TWI753747B - Method and compositions for treating coronavirus infection - Google Patents

Abstract

The present invention provides a method of treating viral infections such as viral infections caused by coronaviruses of the family Coronaviridae. The present invention provides a composition having at least oleandrin for treating viral infection.

Description

Methods and compositions for treating coronavirus infection

The present invention relates to antiviral compositions and their use in the treatment of Arenaviridae, Bunyaviridae, Flaviviridae, Togaviridae infections in mammals , Paramyxoviridae (Paramyxoviridae) infection, Retroviridae (Retroviridae) infection, Coronaviridae (Coronaviridae) infection or Filoviridae (Filoviridae) infection use. Some embodiments relate to the treatment of hemorrhagic viral infections.

Nerium oleander , a member of the genus Nerium species, is an ornamental plant widely distributed in subtropical Asia, the southwestern United States and the Mediterranean. Its medical and toxicological properties have long been recognized and it has been proposed for the treatment of, for example, hemorrhoids, ulcers, leprosy, snake bites, cancer, tumors, neurological disorders, warts and cell proliferative diseases. Zibbu et al. in the literature (J. Chem. Pharm. Res. (2010), 2(6), 351-358) provide an overview of the chemical and pharmacological activities of oleander.

The extraction of plant components from Oleander species is traditionally performed using boiling water, cold water, supercritical fluids or organic solvents.

ANVIRZEL ^™ (US 5,135,745 to Ozel) contains a concentrated or powdered form of a hot water extract of oleander. Muller et al. ( Pharmazie. (1991) September, 46(9), 657-663) disclosed analytical results on aqueous extracts of oleander. They report that the polysaccharide present is mainly galacturonic acid. Other sugars include rhamnose, arabinose and galactose. Newman et al. (J. Herbal Pharmacotherapy, (2001) vol 1, pp. 1-16) have also reported the polysaccharide content and single saccharide composition of polysaccharides in hot water extracts of oleander. Compositional analysis of hot water extract ANVIRZEL ^™ is described in Newman et al. (Anal. Chem. (2000), 72(15), 3547-3552). US Patent No. 5,869,060 to Selvaraj et al. relates to extracts of Oleander species and methods of production. To prepare the extract, the plant material is placed in water and boiled. The crude extract is then separated from the plant matter and sterilized by filtration. The resulting extract is then lyophilized to produce a powder. US Patent No. 6,565,897 (US Pregrant Publication No. 20020114852 to Selvaraj et al. and PCT International Publication No. WO 2000/016793) discloses a hot water extraction method for preparing a substantially sterile aqueous extract. Ishikawa et al. (J. Nutr. Sci. Vitaminol. (2007), 53, 166-173) disclosed a hot water extract of oleander and its fractions obtained by liquid chromatography with a mixture of chloroform, methanol and water, They also report that oleander leaf extract has been used to treat type II diabetes. US20060188585, published August 24, 2006 by Panyosan, discloses a hot water extract of oleander. US 10323055 issued by Smothers on June 18, 2019 discloses a method for extracting plant material with aloe vera and water to provide an extract containing aloe vera and cardiac glycosides. US20070154573 published July 5, 2007 by Rashan et al. discloses cold water extracts of oleander and uses thereof.

Erdemoglu et al. (J. Ethnopharmacol. (2003) Nov, 89(1), 123-129) disclosed comparative results of aqueous and ethanolic extracts of plants including oleander based on plant analgesic and anti-inflammatory activity. Fartyal et al. (J. Sci. Innov. Res. (2014), 3(4), 426-432) disclosed the comparative results of the antibacterial activities of oleander-based methanol, water and petroleum ether extracts.

Adome et al (Afr. Health Sci. (2003) Aug, 3(2), 77-86; ethanolic extract), el-Shazly et al (J. Egypt Soc. Parasitol. (1996) Aug, 26 (2), 461-473; ethanolic extract), Begum et al. (Phytochemistry (1999) February, 50(3), 435-438; methanolic extract), Zia et al. (J. Ethnolpharmacol. (1995) ten Jan, 49(1), 33-39; methanol extract) and Vlasenko et al. (Farmatsiia. (1972) Sep-Oct, 21(5), 46-47; alcohol extract) also revealed that oleander Organic solvent extract. Turkmen et al. (J. Planar Chroma. (2013), 26(3), 279-283) disclosed aqueous ethanolic extracts of oleander leaves and stems. US 3833472, issued by Yamauchi on September 3, 1974, discloses the extraction of safflower oleander SOL (European oleander) leaves with water, an organic solvent, or an aqueous organic solvent, wherein the leaves are heated to 60°-170° C., and then the organic solvent is methanol, Extraction with ethanol, propyl ether or chloroform.

