-10381 Montserrat strain was used. Virus 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 infection and 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 h. On the day of infection, samples were diluted 8-fold in U-bottom plates using MEM with 1% bovine serum albumin (BSA). Test substance dilutions were prepared to a final concentration of 1.25X, and 40 μl were incubated with target cells at 37° C. for 30 minutes. After test substance pre-incubation, 10 μl of virus dilution prepared in MEM with 1% BSA is added to each well (50 μl final volume per well), and the plate is _{incubated in a humidified incubator with 5% CO 2} at 37° C. for 3 hours. cultured. The volume of virus used in the assay was predetermined to produce a signal in a linear range, inhibited by ribavirin and compound A3, a known inhibitor of DENV2. After infection culture, cells were washed with PBS and then washed with MEM containing 2% FBS (MEM2) to remove unbound virus. Then, 50 μl of medium containing inhibitor dilution prepared at 1× concentration in MEM2 was added to each well. Plates were incubated in an incubator (5% CO ₂ ) at 37° C. for 7 days. Virus-free controls (“mock-infected”), infected cells cultured with medium alone, infected cells cultured with vehicle (methanol) alone, and wells without cells (to determine background) were included in the assay plate. Control wells containing 50 μM Ribavirin and 0.5 μM Compound A3 were also included on the assay plate. Seven days after infection, cells were stained neutral red to monitor cell viability. Test substances were evaluated in duplicate using serial 8-fold dilutions in infection medium. Controls included cells cultured without virus (“mock-infected”), infected cells cultured with medium alone, or cells infected in the presence of ribavirin (0.5 μM) or A3 (0.5 μM). Full replication inhibition curves using methanol vehicle only were included on the same assay plate.

Protection of ZIKV-Induced Cell Lesion Effect (CPE) with Neutral Red Readout: For ZIKV antiviral assay, PLCal_ZV strain was used. Vero cells ( epithelial kidney cells derived from Cercopithecus aethiops ) were maintained in MEM with 5% FBS (MEM5). For both infection and viability assays, cells were seeded at 10,000 cells per well in 96-well clear flat bottom plates and maintained at 37 °C for 24 h in MEM5. On the day of infection, samples were diluted 8-fold in U-bottom plates using MEM with 1% bovine serum albumin (BSA). Test substance dilutions were prepared to a final concentration of 1.25X, and 40 μl were incubated with target cells at 37° C. for 30 minutes. After test substance pre-incubation, 10 μl of virus dilution prepared in MEM with 1% BSA was added to each well (50 μl final volume per well), and the plate was incubated at 37° C. for 3 hours in a humidified incubator _{with 5% CO 2 .} did. After incubation of infection, cells were washed with PBS and then washed with MEM containing 2% FBS (MEM2) to remove unbound virus. Then, 50 μl of medium containing inhibitor dilution prepared at 1× concentration in MEM2 was added to each well. Plates were incubated at 37° C. in an incubator (5% CO _{2 ) for 6 days.} Virus-free controls (“mock-infected”), infected cells cultured with medium alone, infected cells cultured with vehicle (methanol) alone, and wells without cells (to determine background) were included in the assay plate. After 6 days of infection, cells were stained neutral red to monitor cell viability. Test substances were evaluated in duplicate using serial 8-fold dilutions in infection medium. Controls included cells cultured without virus (“mock-infected”), infected cells cultured with medium alone, or cells infected in the presence of A3 (0.5 μM). Full replication inhibition curves using methanol vehicle only were included on the same assay plate.

Analysis of CPE-Based Viability Data: For the neutral red assay, cell viability was determined by monitoring absorbance at 490 nm. The average signal obtained in cell-free wells was subtracted from all samples. All data points were then calculated as a percentage of the mean signal observed in 8 wells of mock (uninfected) cells on the same assay plate. Infected cells treated with medium alone reduced the signal by an average of 4.2% (for HRV), 26.9% (for DENV) and 5.1% (for ZIKV) of that observed in uninfected cells. The signal versus background (S/B) for this assay was 2.9 (for DENV) and 7.2 (for ZIKV), which were viability in “mock-infected” cells compared to the viability of infected cells treated with vehicle alone. determined as a percentage.

Viability Assay (XTT) to Assess Compound-Induced Cytotoxicity: Incubate mock-infected cells with inhibitor dilutions (or media alone), using the same experimental setup and inhibitor concentrations used for the corresponding infection assays. did. The incubation temperature and incubation period reflected the conditions of the corresponding infection assay. Cell viability was assessed by the XTT method. The XTT assay measures mitochondrial activity and is based on the cleavage of a yellow tetrazolium salt (XTT) to form an orange formazan dye. The response occurs only in viable cells with active mitochondria. Formazan dye is directly quantified using a scanning multi-well spectrophotometer. Background levels from wells without cells were subtracted from all data points. Controls with methanol vehicle only (at 7 concentrations reflecting the final percent methanol of each oleadrine test well) were included in viability assay plates. The degree of viability was monitored by measuring the absorbance at 490 nm.

Analysis of cytotoxicity data: For the XTT assay, cell viability was determined by monitoring absorbance at 490 nm. The average signal obtained in cell-free wells was subtracted from all samples. All data points were then calculated as a percentage of the mean signal observed in 8 wells of mock (uninfected) cells on the same assay plate. Signal versus background (S/B) for this assay were 29.9 (for IVA), 8.7 (for HRV), 6.5 (for DENV) and 6.7 (for ZIKV), which were observed for cell-free wells. It was determined as the percentage of viability of "mock-infected" cells compared to the signal.

Example 15

Evaluation of antiviral activity against filoviruses (Ebolavirus and Marburg virus)

Method A.

Vero E6 cells were infected with EBOV/Kik (A, MOI = 1) or MARV/Ci67 (B, MOI = 1) in the presence of oleandrine containing plant extracts oleandrine, digoxin or PBI-05204. After 1 hour, the inoculum and compounds were removed and fresh medium was added to the cells. After 48 hours, cells were fixed and immunostained to detect cells infected with EBOV or MARV. Infected cells were enumerated using Operetta. C) Vero E6 was treated with compound as above. ATP levels were measured by CellTiter-Glo as a measure of cell viability.

Method B.

Vero E6 cells were infected with EBOV (A, B) or MARV (C, D). At 2 h post-infection (A, C) or 24 h post-infection (B, D), oleandrine or PBI-05204 was added to cells for 1 h, then discarded and cells returned to culture medium. 48 hours after infection, infected cells were analyzed as in FIG. 1 .

Method C.

Vero E6 cells were infected with EBOV or MARV in the presence of oleandrine or PBI-05204 and cultured for 48 hours. Supernatants from infected cell cultures were passed through fresh Vero E6 cells, incubated for 1 hour, and then discarded (as shown in A). Cells containing the passed supernatant were incubated for 48 hours. Cells infected with EBOV (B) or MARV (C) were detected as described above. Control infection rates were 66% for EBOV and 67% for MARV.

Example 16

Evaluation of antiviral activity against Togaviridae (Togaviridae) virus

(Alphaviruses: VEEV and WEEV)

Vero E6 cells were infected with Venezuelan Equine Encephalitis Virus (A, MOI = 0.01) or Western Equine Encephalitis Virus (B, MOI = 0.1) for 18 h in the presence or absence of the indicated compounds. Infected cells were detected as described herein and enumerated in operetta.

Example 17

Treatment of Paramyxoviridae Infection in a Subject

Exemplary Paramyxoviridae (Paramyxoviridae) and viral infections include Henipavirus genus infection, Nipahvirus infection, or Hendra virus infection.

Method A. Antiviral Composition Therapy

A subject exhibiting a Paramyxoviridae infection is prescribed an antiviral composition, and a therapeutically relevant dose is administered to the subject over a period of time according to a prescribed dosing regimen. The subject's level of response to treatment is determined periodically. A therapeutic response level can be determined by determining a subject's viral titer in blood or plasma. If the level of therapeutic response in one dose is too low, the dose is increased according to a pre-determined schedule of dose escalation until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition may continue as needed, and the dosage or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Other Agents

For the treatment of a Paramyxoviridae infection or its symptoms, Method A above is followed except that one or more other therapeutic agents are prescribed and administered to the subject. One or more other therapeutic agents may then be administered with, or before or after administration of the antiviral composition. Dosage escalation (or reduction) of one or more other therapeutic agents may also be performed.

Example 18

Cell lines and isolation of primary huPBMCs

Virus-producing HTLV-1-transformed (HTLV-1+) SLB1 lymphoma T-cell line (Arnold et al., 2008; P. Green, kindly provided by The Ohio State University-Comprehensive Cancer Center) Iscove ) in Modified Dulbecco's Medium (IMDM; ATCC No. 30-2005) _{, incubated in a humidified incubator at 37° C. under 10% CO 2} , and 10% heat-inactivated fetal bovine serum (FBS; Biowest), 100 U/ml penicillin, 100 μg/ml streptomycin-sulfate and 20 μg/ml gentamicin-sulfate (Life Technologies).