Supercritical fluid extracts of Oleander species are known (US 8394434, US 8187644, US 7402325) and have been used in the treatment of neurological disorders (US 8481086, US 9220778, US 9358293, US 20160243143A1, US 9877979, US 10383886), Its efficacy has been shown in cell proliferative disorders (US 8367363, US 9494589, US 9846156) and some viral infections (US 10596186, WO 2018053123A1, WO2019055119A1).

Triterpenes are known to have various therapeutic activities. Some of the known triterpenes include oleanolic acid, ursolic acid, betulinic acid, bardoxolone, maslinic acid and others. The therapeutic activity of these triterpenes was primarily assessed individually, rather than in combinations of triterpenes.

Oleanolic acid belongs to a class of triterpenoids represented by compounds such as bardoxolone, which have been shown to be potent activators of the innate cell phase 2 detoxification pathway, where activation of the transcription factor Nrf2 results in antioxidant-containing transcription Increased transcriptional programs of antioxidant genes downstream of response elements (AREs). Bardoxolone itself has been extensively studied in clinical trials in inflammatory conditions; however, the Phase 3 clinical trial for chronic kidney disease was discontinued due to several adverse events. In bed studies, the reason may be related to the known cytotoxicity of some triterpenoids containing bardoxolone at high concentrations.

Compositions containing triterpenes combined with other therapeutic ingredients are found as botanical extracts. Fumiko et al. (Biol. Pharm. Bull (2002), 25(11), 1485-1487) disclosed the evaluation of a methanolic extract of rosemary ( Rosmarimus officinalis L.) for the treatment of trypanosomiasis. Addington et al. (US 8481086, US 9220778, US 9358293, US 20160243143 A1) disclose a supercritical fluid extract of oleander (SCF; PBI-05204) containing oleandrin and triterpenes for the treatment of neurological conditions. Addington et al. (US 9011937, US 20150283191 A1) disclose a triterpenoid-containing fraction (PBI-04711) of a supercritical fluid extract of oleander containing oleandrin and triterpenes for the treatment of neurological disorders. Jäger 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 25 Jul 2016) discloses Betula utilis containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components ) bark extract. Wang et al. (Molecules (2016), 21, 139) disclosed a blackboard tree ( Alstonia scholaris ) extract containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components. Le Silva et al. (Molecules (2012), 17, 12197) disclose Eriope blanchetti extracts containing oleanolic acid, ursolic acid, betulinic acid and a mixture of other components. (Int. J. Mol. Sci. (2012), 13, 7648-7662) discloses Eucaplyptus globulus extract containing oleanolic acid, ursolic acid, betulinic acid and mixtures of other components . Ayatollahi et al. (Iran. J. Pharm. Res. (2011), 10(2), 287-294) disclose Euphorbia microsciadia extracts containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components. Wu et al. (Molecules (2011), 16, 1-15) disclosed an extract of Ligustrum species ( 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 a Forsythia viridissima extract 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 listed in PBI-05204 (PBI-23; supercritical fluid extract of oleander) and PBI-04711 (PBI - The three main triterpene components found in the triterpene-containing fractions 0-4) of 05204. We (two of the inventors) previously reported (Van Kanegan et al., in Nature Scientific Reports (2016)) by comparing the neuroprotective activity of triterpenes at similar concentrations in a brain slice glucose-oxygen deprivation (OGD) model assay. May), 6:25626.doi:10.1038/srep25626) Contribution of triterpenes to the efficacy of neuroprotective activity. We found that PBI-05204 (PBI) and PBI-04711 (fractions 0-4) provided neuroprotective activity.

Extracts of Oleander species are known to contain many disparate classes of compounds: cardiac glycosides, glycosomes, steroids, triterpenes, polysaccharides, and others. Specific compounds include oleandrin; oleandroside; odonoside; oleanolic acid; ursolic acid; betulinic acid; oleandrin; diol) (urs-12-ene-3β, 28-diol); 28-norurs-12-en-3β-ol; ursop-12- En-3β-ol; 3β,3β-hydroxy-12-oleanen-28-acid (3β,3β-hydroxy-12-oleanen-28-oic acid); 3β,20α-dihydroxyurso-21- 3β,20α-dihydroxyurs-21-en-28-oic acid; 3β,27-dihydroxy-12-ursene-28-acid (3β,27-dihydroxy-12-ursen-28 -oic acid); 3β,13β-dihydroxyursop-11-ene-28-acid (3β,13β-dihydroxyurs-11-en-28-oic acid); 3β,12α-dihydroxyoleanane-28 ,13β-lactone (3β,12α-dihydroxyoleanan-28,13β-olide); 3β,27-dihydroxy-12-olean-28-oic acid ); and other components.

Viral hemorrhagic fever (VHF) can be caused by five distinct virus families: Arenaviridae, Buniaviridae, Filoviridae, Flaviviridae, and Paramyxoviridae. Filoviruses such as Ebola virus (EBOV) and Marburg virus (MARV) are among the most pathogenic viruses known to humans and are responsible for outbreaks of viral hemorrhagic fever with a mortality rate of up to 90%. Each virion contains A molecule of antisense single-stranded RNA. Apart from supportive care or symptomatic treatment, there are no commercially available therapeutically effective and prophylactic drugs available for the treatment of EBOV (Ebola virus) and MARV (Marburg virus) infections (ie, filovirus infections). Five Ebola virus species have been identified: Taï Forest (formerly Ivory Coast), Sudan, Zaire, Reston and Bundibugyo.

Antisense single-stranded enveloped RNA viruses ((-)-(ss)-envRNAV) include arenaviridae, bunyaviridae (order Bunyaviridae), filoviridae, orthomyxoviridae, paramyxoviridae Viruses in the Viridae and Rhabdoviridae. Antisense viral RNA is complementary to mRNA and in addition must be converted to sense RNA by RNA polymerase prior to translation; therefore, purified RNA of antisense virus is not inherently infectious because it must be converted to sense RNA for replication. Exemplary viruses and infections from the arenaviridae family include Lyssa virus, aseptic meningitis, Guanarito virus, Junin virus, Lujo virus, Maqiupo virus (Machupo virus), Sabia virus (Sabia virus) and Whitewater River virus (Whitewater Arroyo virus). Exemplary viruses and infections from the Buniaviridae family include hantavirus, Crimean-Congo hemorrhagic fever, and orthonarovirus. Exemplary viruses and infections of the Paramyxoviridae family include mumps virus, Nipah virus, Hendra virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV), and NDV. Exemplary viruses and infections from the Orthomyxoviridae family include influenza virus (A to C), Isavirus, Thogotovirus, Quaranjavirus, H1N1, H2N2, H3N2, H1N2 , Spanish flu, Asian flu, Hong Kong flu, Russian flu. Exemplary viruses and infections of the Rhabdoviridae family include rabies virus, vesicular virus, lyssavirus, intracellular rice yellow dwarf shell virus.

Flaviviruses are positive-sense, single-stranded, enveloped RNA viruses ((+)-(ss)-envRNAV). They are found in arthropods, mainly ticks and mosquitoes, and cause extensive morbidity and mortality worldwide. Some mosquito-borne viruses include yellow fever, dengue, Japanese encephalitis, Western Nile virus and Zika virus. Some tick-borne viral infections include tick-borne encephalitis, Kejeldahl disease, Alkhurma disease, and Omsk hemorrhagic fever. Although not a hemorrhagic infection, Powassan virus is a flavivirus. (+)-(ss)-envRNAV includes Coronaviridae (human and animal pathogens), Flaviviridae (human and animal pathogens), Togaviridae (human and animal pathogens) and Arterviridae family ( animal pathogens).

Coronavirus (CoV) is the generic name of the family Coronaviridae. In humans, respiratory infections caused by CoV are usually mild, but are fatal in rarer forms such as SARS (severe acute respiratory syndrome)-CoV, MERS (Middle East respiratory syndrome coronavirus)-CoV, and COVID-19 . CoVs have helical symmetric nucleocapsids and genome sizes ranging 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: receptor binding, cell fusion, major antigen; E-envelope: small, envelope-associated protein; and M-membrane: transmembrane-budding and envelope form. In a few CoV types, a fourth glycoprotein is present: HE-hemagglutinin esterase. The genome has 5' methylated end caps and 3' polyadenylation tails and functions directly as mRNA. CoVs enter human cells by endocytosis and membrane fusion; and replicate in the cytoplasm of cells. CoV is transmitted by aerosols from respiratory secretions, the fecal-oral route, and mechanical transmission. Most viruses grow in epithelial cells. Occasionally, it can infect the liver, kidney, heart or eye, as well as other cell types such as macrophages. In cold-type respiratory infections, growth appears to be limited to the upper respiratory epithelium. Coronavirus infection is very common and occurs around the world, and its infection rate is highly seasonal, with the highest infection rate in children in winter and less common in adults. The number of coronavirus serotypes and the extent of antigenic variation are unknown. The possibility of reinfection over a lifetime means that there are multiple serotypes (at least four are known) and/or antigenic variations of the coronavirus, so immunity against all serotypes with a single vaccine is extremely unlikely. SARS is a viral pneumonia with symptoms including fever, dry cough, dyspnea (shortness of breath), headache and hypoxemia (low blood oxygen concentration). Typical laboratory findings include lymphopenia (decreased number of lymphocytes) and mildly elevated aminotransferase levels (indicating liver damage). Progressive respiratory failure due to alveolar damage can lead to death. The typical clinical course of SARS involves symptomatic improvement in the first week of infection, followed by worsening in the second week. There is still a great need for therapeutics (compositions and methods) that are effective against human coronaviruses.