Primary human peripheral blood mononuclear cells (huPBMC) were isolated from whole blood samples and provided without identifiers by the SMU Memorial Health Center, under a protocol consistent with the declaration of the SMU Institutional Review Board and the Helsinki principle. Briefly, 2 ml of whole blood was mixed with an equal volume of sterile phosphate buffered saline (PBS), pH 7.4 in a polypropylene conical tube (Corning), and the sample was then gently stacked on 3 ml of lymphocyte isolation medium (MP Biomedicals). Samples were centrifuged at 400 x g for 30 min in a swinging bucket rotor at room temperature. Buffy-coated huPBMCs were then aspirated, washed twice in RPMI-1640 medium (ATCC No. 30-2001), and pelleted by centrifugation at 260×g for 7 minutes. Cells were treated with 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin-sulfate, 20 μg/ml gentamicin-sulfate and 50 U/ml recombinant human interleukin-2 (hu-IL-2; Roche Applied Science). was resuspended in RPMI-1640 medium supplemented with They were stimulated with 10ng/ml phytohemagglutinin (PHA; Sigma-Aldrich) for 24 hours and grown at 37° C. in a _{humidified incubator under 10% CO 2 .} The next day, the cells were pelleted by centrifugation at 260 x g for 7 min, washed twice with RPMI-1640 medium to remove PHA, then resuspended in complete medium supplemented with antibiotics and 50 U/ml hu-IL-2 and cultured.

Example 19

Generation of GFP-expressing HTLV-1 + SLB1/pLenti-GFP T-cell clones

To generate GFP-expressing HTLV-1 + SLB1 T-cell clones, 2x10 ⁶ SLB1 cells were plated in 60 mm ² tissue culture dishes (Corning) of IMDM, supplemented with 10% heat-inactivated FBS and antibiotics. Then, it was transduced with lentiviral particles containing the pLenti-6.2/V5-DEST-green fluorescent protein expression vector carrying the blasticidin resistance gene. After 6 h, the transduced cells were pelleted by centrifugation at 260 x g for 7 min at room temperature, washed twice with serum-free IMDM, and complete supplemented with 5 μg/ml blasticidin (Life Technologies). Resuspended in medium and aliquoted into 96-well microtiter plates (Corning). Cultures were maintained with blasticidin-selection for 2 weeks in a humidified incubator at 37° C. and 10% CO _{2 .} GFP-expressing lymphoblasts were screened by fluorescence microscopy and then plated by limiting dilution in 96-well microtiter plates to obtain homogeneous GFP-expressing cell clones. The resulting HTLV-1 + SLB1/pLenti-GFP T-lymphocyte clones were expanded and passed repeatedly; Expression of GFP was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using rabbit polyclonal anti-GFP (FL) antibody (Santa Cruz Biotechnology).

Example 20

Quantification of Virus Production and Particle Infection by Anti-HTLV-1 p19 ^{Gag ELISA}

To determine the effect of oleandrine or N. oleander extracts on HTLV-1 proviral replication and release of newly synthesized extracellular viral particles, HTLV-1 + SLB1 lymphoma T-cell lines were cultured in 96-well microtiter plates. In 300 μl of complete medium supplemented with antibiotics, 2×10 ⁴ cells per well were plated and incubated at 37° C. under _{10% CO 2 .} Purified oleandrine compound and N. oleander extract (see Phoenix Biotechnology; Singh et al., 2013) were prepared in a vehicle solution (MilliQ distillation/deionized H ₂ O with 20% v/v dimethyl in stock concentration of 2 mg/ml). sulfoxide, DMSO) and sterilized using a Luer Lock 0.2 μm syringe filter (Millipore). HTLV-1 + SLB1 cells were treated with oleandrine or N. oleander extract at concentrations of 10, 50 and 100 μg/ml, or increasing amounts of vehicle control (1.5, 7.5 and 15 μl) for 72 hours. The 96-well microtiter plate was centrifuged using an Eppendorf A-2-DWP swing plate rotor at room temperature at 260 x g for 7 min to pellet the cells and HTLV containing ^{extracellular p19 Gag released into the culture supernatant.} Levels of -1 particles were quantified against ^{p19 Gag protein standards by performing colorimetric Anti-p19 Gag} enzyme-linked immunosorbent assays (ELISAs; Zeptometrix). Samples were analyzed in triplicate on a Berthold Tristar LB 941 multimode microplate-reader at 450 nm in absorbance mode.

To evaluate the infectivity of newly synthesized extracellular HTLV-1 particles collected from oleandrine-treated cells, 2x104 HTLV-1 + SLB1 T-lymphoblasts were plated in 300 μl of complete medium supplemented with antibiotics, Cultures were treated with increasing concentrations (10, 50 and 100 μg/ml) of oleandrine or N. oleander extracts or vehicle control (1.5, 7.5 and 15 μl) for 72 hours. Then, 50 μl of the virus-containing supernatant was used to directly infect huPBMCs plated at a density of ^{2×10 4} cells per well in 96-well microtiter plates in complete medium supplemented with antibiotics and hu-IL-2. Oleandrine compounds, N. oleander extracts or vehicle controls were maintained in huPBMC culture medium to control possible re-infection events by newly generated particles. ^{After 72 h, the relative levels of extracellular p19 Gag-} containing HTLV-1 virions released into the culture supernatant by infected huPBMCs were quantified via Anti-HTLV-1 p19 ^{Gag ELISA.}

Example 21

cell death measurement

To evaluate the relative cytotoxicity of oleandrine compounds, N. oleander extracts, or vehicle controls in treated cell cultures, 2x10 ⁴ HTLV-1 + SLB1 lymphoma T-cells or activated/cultured huPBMCs supplemented with 300 antibiotics Plated in μl of complete medium and maintained at 37° C. under _{10% CO 2 in a humidified incubator.} Cultures were treated with increasing concentrations (10, 50 and 100 μg/ml) of oleandrine or N. oleander extract or vehicle control (1.5, 7.5, 15 ml) and incubated for 72 hours. Cyclophosphamide (50 μM; Sigma-Aldrich)-treated cells were included as positive controls for apoptosis. Thereafter, the cells were aspirated and plated on Permanox 8-chamber tissue-culture slides (Nalge) pretreated with a sterile 0.01% solution of Poly- _{L-Lysine and Concanavalin A (1 mg/ml; Sigma-Aldrich).} did. Samples were then stained using a microscopic cell death detection kit with Annexin V conjugated to fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI; BD-Pharmingen) and field The relative percentage of cell death per cell (ie, Annexin V-FITC and/or PI-positive) was quantified in triplicate by confocal fluorescence microscopy using a 20x objective. The total number of cells per field was quantified microscopically using a DIC phase-contrast filter.

Example 22

HTLV-1 propagation and viral synapse formation in co-culture assays

Since the propagation of HTLV-1 usually occurs through direct contact between infected and uninfected target cells via viral synapses (Igakura et al., 2003; Pais-Correia et al., 2010; Gross et al., 2016; Omsland et al., 2018; Majorovits et al., 2008), we found that oleandrine, N. oleander extracts, or vehicle controls were used to form infectious HTLV-1 particles through viral synapse formation and/or cell-to-cell interactions in vitro. tested whether it can affect the propagation of For this experiment 2x10 ⁴ virus-producing HTLV-1 + T cells play a SLB1 in a 96-well microtiter plate in a floating, and, 37 ℃ under 10% CO _2, in complete medium for 2 hours in 300μl mitomycin C ( 100 μg/ml) (Bryja et al., 2006). The culture medium is then removed, the cells are washed twice with serum-free IMDM, and cells at increasing concentrations (10, 50 and 100 μg/ml) of oleandrine or N. oleander extract, or vehicle control ( 1.5, 7.5 and 15 μl) for 15 minutes or 3 hours. _{Alternatively, 2×10 4} of GFP-expressing HTLV-1 + SLB1/pLenti-GFP T cells were plated on 8-chamber tissue culture slides in 300 μl of complete medium, treated with mitomycin C, and treated with serum-free IMDM. Washed twice and treated with oleandrine, N. oleander extract or vehicle control as described for confocal microscopy experiments. Then, the medium was aspirated, and the HTLV-1 + SLB1 cells were washed twice with serum-free medium, in 300 μl of RPMI-1640 medium supplemented with 20% FBS, antibiotics and 50 U/ml hu-IL-2. ^{2x10 4} huPBMCs were added to each well, then visualized viral synapse formation and virus propagation by confocal microscopy using SLB1/pLenti-GFP lymphoblasts (SLB1/pLenti-GFP lymphoblasts) for another 72 h at 37 °C under _{10% CO 2 .} The cells were co-cultured in a humidified incubator for 6 hours. As a negative control, huPBMCs were cultured alone in the absence of virus-producing cells. Oleandrin, N. oleander extract and vehicle were maintained in the co-culture medium. Anti-HTLV-1 p19Gag ELISA was performed to quantify the relative levels of ^{extracellular p19 Gag-} containing HTLV-1 particles released into the co-culture supernatant as a result of intercellular viral propagation. Viral synapses formed between GFP-positive HTLV-1 + SLB/pLenti-GFP cells and huPBMCs were immunized by staining fixed samples with Anti-HTLV-1 gp21 ^Env primary antibody and rhodamine red-conjugated secondary antibody. Visualized using fluorescence-confocal microscopy. Diamidino-2-phenyl-indole, dihydrochloride (DAPI; Molecular Probes) nuclear staining was included for comparison and to visualize uninfected (ie, HTLV-1-negative) cells. Intercellular propagation of HTLV-1 to huPBMCs in co-culture assays was ^{quantified by calculating the relative percentages of HTLV-1 gp21 Env} -positive (and GFP-negative) huPBMCs in 20 fields of view using a 20x objective.

Example 23

microscopic examination

Annexin V-FITC/PI-stained samples for quantifying cell apoptosis and cytotoxicity using Plan-Apochromat 20x/0.8 objective and Zeiss ZEN system software (Carl Zeiss Microscopy), Airyscan detector and stage CO ₂ Visualized with a confocal fluorescence microscope on a Zeiss LSM800 instrument equipped with an incubator. Virological synapses and viral propagation between mitomycin C-treated HTLV-1 + SLB1/pLenti-GFP lymphoblasts and cultured huPBMCs (i.e., as determined by quantifying the relative percentage of ^{Anti-HTLV-1 gp21 Env-positive huPBMCs)} The formation of α was visualized by immunofluorescence-confocal microscopy using a Plan-Apochromat 20x/0.8 objective. Relative fluorescence intensities of DAPI, Anti-HTLV-1 gp21Env-specific (rhodamine red-positive) and GFP signals were quantified graphically using a Zen 2.5D analysis tool (Carl Zeiss Microscopy). GFP-expressing HTLV-1 + SLB1/pLenti-GFP T cell clones using a Plan Fluor 10x/0.30 objective lens and DIC phase-contrast filter (Nikon Instruments), Nikon Eclipse equipped with 633 nm and 543 nm He/Ne and 488 nm Ar lasers. Screened by confocal fluorescence microscopy on a TE2000-U inverted microscope and a D-Eclipse confocal imaging system.

Example 24

statistical analysis

Statistical significance of experimental data sets was determined using unpaired two-tailed Student's t-test (alpha = 0.05), and P-values were calculated using Shapiro-Wilk normality test and Graphpad Prism 7.03 software. P-values were defined as 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). Unless otherwise specified, error bars represent SEMs from at least three independent experiments.

Example 25

Treatment of a Deltaretroviral Infection in a Subject

Exemplary deltaretroviral infections include HTLV-1.

Method A. Antiviral Composition Therapy

A subject exhibiting HTLV-1 infection is prescribed an antiviral composition, and a therapeutically appropriate dose is administered to the subject according to the prescribed dosing regimen for a period of time. The subject's level of response to treatment is determined periodically. The level of therapeutic response can be determined by measuring the HTLV-1 virus titer of the subject in blood or plasma. If the level of therapeutic response is too low in a single dose, 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 the antiviral composition continues as needed, and the dosage or dosing regimen can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition and Other Agents

Method A is followed except that the subject is prescribed and administered one or more other therapeutic agents to treat HTLV-1 infection or symptoms thereof. Thereafter, one or more other therapeutic agents may be administered before, after, or concurrently with administration of the antiviral composition. Dosage escalation (or reduction) of one or more other therapeutic agents may also be performed. Exemplary other therapeutic agents are described herein.

Example 26

Treatment of a CoV Infection in a Subject

Exemplary CoV infections include SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1 and CoV HKU20.

Method A. Antiviral Composition Therapy

A subject exhibiting a CoV infection is prescribed an antiviral composition, and a therapeutically appropriate dose is administered to the subject according to the prescribed dosing regimen for a period of time. The subject's level of response to treatment is determined periodically. The level of therapeutic response can be determined by measuring the subject's CoV virus titer in blood or plasma. If the level of therapeutic response is too low in a single dose, 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 the antiviral composition continues as needed, and the dosage or dosing regimen can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition and Other Agents

Method A is followed except that the subject is prescribed and administered one or more other therapeutic agents to treat the CoV infection or symptoms thereof. Thereafter, one or more other therapeutic agents may be administered before, after, or concurrently with administration of the antiviral composition. Dosage escalation (or reduction) of one or more other therapeutic agents may also be performed. Exemplary other therapeutic agents are described herein.

Example 27

Treating a Subject's COVID-19 Infection Using ANVIRZEL™

To treat symptoms associated with COVID-19, ANVIRZEL™ was administered to children (infants) with COVID-19 as follows. The subject's viral infection worsened prior to administration of ANVIRZEL™. Subjects were prescribed and administered ANVIRZEL™ according to the following dosing regimen: initial dose- 0.25 mL of reconstituted ANVIRZEL™ followed by 0.5 mL of reconstituted ANVIRZEL™ every 12 hours for 2-3 days. The subject's COVID-19 infection resolved, and no drug-related toxicity was observed.

Example 28

In vitro evaluation of oleandrine for COVID-19 virus

The purpose of this study was to determine the effect of oleandrin on the infectivity of progeny virions.

A stock solution of oleandrine in methanol (10 mg oleandrine/mL) was prepared. Stock solutions were culture medium containing DMSO (aqueous culture medium RPMI 1640 and oleandrine (20 μg/mL, 10 μg/mL, 1.0 μg/mL or 0.1 μg/mL) 0.1% or 0.01% v/v) was used to prepare The culture medium is as follows.

Uninfected Vero cells in culture (target initial cell number 1×10 ⁶ ) were cultured in each of the indicated culture media in a vial at 37° C. for 30 minutes. A viral inoculum of SARS-CoV-2 was added to each vial to achieve ^{a target initial viral titer (approximately PFU/mL 1×10 4 ).} Target MOI (multiplicity of infection) of about 0.1. The solution was incubated at 37 °C for an additional 2 h to infect Vero cells. Infected Vero cells were washed with control vehicle to remove extracellular virus and oleandrin. A fresh aliquot of each culture medium was added to each vial of infected Vero cells. Those that received oleandrine in the second aliquot were marked "post-infection + treatment" and those that did not receive oleandrine in the second aliquot were marked "post-infection - treatment" ( FIGS. 23A-23D ). Virus titers for each vial were determined at about 24 and about 48 hours post infection.

As a means to determine the potential toxicity of oleandrine to Vero cells, a very similar culture based on the above was prepared for uninfected Vero cells.

The data obtained included the amount of virus produced, the infectivity of the progeny virus, and the relative safety (non-toxic) of oleandrine in infected and uninfected cells.

Example 29

In vitro evaluation of oleandrine for COVID-19 virus

The purpose of this assay was to determine the direct antiviral activity of oleandrine against SARS-CoV-2.

Growth medium was removed from confluent monolayers of ^{approximately 10 6 Vero CCL81 cells in 6-well plates.} Oleandrin was serially diluted in culture medium and added to Vero-E6 cells seeded in 96 well plates. Growth medium was replaced with 200 μl of maintenance medium containing either 1.0 μg/ml, 0.5 μg/ml, 100 ng/ml, 50 ng/ml, 10 ng/ml or 5 ng/ml of oleandrine, or a matching DMSO-only control. Plates were incubated at 37 °C for about 30 min prior to virus addition.

SARS-CoV-2 virus was added to oleandrine treated and untreated cells at an MOI (multiplicity of infection) of 0.4 (influx assay) or 0.02 (replication assay). Oleandrin remained in the wells for 1 h incubation at 37 °C.

After 1 hour absorption, the inoculation medium was removed and washed once with PBS (standard phosphate buffered saline).

Media alone (no oleandrine) was added back to the oleandrine treated wells designated as "pretreatment" in the data slides. Medium with the indicated concentrations of oleandrine was added back to the wells designated as "duration" in the data slides.

Plates were fixed at 24 (influx assay) or 48 (replication assay) hours post infection and immunostained with virus-specific antibody and fluorescently labeled secondary antibody.

Cells were imaged using Operetta and data analyzed using a custom algorithm in Harmonia software to determine the percentage of infected cells in each well.

The results are shown in FIGS. 24A and 24B .

Example 30

In vitro evaluation of oleandrine toxicity to Vero-E6 cells

The purpose of this assay was to determine the relative potential toxicity of oleandrine on Vero-E6 cells.

Oleandrin was serially diluted in culture medium, added to Vero-E6 cells seeded in 96-well plates, and incubated at 37°C for about 24 hours. Cell number was obtained using CellTiter Glo assay.

The results are shown in FIG. 25 .

Example 31

In vitro evaluation of oleandrine for COVID-19 virus

The purpose of this study was to determine the dose response of COVID-19 virus to oleandrine treatment.

The procedure of Example 28 was repeated except that low concentrations of 1 μg/mL, 0.5 μg/mL, 0.1 μg/mL, 0.05 μg/mL, 0.01 μg/mL and 0.005 μg/mL of oleandrine were used. . In addition, VERO CCL-81 cells were used instead of VERO E6 cells.

Virus titers were determined according to Example 28, and fold reductions in viral titers were calculated compared to control samples. Results are shown in 26a-26d, 27a-27d, 28a and 28b.

Example 32

Sublingual liquid formulation

Sublingual formulations comprising oleandrine include oleandrine or an oleandrine-containing composition (e.g., oleandrine-containing extract; 2 wt %) with medium chain triglycerides (95 wt %) and a flavoring agent (3 wt %). ) and was prepared by mixing. The oleandrine content of the formulation was about 25 μg/mL.

Example 33

Preparation of Subcritical Fluid Extracts of Nerium Oleander

An improved process for the preparation of oleandrine containing extracts was developed using subcritical liquid extraction rather than supercritical fluid extraction of Nerium oleander biomass.