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

Oleandrin has shown anti-HIV activity but has not been evaluated against multiple viruses. The triterpenes oleanolic acid, betulinic acid, and ursolic acid have been reported to exhibit varying levels of antiviral activity, but have not been evaluated against multiple viruses. Betulinic acid has shown partial antiviral activity against HSV-1 1C strain, influenza A H7N1, ECHO 6 and HIV-1. Oleanolic acid has shown partial antiviral activity against HIV-1, HEP C and HCV H strain NS5B. Ursolic acid has shown partial 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 active. The antiviral activity of oleandrin, oleanolic acid, ursolic acid and betulinic acid is unpredictable in terms of efficacy against specific viruses. The presence of viruses for which oleandrin, oleanolic acid, ursolic acid and/or betulinic acid has little antiviral activity means that oleandrin, oleanolic acid, ursolic acid and/or betulinic acid cannot be predicted a priori Whether it exhibits antiviral activity against a specific virus genus.

Barrows et al. (“A screen of FDA-approved drugs for inhibitors of Zikavirus infection” in Cell Host Microbe (2016), 20, 259-270) reported that digitonin showed antiviral activity against Zika virus, but the doses were too high High and potentially toxic. 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.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus belonging to the family Retroviridae and the genus Delta retrovirus. It has a sense RNA genome that can be reverse transcribed into DNA and then integrated into cellular DNA. Once integrated, HTLV-1 persists only in a proviral form that spreads from cell to cell via viral synapses. Even when free virions are present, only small amounts are produced, and although the virus is present in genital secretions, there is usually no detectable virus in plasma. HTLV-1 primarily infects CD4 ⁺ T lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL) - a rare but aggressive hematological malignancy that, in addition to certain autoimmune/inflammatory conditions, involves infectious skin Inflammation, rheumatoid arthritis, uveitis, keratoconjunctivitis, Sjögren's syndrome, Sjögren's syndrome, and HAM/TSP, among others, also have high rates of treatment resistance and generally poorer clinical outcomes. The clinical features of HAM/TSP are chronic progressive spastic paresis, urinary incontinence, and mild sensory disturbances. Although ATLL is aetiologically related to viral latency, oncogenic transformation, and selective expansion of HTLV-1-infected cells, inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paresis (HAM/TSP) are caused by Caused by autoimmunity and/or immunopathological response to proviral replication and viral antigen expression. HAM/TSP is a progressive neuroinflammatory disease that causes degeneration and demyelination of the lower spinal cord. HTLV-1-infected circulating T cells invade the central nervous system (CNS) and elicit an immunopathogenic response against the virus and possibly CNS components. Nerve damage and subsequent degeneration can lead to severe disability in HAM/TSP patients. Persistence of proviral replication and proliferation of HTLV-1-infected cells in the CNS leads to cytotoxic T-cell responses against viral antigens, which may be responsible for autoimmune destruction of neural tissue.

Although cardiac glycosides have been shown to exhibit partial antiviral activity against a small number of viruses, specific compounds exhibit very different levels of antiviral activity against different viruses, meaning that when the same disease When evaluating the virus(s), some show very poor antiviral activity and some show good antiviral activity.

There remains a need for improvement in pharmaceutical compositions comprising oleandrin, oleanolic acid, ursolic acid, betulinic acid, or any combination thereof, which are therapeutically active against specific viral infections.

【Prior technical literature】 None

The present invention provides a pharmaceutical composition and method for treating and/or preventing viral infection in mammalian subjects. The present invention also provides a pharmaceutical composition and method for treating viral infections such as viral hemorrhagic fever (VHF) infection in mammalian subjects. The present invention also provides a method of treating a viral infection in a mammal by administering the aforementioned pharmaceutical composition. The present inventors have successfully prepared antiviral compositions that exhibit sufficient antiviral activity to justify their use in the treatment of viral infections in humans and animals. The inventors have developed corresponding methods of treatment with specific dosing regimens. The present invention also provides a prophylactic method of treating a subject at risk of viral infection, the method comprising chronically administering to the subject one or more in a repeated manner over an extended treatment period before the subject becomes infected with the virus A dose of an antiviral composition to prevent a subject from contracting a virus; wherein the aforementioned antiviral composition comprises oleandrin.

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

In some embodiments, the viral infection is caused by any of the following viral families: Arenaviridae, Arterivirus, Buniaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae (especially Deltaretroviridae), Coronaviridae or Togaviridae. In some embodiments, viral infection is caused by (+)-ss-envRNAV or (-)-ss-envRNAV.

Some embodiments of the present invention relate to compositions and methods for treating filovirus, flavivirus, Hennevirus, alphavirus, or togavirus infections. Treatable viral infections include at least Ebola, Marburg, Alpha, Flavivirus, Yellow Fever, Dengue, Japanese Encephalitis, West Nile Virus, Zika Virus, Venezuelan Equine Encephalomyelitis (Encephalitis) (VEE ) virus, Chikungunya virus, Western equine encephalomyelitis (encephalitis) (WEE) virus, Eastern equine encephalomyelitis (encephalitis) (EEE) virus, tick-borne encephalitis, Kejeldahl disease, Alkhurma disease, Om Skunk haemorrhagic fever, Hendra virus, Nipah virus, Delta retrovirus, HTLV-1 virus and its species.

Some embodiments of the present invention relate to compositions and methods for the treatment of viral infections from the following viruses: Arenaviridae, Arteriviridae, Buniaviridae, Filoviridae, Flaviviridae (Flaviridae) genus), Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae (Deltaretroviridae), Coronaviridae, (+)-ss-envRNAV, (-)-ss-envRNAV or Togaviridae.

Some embodiments of the invention relate to viruses for the treatment of viruses from the genera Hennevirus, Ebolavirus, Flavivirus, Marburg virus, Deltaretrovirus, Coronavirus (CoV) or Alphavirus Compositions and methods of infection.

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

In some embodiments, the (+)-ss-envRNAV is a coronavirus 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 selected from the group consisting of Groups of viruses: Flavivirus, Yellow fever virus, Dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, Tick-borne encephalitis virus, Kejelda virus, Alkhurma disease virus, Omsk hemorrhagic fever Viruses and Poisson Viruses.

In some embodiments, (+)-ss-envRNAV is a Togaviridae virus selected from the group consisting of: arborvirus, eastern equine encephalomyelitis virus (EEEV), western equine cerebrospinal Inflammation virus (WEEV), Venezuelan equine encephalomyelitis virus (VEEV), Chikungunya virus (CHIKV), Anion virus (ONNV), Pogosta disease virus, Sindbis virus, Ross River fever virus (RRV) and Semliki Forest virus.

In some embodiments, (-)-ss-envRNAV is a virus selected from the group consisting of Arenaviridae, Buniaviridae (Bunyaviridae), Filoviridae, Orthomyxoviridae, Paramyxoviridae or Rhabdoviridae.

In some embodiments, the arenaviridae virus is selected from the group consisting of: Lysa virus, aseptic meningitis, Guanarito virus, Junin virus, Luyo virus, Machubo virus, Sabia Virus and Whitewater River Virus.

In some embodiments, the Buniaviridae virus is selected from the group consisting of hantavirus, Crimean-Congo hemorrhagic fever, and orthonarovirus.

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

In some embodiments, the Orthomyxoviridae virus is selected from the group consisting of: Influenza Viruses (A to C), Salmon Anemia Virus, Togokato Virus, Quaranza Virus, H1N1 Virus, H2N2 Virus, H3N2 Virus , H1N2 virus, Spanish flu virus, Asian flu virus, Hong Kong flu virus and Russian flu virus.

In some embodiments, the Rhabdoviridae is selected from the group consisting of: Rabies virus, vesicular virus, lyssavirus and intracellular rice yellow dwarf shell virus.

The present invention also provides embodiments for the treatment of HTLV-1 related conditions or neuroinflammatory diseases. In some embodiments, the HTLV-1 related condition or neuroinflammatory disease is selected from the group consisting of: myelopathy/tropical spastic paresis (HAM/TSP), adult T-cell leukemia/lymphoma (ATLL), autologous Immune diseases, inflammatory diseases, infectious dermatitis, rheumatoid arthritis, uveitis, keratoconjunctivitis, Sjögren's syndrome, Sjogren's disease and D. faecalis.

The present invention also provides a method of inhibiting infectious release of HTLV-1 particles into treated cell culture supernatant and reducing HTLV-1 cell-to-cell spread by inhibiting Env-dependent formation of viral synapses, the aforementioned methods comprising An effective amount of the antiviral composition is administered to a subject in need thereof.

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

One aspect of the present invention provides a method of treating a viral infection in a subject by chronically administering to the subject an antiviral composition, by chronically administering to the subject a therapeutically effective amount (a therapeutically relevant dose) of the composition The subject is treated to provide relief of symptoms associated with the viral infection or amelioration of the viral infection. Administration of the antiviral composition can begin immediately after infection, or at any time from 1 day to 5 days after infection, or at the earliest time after viral infection is definitively diagnosed to the subject. The virus may be any virus described in this specification.

Accordingly, the present invention also provides a method of treating a viral infection in a mammal, the aforementioned method comprising administering to the mammal one or more therapeutically effective doses of an antiviral composition. One or more doses are administered daily, weekly or monthly. One or more doses may be administered per day. The virus may be any virus described in this specification.

The present invention also provides a method for treating a viral infection in a subject in need thereof A method comprising: determining whether a subject has a viral infection; instructing administration of an antiviral composition; administering to the subject an initial dose of the antiviral composition for a period of time according to a prescribed initial dosing regimen; periodically determining the subject Appropriateness of 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, continued administration of the desired antiviral composition treatment until desired or if the clinical response and/or therapeutic response of the subject at the initial dose and initial dosing regimen is inappropriate, escalate or decrease the dose until the desired clinical response and/or treatment response.