The dried and powdered biomass was placed in an extraction chamber and then sealed. Carbon dioxide (about 95 % wt) and alcohol (about 5 % wt; methanol or ethanol) were injected into the chamber. The temperature and pressure inside the chamber is such that the extraction medium is at a temperature of from about 2° C. to about 16° C. (about 7° C. to about 8° C.) and a pressure from about 115 to about 135 bar (about 124 bar), to be maintained in the subcritical liquid phase and not in the supercritical fluid phase. The extraction period was from about 4 hours to about 12 hours (about 6 to about 10 hours). The extraction environment was filtered and the supernatant was collected. Carbon dioxide was drained from the supernatant, and the resulting crude extract was diluted with ethanol (about 9 parts ethanol:about 1 part extract) and frozen at about -50°C for at least 12 hours. 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 diluted with 50% aqueous ethanol to a concentration of about 1.5 mg of extract per mL of solution.

The resulting subcritical liquid (SbCL) extract comprises oleandrine and one or more other compounds extractable from Nerium oleander, said one or more other compounds as defined herein.

Example 34

In vitro evaluation of oleandrine for COVID-19 virus

The purpose of this study was to determine the effect of oleandrine on the infectivity of progeny virions without oleandrine pretreatment (according to Example 28).

The procedure of Example 28 was repeated except that the cells were not pretreated with oleandrine prior to infection. Instead, infected cells were treated with oleandrine or control vehicle at 12 and 24 hours post infection. Moreover, VERO CCL-81 cells were used instead of VERO E6 cells, and lower concentrations of oleandrin: 1 μg/mL, 0.5 μg/mL, 0.1 μg/mL and 0.05 μg/mL were used. Data is shown in Figures 29A and 29B.

Example 35

In vivo evaluation of oleandrine for COVID-19 virus

The purpose of this study was to determine the efficacy of an oleandrine-containing extract (OCE) in treating subjects already infected with the COVID-19 virus.

Subjects exhibiting a broad demographic distribution and exhibiting COVID-19 infection were evaluated to determine the clinical status prior to sublingual, buccal, or buccal administration of OCEs prepared according to the formulation of Example 32. The composition was safely administered to a subject by dropping a liquid drop into the subject's mouth. The dosing regimen was about 0.5 mL at the time of administration and 4 times a day (about once every 6 hours), which corresponds to about 50 μg of oleandrine per day. Alternatively, half of the total daily dose was administered. All subjects experienced full recovery.

Example 36

Preparation of ethanol extract of Nerium oleander

The purpose of this was to prepare an ethanolic extract by extracting Nerium oleander biomass with aqueous ethanol.

The crushed dry leaves were repeatedly treated with aqueous ethanol (90 % v/v ethanol; 10 % v/v water). The combined ethanol supernatant was filtered, and then concentrated by evaporation in vacuo to reduce the amount of ethanol and water therein, and crude ethanolic extract containing about 25 mg of oleandrine/mL of the extract (about 50 % v/v ethanol content) was provided.

Example 37

Preparation of Formulations Comprising Combination of Nerium Oleander Extracts

The purpose of this was to combine a portion (1 wt %) of the ethanol extract of Example 36 with a portion (1 wt %) of the SbCL extract of Example 33, medium chain triglycerides (95 wt %) and a flavoring agent (3 wt %). except that it was intended to prepare a formulation according to Example 32.

Example 38

In vivo evaluation of digoxin against COVID-19 virus

The purpose of this study is to determine the efficacy of a composition containing digoxin (DCC) in treating subjects already infected with the COVID-19 virus. A commercial formulation containing digoxin was purchased.

Subjects with COVID-19 infection were evaluated to determine clinical status prior to oral or systemic administration of DCC. Commercially available compositions are described herein. Safe dosing regimens for each are described in the appropriate NDA and package insert. The composition is safely administered to each subject according to the intended route of administration. Clinical monitoring was performed to determine treatment response and dose titrated accordingly.

Example 39

Determination of Genome-to-Infectious Particle Ratio in SARS-CoV-2 Infection Treated with Oleandrine

The purpose of this study was to determine whether inhibition of SARS-CoV-2 by oleandrine was at the level of total or infectious particle production.

To quantify genomic copies for a sample, 200 μl of sample was extracted in a 5:1 volume ratio of TRIzol LS (Ambion, Carlsbad, CA) using standard manufacturer protocols and resuspended in 11 μl water. The extracted RNA was tested for SARS-CoV-2 by qRT-PCR according to a previously published assay (26). Briefly, the N gene was amplified using the following primers and probes: 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 20 μl reaction mixture was prepared using the iTaq Universal probes One-Step kit (BioRad, Hercules, CA) according to the manufacturer's instructions. Reaction mixture (2x:10 µL), iScript reverse transcriptase (0.5 µL), primers (10 µM: 1.0 µL), probe (10 µM: 0.5 µL), extracted RNA (4 µL) and water (3 µL). qRT-PCR reactions were performed using a Thermocycler StepOnePlus™ Real-Time PCR system (Applied Biosystems). The reaction was incubated at 50° C. for 5 min, 95° C. for 20 sec, followed by 40 cycles of 95° C. for 5 sec and 60° C. for 30 sec. A positive control RNA sequence (nucleotides 26,044-29,883 of the COVID-2019 genome) was used to estimate the number of RNA copies of the N gene in the sample under evaluation.

As used herein, the term “about” or “approximately” is intended to mean ±10%, ±5%, ±2.5%, or ±1% of the specified value. As used herein, the term “substantially” is intended to mean “a majority” or “at least a majority” or “greater than 50%”.

The above description is a detailed description of a specific embodiment of the present invention. Although specific embodiments of the invention have been described herein for purposes of illustration, it will be understood that various changes may be made therein without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims. All embodiments disclosed and claimed herein can be made and practiced without undue experimentation in light of this disclosure.

SEQUENCE LISTING <110> PHOENIX BIOTECHNOLOGY, INC. <120> Method and Compositions for Treating Coronavirus Infection <130> PBI-22-PCT9 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward (5' to 3') primer sequence <400> 1 taatcagaca aggaactgat ta 22 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse (5' to 3') primer sequence <400> 2 cgaaggtgtg acttccat 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence (5'-FAM-sequence-TAMRA-3'); FAM is 6-fluorescein amidite; TAMRA is Carboxytetramethylrhodamine <400> 3 gcaaattgtg caatttgcgg 20

Claims (114)

A method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject one or more doses of an antiviral composition comprising oleandrine, digoxin or a combination thereof, the virus A method in which infection is caused by a positive-sense single-stranded enveloped RNA virus ((+)-ss-envRNA-V). A method of prophylaxis for treating a subject at risk of contracting a viral infection, the method comprising administering to the subject one or more doses of an antiviral composition repeatedly over an extended treatment period prior to the subject becoming afflicted with a viral infection chronically to the subject. administering to prevent the subject from contracting a viral infection; wherein the antiviral composition comprises oleandrine, digoxin or a combination thereof, wherein the viral infection is caused by a positive-sense single-stranded enveloped RNA virus ((+)-ss-envRNA-V). The method of claim 1,
determining whether the subject is infected with the virus;
directing administration of the antiviral composition;
administering to the subject an initial dose of the antiviral composition according to a prescribed initial dosing regimen for a period of time;
periodically determining the adequacy of the subject's clinical response and/or therapeutic response to treatment with the antiviral composition; and
if the subject's clinical response and/or therapeutic response is appropriate, continuing treatment with the antiviral composition as needed until a desired clinical endpoint is reached; or
if the subject's clinical and/or therapeutic response is inadequate at the initial dosage and initial dosing regimen, increasing or decreasing the dosage until the desired clinical and/or therapeutic response in the subject is achieved;
How to include. The method of any one of the preceding claims, wherein the dosage comprises about 100-1000 mg or about 100-1000 μg of the antiviral composition per kg body weight of the subject. The method of any one of the preceding claims, wherein the amount of oleandrine as part of the composition administered daily is between 140 μg and 315 μg, between 20 μg and 750 μg, between 12 μg and 300 μg, between 12 μg and 120 μg, between 0.01 μg and 100 μg. mg, 0.01 μg to 100 μg, about 0.5 to about 100 μg, about 1 to about 80 μg, about 1.5 to about 60 μg, about 1.8 to about 60 μg, or about 1.8 to about 40 μg. . The method according to any one of the preceding claims, wherein the dose of the antiviral composition is administered twice a day or about every 12 hours, and the amount of oleandrine in the dose is about 0.25 to about 50 μg or about 0.9 to 5 μg. how to be. The method according to any one of the preceding claims, wherein the dosage of the antiviral composition is about 0.05-0.5 mg/kg/day, about 0.05-0.35 mg, based on the unit amount of the antiviral composition per kg body weight of the subject per day. /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 μg/kg/day, about 0.05-0.35 μg /kg/day, about 0.05-0.22 μg/kg/day, about 0.05-0.4 μg/kg/day, or about 0.05-0.3 μg/kg/day. . The method according to any one of the preceding claims, wherein a) the daily dose of oleandrine in the antiviral composition is up to about 100 μg/day, about 80 μg/day, about 60 μg/day, about 40 μg/day , about 38.4 μg/day or about 30 μg/day; and/or b) the daily dose of oleandrine in the antiviral composition is at least about 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day , or about 5 μg/day. The method according to any one of the preceding claims, wherein the cardiac glycoside is administered in at least two administration phases: a loading phase and a maintenance phase. 10. The method according to claim 9, wherein the cardiac glycoside is digoxin and the dosing regimen for the loading phase and the maintenance phase is as follows:

The method of any one of the preceding claims, wherein after administration of the one or more doses, the plasma concentration of oleandrine in the subject is about 0.05 to about 2 ng/ml, about 0.005 in an amount of oleandrine per mL of plasma. to about 10 ng, 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 0.05 to about 2.5 Methods in the ng/mL range. A method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject one or more doses of an antiviral composition comprising oleandrine, wherein the viral infection is positive-sense single caused by strand enveloped RNA virus ((+)-ss-envRNA-V),
The dosage includes about 100-1000 mg or about 100-1000 μg of the antiviral composition per kg of the subject's body weight, based on the unit amount of the antiviral composition per kg of the subject's body weight per day, or is administered per day The amount of oleandrine used is 140 μg to 315 μg, 20 μg to 750 μg, 12 μg to 300 μg, 12 μg to 120 μg, 0.01 μg to 100 mg, 0.01 μg to 100 μg, about 0.5 to about 100 μg, about 1 to about 80 μg, about 1.5 to about 60 μg, about 1.8 to about 60 μg, or about 1.8 to about 40 μg, or the dosage of the antiviral composition is 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 μg/kg /day, about 0.05-0.35 μg/kg/day, about 0.05-0.22 μg/kg/day, about 0.05-0.4 μg/kg/day, or about 0.05-0.3 μg/kg/day; and
After administration of the one or more doses, the plasma concentration of oleandrine in the subject is, in an amount of oleandrine per mL of plasma, from about 0.05 to about 2 ng/ml, from about 0.005 to about 10 ng/mL, from about 0.005 to about 8 ng /mL, about 0.01 to about 7ng/mL, about 0.02 to about 7ng/mL, about 0.03 to about 6ng/mL, about 0.04 to about 5ng/mL, or about 0.05 to about 2.5ng/mL. The method according to any one of the preceding claims, wherein the (+)-ss-envRNAV is a virus selected from the group consisting of Coronaviridae, Flaviviridae, Togaviridae and Arterviridae. The method according to any one of the preceding claims, wherein the (+)-ss-envRNAV is a coronavirus pathogenic to humans. 15. The method of claim 14, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU20. . The method according to any one of claims 1 to 13, wherein the (+)-ss-envRNAV is flavivirus, yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, tick-borne encephalitis virus, A method selected from the group consisting of Sanur Forest Disease Virus, Alkhurma Disease Virus, Omsk Hemorrhagic Fever Virus and Powasan Virus. 14. The method of any one of claims 1 to 13, wherein the (+)-ss-envRNAV is arbovirus, eastern equine encephalomyelitis virus (EEEV), western equine encephalomyelitis virus (WEEV), Venezuelan equine encephalomyelitis virus (VEEV), A method selected from the group consisting of Chikungunya Virus (CHIKV), Oningin Virus (ONNV), Pogosta Disease Virus, Sindbis Virus, Ross River Fever Virus (RRV) and Semliki Forest Virus. A method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject one or more doses of an antiviral composition comprising oleandrine, digoxin or a combination thereof, the virus A method in which infection is caused by a negative-sense single-stranded enveloped RNA virus ((-)-ss-envRNA-V). A method of prophylaxis for treating a subject at risk of contracting a viral infection, the method comprising administering to the subject one or more doses of an antiviral composition repeatedly over an extended treatment period prior to the subject becoming afflicted with a viral infection chronically to the subject. administering to prevent the subject from contracting a viral infection; wherein the antiviral composition comprises oleandrine, digoxin or a combination thereof, wherein the viral infection is caused by a negative-sense single-stranded enveloped RNA virus ((-)-ss-envRNA-V). 19. The method of claim 18,
determining whether the subject is infected with the virus;
directing administration of the antiviral composition;
administering to the subject an initial dose of the antiviral composition according to a prescribed initial dosing regimen for a period of time;
periodically determining the adequacy of the subject's clinical response and/or therapeutic response to treatment with the antiviral composition; and
if the subject's clinical response and/or therapeutic response is appropriate, continuing treatment with the antiviral composition as needed until a desired clinical endpoint is reached; or
if the subject's clinical and/or therapeutic response is inadequate at the initial dosage and initial dosing regimen, increasing or decreasing the dosage until the desired clinical and/or therapeutic response in the subject is achieved;
How to include. 21. The method of any one of claims 18-20, wherein the dosage comprises about 100-1000 mg or about 100-1000 μg of the antiviral composition per kg body weight of said subject. 22. The method according to any one of claims 18 to 21, wherein the amount of oleandrine as part of the composition administered daily is between 140 μg and 315 μg, between 20 μg and 750 μg, between 12 μg and 300 μg, between 12 μg and 120 μg. , 0.01 μg to 100 mg, 0.01 μg to 100 μg, about 0.5 to about 100 μg, about 1 to about 80 μg, about 1.5 to about 60 μg, about 1.8 to about 60 μg, or about 1.8 to about 40 μg a method selected from the group. 23. The method of any one of claims 18 to 22, wherein the dosage of the antiviral composition is administered twice a day or about every 12 hours, and the amount of oleandrine in the dosage is about 0.25 to about 50 μg or about 0.9 to 5 μg. 24. The method according to any one of claims 18 to 23, wherein the dosage of the antiviral composition is about 0.05-0.5 mg/kg/day, based on the unit amount of the antiviral composition per kg body weight of the subject per 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 μg/kg/day, about 0.05-0.35 μg/kg/day, about 0.05-0.22 μg/kg/day, about 0.05-0.4 μg/kg/day, or about 0.05-0.3 μg/kg/day. . 25. The method of any one of claims 18 to 24, wherein a) the daily dose of oleandrine in the antiviral composition is up to about 100 μg/day, about 80 μg/day, about 60 μg/day, about 40 μg/day, about 38.4 μg/day, or about 30 μg/day; and/or b) the daily dose of oleandrine in the antiviral composition is at least about 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day , or about 5 μg/day. 26. The method of any one of claims 18 to 25, wherein after administration of the one or more doses, the plasma concentration of oleandrine in the subject is from about 0.05 to about 2 ng in an amount of oleandrine per mL of plasma. /ml, about 0.005 to about 10 ng, 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 0.05 to about 2.5 ng/mL. A method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject one or more doses of an antiviral composition comprising oleandrine, wherein the viral infection is a negative-sense single caused by a strand enveloped RNA virus ((-)-ss-envRNA-V),
The dosage includes about 100-1000 mg or about 100-1000 μg of the antiviral composition per kg of the subject's body weight, based on the unit amount of the antiviral composition per kg of the subject's body weight per day, or is administered per day The amount of oleandrine used is 140 μg to 315 μg, 20 μg to 750 μg, 12 μg to 300 μg, 12 μg to 120 μg, 0.01 μg to 100 mg, 0.01 μg to 100 μg, about 0.5 to about 100 μg, about 1 to about 80 μg, about 1.5 to about 60 μg, about 1.8 to about 60 μg, or about 1.8 to about 40 μg, or the dosage of the antiviral composition is 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 μg/kg /day, about 0.05-0.35 μg/kg/day, about 0.05-0.22 μg/kg/day, about 0.05-0.4 μg/kg/day, or about 0.05-0.3 μg/kg/day; and
After administration of the one or more doses, the plasma concentration of oleandrine in the subject is, in an amount of oleandrine per mL of plasma, from about 0.05 to about 2 ng/ml, from about 0.005 to about 10 ng/mL, from about 0.005 to about 8 ng /mL, about 0.01 to about 7ng/mL, about 0.02 to about 7ng/mL, about 0.03 to about 6ng/mL, about 0.04 to about 5ng/mL, or about 0.05 to about 2.5ng/mL. 28. The method according to any one of claims 18 to 27, wherein the (-)-ss-envRNAV is a virus of the family Arenaviridae, a virus of the Bunyavirales order, a virus of the family Philoviridae, a family of Orthomyxoviridae. A method which is a virus selected from the group consisting of a virus, a Paramyxoviridae family virus, and a Rhabdoviridae family virus. 29. The method according to any one of claims 18 to 28, wherein said (-)-ss-envRNAV is Lassa virus, aseptic meningitis, Guanarito virus, Junin virus, Lujo ) virus, Machupo virus, Sabia virus and Whitewater Arroyo virus. 29. The bunia according to any one of claims 18 to 28, wherein the (-)-ss-envRNAV is selected from the group consisting of Hantavirus, Crimean-Congo hemorrhagic fever orthonairovirus. Viridae and how it is a virus. 29. The method according to any one of claims 18 to 28, which is a virus of the family Paramyxoviridae selected from mumps virus, nipa virus, Hendra virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) and NDV. . The method according to any one of claims 18 to 28, wherein the (-)-ss-envRNAV is influenza virus (A to C), isa virus, togotovirus, quaranzavirus, H1N1, H2N2, H3N2, H1N2, Orthomyxoviridae Kwak virus selected from Spanish flu, Asian flu, Hong Kong flu, and Russian flu. 29. The method according to any one of claims 18 to 28, wherein the (-)-ss-envRNAV is a virus of the family Rhabdoviridae selected from rabies virus, vesiculovirus, lyssavirus, cytorhabdovirus. A method of treating a virus according to any one of the preceding claims, comprising sublingually administering to a subject having a viral infection one or more doses of the cardiac glycoside comprising oleandrine, digoxin or a combination thereof. A method of treating a coronavirus infection comprising administering to a subject having said infection a plurality of therapeutically effective amounts of oleandrine, digoxin, or a combination thereof. 36. The method of claim 34 or 35, wherein the multiple doses are one or more doses administered per day for at least 2 days per week. 37. The method of claim 36, wherein the administration continues for at least one week per month. 38. The method of claim 37, wherein the administration continues for at least one month per year. 39. The method according to any one of claims 34 to 38, wherein said coronavirus is pathogenic to humans. 40. The method according to any one of claims 34 to 39, wherein the coronavirus is SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1 and CoV. A method selected from the group consisting of HKU20. 41. The method of any one of claims 34-40, wherein the concentration of oleandrine in plasma of the subject after administration of oleandrine is an amount of oleandrine per mL of plasma: a) about 10 μg/mL or less, about 5 μg/mL or less, about 2.5 μg/mL or less, about 2 μg/mL or less, or about 1 μg/mL or less; b) at least about 0.0001 μg/mL, at least about 0.0005 μg/mL, at least about 0.001 μg/mL, at least about 0.0015 μg/mL, at least about 0.01 μg/mL, at least about 0.015 μg/mL, at least about 0.1 μg/mL , at least about 0.15 μg/mL, at least about 0.05 μg/mL, or at least about 0.075 μg/mL; c) a combination of the upper limit of item a) and the lower limit of item c), or d) 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. 41. The method of any one of claims 34-40, wherein the dosage comprises about 100-1000 mg or about 100-1000 μg of the antiviral composition per kg body weight of said subject. 43. The method according to any one of claims 34 to 42, wherein the amount of oleandrine as part of the composition administered daily is between 140 μg and 315 μg, between 20 μg and 750 μg, between 12 μg and 300 μg, between 12 μg and 120 μg. , 0.01 μg to 100 mg, 0.01 μg to 100 μg, about 0.5 to about 100 μg, about 1 to about 80 μg, about 1.5 to about 60 μg, about 1.8 to about 60 μg, or about 1.8 to about 40 μg a method selected from the group. 44. The method of any one of claims 34 to 43, wherein the dose of the antiviral composition is administered twice a day or about every 12 hours, and the amount of oleandrine in the dose is from about 0.25 to about 50 μg or about 0.9 to 5 μg. 45. The method according to any one of claims 34 to 44, wherein the dosage of the antiviral composition is about 0.05-0.5 mg/kg/day, based on the unit amount of the antiviral composition per kg body weight of the subject per 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 μg/kg/day, about 0.05-0.35 μg/kg/day, about 0.05-0.22 μg/kg/day, about 0.05-0.4 μg/kg/day, or about 0.05-0.3 μg/kg/day. 46. The method of any one of claims 34 to 45, wherein a) the daily dose of oleandrine in the antiviral composition is up to about 100 μg/day, about 80 μg/day, about 60 μg/day, about 40 μg/day, about 38.4 μg/day, or about 30 μg/day; and/or b) the daily dose of oleandrine in the antiviral composition is at least about 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day , or about 5 μg/day. 48. The method of any one of claims 1-47, wherein the cardiac glycoside is administered in at least two administration phases: a loading phase and a maintenance phase. 48. The method of claim 47, wherein said cardiac glycoside is digoxin and said dosing regimen for the loading phase and the maintenance phase is as follows:

The composition of any one of the preceding claims, wherein the antiviral composition comprises: a) oleandrine; b) a combination of oleandrine, oleanolic acid (free acid, salt or prodrug) and ursolic acid (free acid, salt or prodrug); c) a combination of oleandrine, oleanolic acid (free acid, salt or prodrug) and betulinic acid (free acid, salt or prodrug); d) a combination of oleandrine, oleanolic acid (free acid, salt or prodrug), ursolic acid (free acid, salt or prodrug) and betulinic acid (free acid, salt or prodrug); e) a combination of oleandrine, oleanolic acid (free acid or salt thereof), ursolic acid (free acid or salt thereof) and betulinic acid (free acid or salt thereof); or f) olean and at least two triterpenes selected from the group consisting of oleanolic acid (free acid, salt or prodrug), ursolic acid (free acid, salt or prodrug), betulinic acid (free acid, salt or prodrug) A method comprising a combination of drin. 50. The method of claim 49, wherein in administering the antiviral composition, the molar ratio of oleandrine to said triterpene is selected from any of the following.

50. The method of claim 49, wherein the molar ratio of total triterpene content (oleanolic acid + ursolic acid + betulinic acid) to oleandrine is from about 15:1 to about 5:1, or from 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 in the range of about 10:1. 50. The method of claim 49, wherein the molar ratio of the individual triterpenes (oleanolic acid (OA):ursolic acid (UA):betulinic acid (BA)) to oleandrine (OL) is 2-8(OA):2-8(UA) :0.1-1 (BA):0.5-1.5 (OL); or 3-6(OA):3-6(UA):0.3-8(BA):0.7-1.2(OL); or 4-5(OA):4-5(UA):0.4-0.7(BA):0.9-1.1(OL); 4.6(OA):4.4(UA):0.6(BA):1(OL); about 9-12 (OA): up to about 2 (UA): up to about 2 or about 10 (OA): about 1 (UA): about 1 or about 9-12 (OA): about 0.1-2 (UA): about 0.1-2 (BA) or about 9-11 (OA): about 0.5-1.5 (UA): about 0.5-1.5 (BA), or about 9.5-10.5 (OA): about 0.75-1.25 (UA): about 0.75-1.25 (BA), or about 9.5-10.5 (OA): about 0.8-1.2 (UA): about 0.8-1.2 (BA), or about 9.75-10.5 (OA): about 0.9-1.1 (UA): about Methods ranging from 0.9-1.1 (BA). A method according to any one of the preceding claims, wherein the antiviral composition comprises an extract of biomass. 54. The method of claim 53, wherein the extract is prepared by hot water extraction, cold water extraction, organic solvent extraction, supercritical fluid extraction, subcritical liquid extraction, or a combination thereof. 55. The method according to claim 53 or 54, wherein said biomass is plant material from Nerium spp. or Agrobacterium spp. biomass. 56. The method of any one of claims 53-55, wherein said extract comprises a combination of oleandrine and one or more compounds extracted from said biomass. 57. The method of any one of claims 53-56, wherein the extract further comprises one or more cardiac glycoside precursors, one or more glycoside components of one or more cardiac glycosides, or a combination thereof. 58. The method according to any one of claims 53 to 57, wherein the extract comprises oleandrine and cardiac glycosides, glycosides, non-saccharides, steroids, triterpenes, polysaccharides, saccharides, alkaloids, fats, proteins, neritalos. Side, odoroside, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-ene-3β,28-diol), 28-norurs-12-en-3β- ol, urs-12-en-3β-ol, 3β,3β-hydroxy-12-oleanene-28-oic acid, 3β,20α-dihydroxyurs-21-en-28-oic acid, 3β,27 -dihydroxy-12-ursen-28-oic acid, 3β,13β-dihydroxyurs-11-en-28-oic acid, 3β,12α-dihydroxyoleanane-28,13β-olide, 3β, 27-dihydroxy-12-oleanane-28-oic acid, homopolygalacturonan, arabinogalacturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumaroyl-CoA, 3-O -caffeoylquinic acid, 5-O-caffeoylquinic acid, cardenolide B-1, cardenolide B-2, oleagenin, neridizinoside, nerizoside, orodoside-H, 3- beta-O-(D-digitinosyl)-5-beta, galacturonic acid, rhamnose, arabi having a MW in the range of 17000-120000 D or a MW of about 35000 D, about 3000 D, about 5500 D or about 12000 D 14 beta-dihydroxy-card-20(22)-enolide consisting of glucose, xylose and galactose, polysaccharides pectic polysaccharide, cardenolide monoglycoside, cardenolide N-1, cardenolide N-2, cardenolide N-3, cardenolide N-4, pregnan, 4,6-diene-3,12,20-trione, 20R-hydroxypregna-4,6-diene -3,12-dione, 16beta,17 beta-epoxy-12beta-hydroxypregna-4,6-diene-3,20-dione, 12beta-hydroxypregna-4,6,16-tri N-3,20-dione (neridienone A), 20S,21-dihydroxypregna-4,6-diene-3,12- Dion (neridion B), neriucoumaric acid, isoneriucoumaric acid, oleanderoic acid, oleanderen, 8 alpha-methoxylabdan -18-oic acid, 12-urcene, canneroside, neriumoside, 3β- O- (D-digitinosyl)-2α-hydroxy-8,14β-epoxy-5β-carda-16:17, 20:22 -dienolide, 3β-O- (D-digitinosyl)-2α,14β-dihydroxy-5β-carda-16:17,20:22-dienolide, 3β,27-dihydro roxy-urs-18-en-13,28-olide, 3β,22α,28-trihydroxy-25-nor-lup-1(10),20(29)-dien-2-one, cis -karenine ( 3β-hydroxy-28-Zp-coumaroyloxy-urs-12-en-27-oic acid), trans -karenine (3-β-hydroxy-28-Ep-coumaroyloxy-urs-12- en-27-oic acid), 3beta-hydroxy-5alpha-carda-14(15),20(22)-dienolide (beta-anhydroepidigitoxigenin, 3beta- O-(D-digitalosyl)-21-hydroxyl-5beta-carda-8,14,16,20(22)-tetraenolide (nerium mogenin-A-3beta-D-digital) loside), proseragenin, neridienone A, 3beta,27-dihydroxy-12-urcene-28-oic acid, 3beta,13beta-dihydroxyurate-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-oleanene-28-oic acid, (20S, 24R)-epoxydammaran-3beta,25-diol, 20beta,28-epoxy-28alpha- Methoxytaraxasteran-3beta-ol, 20beta,28-epoxytaraxaster-21-en-3beta-ol, 28-nor-urs-12-ene-3beta,17beta-diol, 3beta -Hydroxyurus-1 2-en-28-aldehyde, alpha-neriursate, beta-neriursate, 3alpha-acetophenoxy-urs-12-en-28-oic acid, 3beta-acetophenoxy-urs- 12-en-28-oic acid, oleanderoic acid, Kanerodione, 3β- p -hydroxyphenoxy-11α-methoxy-12α-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-dizzy North, neridinoside, nerizoside, isoricinoleic acid, gentiobiosylnerigoside, gentiobiosylbeaumontoside, gentiobiosyloleandrine ( gentiobiosyloleandrin), polynerin (folinerin), 12β-hydroxy-5β-carda-8,14,16,20(22)-tetraenolide, 8β-hydroxy-digitoxigenin, Δ16-8β-hydroxy-digitoxygenin, Δ16-neriagenin, uvaol, ursolic aldehyde, 27(p-coumaroyloxy)ursolic acid, oleanderol ( oleanderol), 16-anhydrous-decyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, adigoside, adinerine, alpha-amirin, beta-sitosterol (beta-sitosterol) ), campestrol, natural rubber (caoutchouc), capric acid, caprylic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl- nerigoside (diacetyl-nerigoside), polyandrin (foliandrin), pseudocuramine (pseudocuramine), quercetin (quercetin), quercetin (qu ercetin)-3-hamnoglucoside, quercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospeside, urea A method comprising at least one compound selected from the group consisting of urehitoxin and uzarigenin. 59. The method of any one of claims 53-58, wherein the daily dose of the extract is up to about 100 μg/day, about 80 μg/day, about 60 μg/day, about 40 μg/day, about 38.4 μg/day. /day or about 30 μg/day. 59. The method of any one of claims 52-58, wherein the daily dose of the extract is at least about 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day. day, or about 5 μg/day. The method of any one of the preceding claims, wherein as a result of the treatment, the concentration of the virus in the blood or plasma of the subject does not decrease or increase. 63. The method of any one of claims 62, wherein said administration is systemic, parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, peroral, oral administration, or a combination thereof. The method of any one of the preceding claims, wherein the one or more doses are administered on a daily, weekly or monthly basis. The method according to any one of the preceding claims, wherein the antiviral composition is administered immediately after infection or within 1 to 5 days after infection or at the earliest time after definitive diagnosis of viral infection. The method according to any one of the preceding claims, wherein the antiviral composition is administered as a primary antiviral therapy, an adjuvant antiviral therapy or a co-antiviral therapy. The method according to any one of the preceding claims, wherein said administering comprises administering said antiviral composition separately or concurrently with at least one other antiviral composition or at least one other composition to treat a symptom associated with said viral infection. How to include. 67. The method of claim 66, wherein said symptom is selected from the group consisting of inflammation, vomiting, nausea, headache, fever, diarrhea, nausea, urticaria, conjunctivitis, malaise, myalgia, arthralgia, seizures and paralysis. A sublingual formulation comprising oleandrine, digoxin, or a combination thereof. 69. The formulation of claim 68, further comprising one or more liquid carriers. An extract comprising oleandrine, wherein the extract is an extract prepared by liquid extraction of Critical Mian of biomass of Nerium species or Thevetia species. 71. The extract of claim 70, wherein said subcritical liquid comprises carbon dioxide and optionally further comprises an alcohol. A method for preparing an extract comprising oleandrine, the method comprising:
extracting the oleandrine-containing biomass with a subcritical liquid carbon dioxide optionally comprising alcohol for a time sufficient to extract oleandrin from the biomass. A method for preparing an extract comprising oleandrine, the method comprising:
extracting the oleandrin-containing biomass with an extraction liquid comprising subcritical liquid carbon dioxide for a time sufficient to extract oleandrin from the biomass;
separating the biomass from the extraction liquid while retaining the extraction liquid; and
removing the extract to form the extract;
How to include. A method for preparing a combination oleandrine-containing combination extract, said method comprising:
subjecting the first biomass containing oleandrin to organic solvent extraction to provide an organic solvent extract containing oleandrin;
subjecting the oleandrine-containing second biomass to subcritical liquid (SbCL) extraction (SbCLE) to provide an oleandrine-containing SbCL extract; and
providing the combined oleandrine-containing extract by combining the oleandrine-containing organic solvent extract and the oleandrine-containing SbCL extract;
How to include. An improved oleandrine-containing composition comprising a portion of an oleandrine-containing organic solvent extract as defined herein and a portion of an oleandrine-containing SbCL extract as defined herein. A method of treating a coronavirus infection, said method comprising:
administering to a subject in need thereof a plurality of doses of oleandrine per day for a treatment period of at least about 5 days to about 1 month, wherein the oleandrine is at least one obtained from an oleandrine containing biomass. A method of presenting as a pharmaceutical composition comprising an extract of 77. The method of claim 76, wherein the total dosage of oleandrine per day is, independently in each occurrence, from about 1 μg to about 180 μg, from about 1 μg to about 120 μg, from about 5 μg to about 100 μg, about 10 μg. how to be chosen. about 80 μg, about 10 μg to about 75 μg, about 15 μg to about 120 μg, about 15 μg to about 100 μg, about 15 μg to about 80 μg, about 30 μg to about 120 μg, about 30 μg to about 100 μg, about 30 μg to about 90 μg, about 40 μg to about 70 μg, about 1 μg, about 5 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, or about 180 μg how to be 78. The method of claim 76 or 77, wherein said multiple doses of oleandrine per day are 2 to 10 times a day, 2 to 8 times a day, 2 to 6 times a day, 2 to 4 times a day, 1 A method that is two doses at a time. 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or 10 times a day. 79. A method according to any one of claims 76 to 78, wherein said pharmaceutical composition comprises at least two extracts obtained from Nerium species. 80. The method of claim 79, wherein the extract is independently selected at each occurrence from the group consisting of a hydrothermal extract, a solvent extract, a supercritical fluid extract, and a subcritical liquid extract. A method of treating a viral infection described herein. A composition as described herein. An antiviral composition as described herein. An oleandrine containing composition as described herein. A method of treating a coronavirus infection, said method comprising:
A method comprising administering to a subject in need thereof a dose of at least one oleandrine once per day for a period of treatment from at least about 5 days to at least about 1 month, wherein said coronavirus infection is pathogenic to a human. 86. The method of claim 85, wherein the coronavirus is selected from SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU20. 86. The method of claim 85, wherein the total dosage of oleandrine per day is, in each occurrence, independently about 1 μg to about 180 μg, about 1 μg to about 120 μg, about 5 μg to about 100 μg, about 10 μg to about 80 μg, about 10 μg to about 75 μg, about 15 μg to about 120 μg, about 15 μg to about 100 μg, about 15 μg to about 80 μg, about 30 μg to about 120 μg, about 30 μg to about 100 μg, about 30 μg to about 90 μg, about 40 μg to about 70 μg, about 1 μg, about 5 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg , about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 140 μg to about 315 μg, about 20 μg to about 750 μg, about 12 μg to about 300 μg, about 12 μg to about 120 μg, about 0.01 μg to about 100 mg, about 0.01 μg to about 100 μg, about 0.5 to about 100 μg, about 1 to about 80 μg, about 1.5 to about 60 μg, about 1.8 to about 60 μg, and about 1.8 to about 40 μg. 86. The method of claim 85, wherein the dosage is about 0.05 0.5 mg/kg/day, about 0.05-0.35 mg/kg/day, about 0.05-0.22 mg, based on the unit amount of oleandrine per kg body weight of the subject per day. /kg/day, about 0.05-0.4 mg/kg/day, about 0.05 0.3 mg/kg/day, about 0.05-0.5 μg/kg/day, about 0.05-0.35 μg/kg/day, about 0.05-0.22 μg/day kg/day, about 0.05-0.4 μg/kg/day, or about 0.05-0.3 μg/kg/day. 86. The method of claim 85, wherein a) the daily dose of oleandrine is up to about 100 μg/day, about 80 μg/day, about 60 μg/day, about 40 μg/day, about 38.4 μg/day, or about 30 μg/day; and/or b) the daily dose of oleandrine is at least about 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day, or about 5 μg/day One way. 86. The method of claim 85, wherein the dosage is administered twice a day or about every 12 hours, and wherein the amount of oleandrine in the dosage is about 0.25 to about 50 μg or about 0.9 to 15 μg. 86. The method of claim 85, wherein the one or more daily doses of oleandrine are administered 2 to 10 times a day, 2 to 8 times a day, 2 to 6 times a day, 2 to 4 times a day. , 2 times a day, 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or A method of dosing 10 times a day. 86. The method of claim 85, wherein the oleandrine is present in a composition comprising at least one extract obtained from an oleandrine-containing biomass. 93. The method of claim 92, wherein the extract is independently selected at each occurrence from the group consisting of a hydrothermal extract, a solvent extract and a supercritical fluid extract. 86. The method of claim 85, wherein after administration of the one or more doses, the plasma concentration of oleandrine in the subject is from about 0.05 to about 2 ng/ml, from about 0.005 to about 10 ng/ml, in an amount of oleandrine per mL of plasma. 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. A method of treating a coronavirus infection comprising administering to a subject having said infection a plurality of therapeutically effective doses of oleandrine or an oleandrine-containing composition. 96. The method of claim 95, wherein said plurality of therapeutically effective doses are one or more doses administered daily for at least two days per week. 97. The method of claim 96, wherein administration continues for at least one week per month. 