Continue to administer the desired antiviral composition to the subject for treatment, with dose or dosing regimen adjusted as needed until the patient achieves the desired clinical endpoint(s), such as reduction or alleviation of specific symptoms associated with viral infection . Determination of appropriateness of clinical response and/or therapeutic response can be made by clinicians familiar with viral infections.

The various steps of the method of the present invention can be carried out in separate facilities or in the same facility.

The present invention provides alternative embodiments in which digitonin is used in place of oleandrin, or in which digitonin is used in combination with all embodiments described in this specification. The method of the present invention can employ oleandrin, digitonin or a combination of oleandrin and digitonin. Accordingly, oleandrin, digitonin, compositions containing oleandrin, compositions containing digitonin, or compositions containing oleandrin and digitonin can be used in the method of the present invention. Cardiac glycosides can be considered as oleandrin, digitonin or its combination. The cardiac glycoside-containing composition comprises oleandrin, digitonin, or a combination thereof.

The present invention also provides a method of treating a coronavirus infection, particularly an infection by a coronavirus pathogenic to humans (eg, SARS-CoV-2 infection), the aforementioned method comprising chronic administration to a subject suffering from the aforementioned infection A therapeutically effective dose of cardiac glycosides (compositions containing cardiac glycosides).

The present invention also provides a two-way method of treating a coronavirus infection, particularly a coronavirus infection that is pathogenic in humans, such as SARS-CoV-2 infection, the aforementioned method comprising chronic administration of a treatment to a subject suffering from the aforementioned infection An effective dose of cardiac glycosides (the composition containing cardiac glycosides), thereby inhibiting the viral replication of the aforementioned coronavirus and reducing the infectivity of the descendant viruses of the aforementioned coronavirus.

The present invention also provides a method of treating a coronavirus infection, particularly a SARS-CoV-2 infection, by repeated administration (by any of the modes of administration discussed in this specification) to a subject suffering from the aforementioned infection in a plurality of A therapeutically effective dose of cardiac glycosides (compositions containing cardiac glycosides). One or more doses may be administered daily on one or more days of the week and optionally for one or more weeks of each month and optionally for one or more months of the year.

The present invention also provides a method for treating human coronavirus infection, the method comprising administering to a subject 1-10 doses of cardiac glycosides (a composition containing cardiac glycosides per day for a treatment period of 2 days to about 2 months) ). During the treatment period, 2 to 8, 2 to 6, or 4 doses can be administered per day. Doses can be administered for 2 days to about 60 days, 2 days to about 45 days, 2 days to about 30 days, 2 days to about 21 days, or 2 days to about 14 days. The foregoing administration can be carried out by any of the modes of administration discussed in this specification. Systemic administration is ideal to provide therapeutically effective plasma levels of oleandrin and/or digitonin in the aforementioned subjects.

In some embodiments, one or more doses of oleandrin are administered daily for multiple days until the viral infection is cured. In some embodiments, one or more are administered daily Doses of cardiac glycosides (compositions containing cardiac glycosides) are administered for multiple days and weeks until the viral infection is cured. One or more doses can be administered in a day. One, two, three, four, five, six or more doses can be administered per day.

In some embodiments, the concentration of oleandrin and/or digitonin 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 oleandrin and/or digitoxin in the plasma of a subject treated with a coronavirus infection is about 0.0001 μg/mL or more, about 0.0005 μg/mL or more, about 0.001 μg/mL or more, about 0.0015 μg/mL or more, about 0.01 μg/mL or more, about 0.015 μg/mL or more, about 0.1 μg/mL or more, about 0.15 μg/mL or more, about 0.05 μg/mL or more, or about 0.075 μg/mL or more. In some embodiments, the concentration of oleandrin and/or digitonin in the plasma of the subject treated for infection is from about 10 μg/mL to about 0.0001 μg/mL, from about 5 μg/mL to about 0.0005 μg/mL mL, about 1 μg/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 encompasses all combinations and selections of the plasma concentration ranges described in this specification.

The antiviral composition can be administered chronically, i.e. in a repetitive manner, eg, every day, every other day, every two days, every third day, every fourth day, every five days, every six days, every week, every other week, every two weeks , every three weeks, every month, every two months (bimonthly), every half month, every other month, every two months, every quarter, every other quarter, every three months, seasonally, every Semi-annual and/or annual application. The treatment period is one or more weeks, one or more months, one or more quarters, and/or one or more years. An effective dose of cardiac glycosides (compositions containing cardiac glycosides) is administered one or more times a day.