98. The method of claim 97, wherein administration continues for at least one month per year. 96. The method of claim 95, wherein said coronavirus is pathogenic to humans. 101. The method of claim 99, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU20. Way. 96. The method of claim 95, wherein the oleandrine is present in a composition comprising at least one extract obtained from an oleandrine containing biomass. 102. The method of claim 101, wherein the extract is independently selected at each occurrence from the group consisting of a hydrothermal extract, a solvent extract and a supercritical fluid extract. 102. The method of claim 101, wherein said extract comprises a combination of oleandrine and one or more compounds extracted from said biomass. 104. The method of claim 103, wherein the one or more compounds comprises one or more cardiac glycoside precursors, one or more cardiac glycoside glycoside components, or a combination thereof. 96. The method of claim 95, wherein said administration is systemic, parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, peroral, oral administration, or a combination thereof. 96. The method of claim 95, wherein the antiviral composition is administered immediately after infection or within 1 to 5 days after infection or at the earliest time after diagnosis of viral infection. 96. The method of claim 95, wherein the antiviral composition is administered as a primary antiviral therapy, an adjuvant antiviral therapy or a co-antiviral therapy, or wherein the administration comprises administering the composition in combination with or with at least one other antiviral composition. A method comprising separate administration or co-administration. One or more other compositions for treating symptoms associated with said viral infection. 96. The method of claim 95, wherein the total dosage of oleandrine per day is, independently in each occurrence, from about 1 μg to about 180 μg, from about 1 μg to about 120 μg, from about 5 μg to about 100 μg, about 10 μg to about 80 μg, about 10 μg to about 75 μg, about 15 μg to about 120 μg, about 15 μg to about 100 μg, about 15 μg to about 80 μg, about 30 μg to about 120 μg, about 30 μg to about 100 μg, about 30 μg to about 90 μg, about 40 μg to about 70 μg, about 1 μg, about 5 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg , about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 140 μg to about 315 μg, about 20 μg to about 750 μg, about 12 μg to about 300 μg, about 12 μg to about 120 μg, about 0.01 μg to about 100 mg, about 0.01 μg to about 100 μg, about 0.5 to about 100 μg, about 1 to about 80 μg, about 1.5 to about 60 μg, about 1.8 to about 60 μg, and about 1.8 to about 40 μg. 96. The method of claim 95, wherein the dosage is about 0.05 0.5 mg/kg/day, about 0.05-0.35 mg/kg/day, about 0.05-0.22 mg, based on the unit amount of oleandrine per kg body weight of the subject per day. /kg/day, about 0.05-0.4 mg/kg/day, about 0.05 0.3 mg/kg/day, about 0.05-0.5 μg/kg/day, about 0.05-0.35 μg/kg/day, about 0.05-0.22 μg/day kg/day, about 0.05-0.4 μg/kg/day, or about 0.05-0.3 μg/kg/day. 96. The method of claim 95, wherein a) the daily dose of oleandrine is up to about 100 μg/day, about 80 μg/day, about 60 μg/day, about 40 μg/day, about 38.4 μg/day, or about 30 μg/day; and/or b) the daily dose of oleandrine is at least about 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day, or about 5 μg/day One way. 96. The method of claim 95, wherein the dose is administered twice a day or about every 12 hours, and wherein the amount of oleandrine in the dose is from about 0.25 to about 50 μg or from about 0.9 to 15 μg. 96. The method of claim 95, wherein the one or more daily doses of oleandrine are administered 2 to 10 times a day, 2 to 8 times a day, 2 to 6 times a day, 2 to 4 times a day. , 2 times a day, 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or A method of dosing 10 times a day. 96. The method of claim 95, wherein after administration of said one or more doses, the plasma concentration of oleandrine in said subject is in an amount of oleandrine per mL of plasma, from about 0.05 to about 2 ng/ml, from about 0.005 to about 10 ng/ml. 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. 114. The method of claim 113, wherein the extract contains oleandrine and cardiac glycosides, sugars, non-saccharides, steroids, triterpenes, polysaccharides, saccharides, alkaloids, fats, proteins, neritaloside, odoroside, oleanolic acid, ursolic acid, Betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-ene-3β,28-diol), 28-norurs-12-en-3β-ol, urs-12-en-3β-ol , 3β,3β-hydroxy-12-oleanene-28-oic acid, 3β,20α-dihydroxyurs-21-en-28-oic acid, 3β,27-dihydroxy-12-ursen-28- oic acid, 3β,13β-dihydroxyurus-11-en-28-oic acid, 3β,12α-dihydroxyoleanane-28,13β-olide, 3β,27-dihydroxy-12-oleanane- 28-oic acid, homopolygalacturonan, arabinogalacturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumaroyl-CoA, 3-O-caffeoylquinic acid, 5-O-caffeoyl Quinic acid, cardenolide B-1, cardenolide B-2, oleagenin, neridizinoside, nerizoside, orodoside-H, 3-beta-O-(D-digitinosyl)- 5-beta, consisting of galacturonic acid, rhamnose, arabinose, xylose and galactose, polysaccharides having a MW in the range of 17000-120000 D or about 35000 D, about 3000 D, about 5500 D or about 12000 D Beta-dihydroxy-card-20(22)-enolide pectic polysaccharide, cardenolide monoglycoside, cardenolide N-1, cardenolide N-2, cardenolide N-3 , cardenolide N-4, pregnan, 4,6-diene-3,12,20-trione, 20R-hydroxypregna-4,6-diene-3,12-dione, 16beta,17 Beta-epoxy-12beta-hydroxypregna-4,6-diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A), 20S,21-dihydroxypregna-4,6-diene-3,12-dione (neridione B), neriucoumaric acid (neriu) coumaric acid), isoneriucoumaric acid, oleanderoic acid, oleanderen, 8-alpha-methoxyrapdan-18-oic acid, 12-ursen, cannero side, neriumoside, 3β- O- (D-digitinosyl)-2α-hydroxy-8,14β-epoxy-5β-carda-16:17, 20:22-dienolide, 3β- O- ( D-digitinosyl)-2α,14β-dihydroxy-5β-carda-16:17,20:22-dienolide, 3β,27-dihydroxy-urs-18-en-13,28-olide , 3β,22α,28-trihydroxy-25-nor-lup-1(10),20(29)-dien-2-one, cis -karenine (3β-hydroxy-28-Zp-coumaroyloxy -urs-12-en-27-oic acid), trans -karenine (3-β-hydroxy-28-Ep-coumaroyloxy-urs-12-en-27-oic acid), 3beta-hydroxy -5alpha-carda-14(15),20(22)-dienolide (beta-anhydroepidigitoxigenin, 3beta-O-(D-digitalosyl)-21-hydro Roxy-5beta-carda-8,14,16,20(22)-tetraenolide (nerium mogenin-A-3beta-D-digitaloside), proseragenin, neridienone A, 3beta,27-dihydroxyl-12-ursene-28-oic acid, 3beta,13beta-dihydroxyurus-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-oleanene-28-oic acid, (20S, 24R)-epoxydammaran-3beta,25-diol, 20beta,28-epoxy-28alpha-methoxytaraxasteran-3beta-ol, 20beta , 28-epoxytaraxaster-21-en-3beta-ol, 28-nor-urs-12-ene-3beta,17beta-diol, 3beta-hydroxyurs-12-en-28-aldehyde, Alpha-Neriurate (n eriursate), beta-neriursate, 3 alpha-acetophenoxy-urs-12-en-28-oic acid, 3beta-acetophenoxy-urs-12-en-28-oic acid, oleanderoic acid, Kanerodione, 3β- p -hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursen-28-oic acid, 28-hydroxy-20(29)-lupen-3,7- Dion, kanerocin, 3 alpha-hydroxy-urs-18,20-dien-28-oic acid, D-sarmentose, D-digitinose, neridizinoside, nerizoside, isoricinore isoricinoleic acid, gentiobiosylnerigoside, gentiobiosylbeaumontoside, gentiobiosyloleandrin, polynerin, 12β-hydroxy- 5β-carda-8,14,16,20(22)-tetraenolide, 8β-hydroxy-digitoxigenin, Δ16-8β-hydroxy-digitoxygenin, Δ16- neriagenin, uvaol, ursolic aldehyde, 27(p-coumaroyloxy)ursolic acid, oleanderol, 16-anhydride-deactyl-nerigoside , 9-D-hydroxy-cis-12-octadecanoic acid, adigoside, adinerine, alpha-amirin, beta-sitosterol, campestrol, natural rubber (caoutchouc) , capric acid, caprylic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl-nerigoside, polyandrin ), pseudocuramine, quercetin, quercetin-3-hamnoglucoside (rhamno) glucoside), quercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospeside, urehitoxin and uzarigenin A method comprising at least one compound selected from the group consisting of (uzarigenin).

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