In some embodiments, the subject is administered cardiac glycosides from 140 μg daily to 315 μg. In some embodiments, the dose comprises 20 μg to 750 μg, 12 μg to 300 μg, or 12 μg to 120 μg of cardiac glycoside. The daily dose of cardiac glycoside may be 20 μg to 750 μg, 0.01 μg to 100 mg, or 0.01 μg to 100 μg of cardiac glycoside per day. The recommended daily dose of oleandrin present in the SCF extract is generally about 0.25 to about 50 μg twice daily, or about 0.9 to 5 μg twice daily or about every 12 hours. Dosages may be about 0.5 to about 100 μg/day, about 1 to about 80 μg/day, about 1.5 to about 60 μg/day, about 1.8 to about 60 μg/day, about 1.8 to about 40 μg/day. The maximum tolerated dose may be 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 of oleandrin-containing oleander extract, and the minimum effective dose may be 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. Suitable dosages containing cardiac glycosides and triterpenes may be about 0.05-0.5 mg/kg/day, about 0.05-0.35 mg/kg/day, about 0.05-0.22 mg/kg/day, about 0.05-0.4 mg/kg/day , about 0.05~0.3mg/kg/day, about 0.05~0.5μg/kg/day, about 0.05~0.35μg/kg/day, about 0.05~0.22μg/kg/day, about 0.05~0.4μg/kg/day , or about 0.05~0.3 μg/kg/day. In some embodiments, the dosage of oleandrin is about 1 mg to about 0.05 mg, about 0.9 mg to about 0.07 mg, about 0.7 mg to about 0.1 mg, about 0.5 mg to about 0.1 mg, about 0.4 mg to about 0.1 mg, About 0.3 mg to about 0.1 mg, about 0.2 mg. The present invention encompasses all combinations of doses recited in this specification.

In some embodiments, the cardiac glycosides are administered in at least two administration phases: an implementation phase and a maintenance phase. The implementation phase continues until approximately steady-state plasma levels of cardiac glycosides are reached. The maintenance phase begins approximately after the initiation of treatment or the implementation phase. Dose titration can occur during the implementation phase and/or the maintenance phase.

All dosing regimens, dosing schedules and dosages described in this specification are considered appropriate; however, some dosing regimens, dosing schedules and dosages are more suitable for some subjects than others. Targeted clinical indicators are used as the basis for the aforementioned dosing.

The aforementioned antiviral compositions can be administered systemically. Systemic administration includes non-toxic Translucent, buccal, enteral, intramuscular, subcutaneous, sublingual, oral, pulmonary or oral. The aforementioned compositions can also be administered by injection or intravenously. The compositions can 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 modes of administration selected from the group consisting of parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual , Oral, pulmonary and oral.

The present invention also provides a sublingual dosage form comprising oleandrin and a liquid carrier. The present invention also provides a method of treating a viral infection, particularly a coronavirus infection (such as defined herein), the aforementioned method comprising sublingually administering to a subject suffering from the aforementioned viral infection a plurality of oleandrin-containing Dosage of the retinoside) composition. One or more doses may be administered per day on two or more days per week and in one or more weeks per month, optionally in one or more months per year.

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

If present in the antiviral composition, the additional cardiac glycosides may further comprise: odonoside, oleandroside or oleandrin. In some embodiments, the composition further comprises: a) one or more triterpenes; b) one or more steroids; c) one or more triterpenoid derivatives; 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 triterpenoid derivatives; c) two or three triterpenoid salts; or d) a combination thereof. In some embodiments, the triterpenes are selected from the group consisting of oleanolic acid, ursolic acid, betulinic acid, and salts or derivatives thereof.

Some embodiments of the present invention include such pharmaceutical compositions 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 triterpenoid; b) at least one cardiac glycoside and at least two triterpenes; c) at least one cardiac glycoside and at least three triterpenes; d) at least two triterpenes and no cardiac glycosides; e) at least three triterpenes and no cardiac glycosides; or f) at least one cardiac glycoside, eg, oleandrin, digitonin. As used in this specification, unless otherwise indicated, the generic terms triterpenes and cardiac glycosides also include salts and derivatives thereof.

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

In some embodiments, the oleandrin-containing extract is obtained by extraction of plant material. The extract may comprise a hot water extract, a cold water extract, a supercritical fluid (SCF) extract, a subcritical fluid extract, an organic solvent extract, or a combination thereof, of the plant material. In some embodiments, the extract (biomass) has been prepared by extracting oleander material (biomass) using an extraction fluid optionally containing alcohols or a subcritical fluid carbon dioxide. In some embodiments, the oleandrin-containing composition comprises two or more distinct types of oleandrin-containing extracts.

Embodiments of the present invention include Nerium sp. biomass (plant material) in which oleandrin is contained: Nerium oleander ( Nerium oleander L) ( Apocynaceae ), Nerium odorum, White oleander , Pink oleander, Thevetia sp., Thevetia peruviana , Yellow oleander, Thevetia nerifolia , Agrobacterium tumefaciens , a cell culture (cell mass) of any of the foregoing species ) or a combination thereof. In some embodiments, the biomass comprises leaves, stems, flowers, bark, fruits, seeds, sap, and/or pods.

In some embodiments, the extract contains at least one other pharmaceutically active agent obtained with the cardiac glycoside upon extraction, which contributes to the therapeutic efficacy of the cardiac glycoside when the extract is administered to a subject. In some embodiments, the composition further comprises one or more other non-cardiac glycoside therapeutically effective agents, ie, one or more agents that are not cardiac glycosides. In some embodiments, the composition further comprises one or more antiviral compounds. In some embodiments, the antiviral composition does not comprise a pharmaceutically active polysaccharide.

In some embodiments, the extract comprises one or more cardiac glycosides and one or more cardiac glycoside precursors (eg, cardiac steroids, cardadienolides, and cardatrienolides, all of which are cardiac glycosides) aglycone components, for example, digoxigenin, acetyl digoxigenin, digitoxin, digitonin, acetyl digitonin, digitonin Aglycone, digitonin, sauerkraut, magmaside, ouabain, or sauerkraut aglycin). The extract may further comprise one or more glycone components of cardiac glycosides (eg, glucoside, fructoside and/or glucuronide) as cardiac glycoside precursors. Accordingly, the antiviral composition may comprise one or more cardiac glycosides and two or more cardiac glycoside precursors selected from the group consisting of one or more aglycone components and one or more glycosome components. The extract may also contain one or more other non-cardiotropic therapeutically effective agents obtained from plant material of Oleander spp. or Oleander spp.

In some embodiments, compositions containing oleandrin (OL), oleanolic acid (OA), ursolic acid (UA), and betulinic acid (BA) are more effective than pure oleander when comparing equivalent doses based on oleandrin content glycosides are more effective.

In some embodiments, the molar ratio of total triterpene content (OA+UA+BA) to oleandrin ranges from about 15:1 to about 5:1; or about 12:1 to about 8:1; or about 100:1 to about 15:1; or about 100:1 to about 50:1; or about 100:1 to about 75:1; or about 100:1 to about 80:1; or about 100:1 to about 90:1; or about 10:1.

In some embodiments, the molar ratio of the single triterpenes to oleandrin 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, other therapeutic agents, such as those obtained by extraction of plant material of Oleander spp. or Oleander spp., are not polysaccharides from which the extracts are prepared, meaning they are not acidic homogalactose homopolygalacturonan or arabinogalaturonan. In some embodiments, the extract contains no other therapeutic agents and/or no homogalacturonic acid or arabinogalacturonic acid from which the extract was prepared.

In some embodiments, other therapeutic agents, such as those obtained by extraction of plant material of Oleander spp. or Oleander spp. are polysaccharides obtained during the preparation of the extract, such as acidic homogalacturonic acid or arabinogalacturonic acid. In some embodiments, the extract comprises another therapeutic agent and/or comprises acidic homogalacturonic acid or arabinogalacturonic acid obtained during preparation of the extract from the aforementioned plant material.

In some embodiments, the extract comprises oleandrin and at least one other compound selected from the group consisting of cardiac glycosides, glycosides, aglycones, steroids, triterpenes, polysaccharides, carbohydrates, alkaloids, fats , protein, oleandroside, odonoside, oleanolic acid, ursolic acid, betulinic acid, oleandrin, oleandrin A, betulin (urso-12-ene-3β,28-diol) , 28-norursop-12-en-3β-ol, ursop-12-en-3β-ol, 3β,3β-hydroxy-12-oleanene-28-acid, 3β,20α-dihydroxy Ursop-21-en-28-acid, 3β,27-dihydroxy-12-ursene-28-acid, 3β,13β-dihydroxyursop-11-en-28-acid, 3β,12α-dihydroxy Hydroxyoleanane-28,13β-lactone, 3β,27-dihydroxy-12-olean-28-acid, homogalacturonic acid, arabinogalacturonic acid, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumarin coenzyme A, 3-O-caffeoquinic acid, 5-O-caffeoquinic acid, cardiac glycoside B-1, cardiac glycoside B-2, oleandrin oleagenin, neridiginoside, nerizoside, odonoside-H, 3-beta- galacturonic acid, rhamnose, arabinose, xylose and galactose O-(D-geigenoside)-5-β,14β-dihydroxy-cardiosta-20(22)-enolactone pectin polysaccharide, MW in the range of 17000~120000D or MW about 35000D, about 3000D , about 5500D or about 12000D polysaccharide, cardiac glycoside monoglycoside (cardenolide monoglycoside), cardiac glycoside N-1, cardiac glycoside N-2, cardiac glycoside N-3, cardiac glycoside N-4, pregnane, 4,6-di ene-3,12,20-trione, 20R-hydroxypregn-4,6-diene-3,12-dione, 16β,17β-epoxy-12β-hydroxypregn-4,6-diene -3,20-Dione, 12β-Hydroxypregn-4,6,16-triene-3,20-dione (Oildienone A), 20S,21-dihydroxypregn-4,6 - Diene-3,12-dione (Oildienone B), neriucoumaric acid, isoneriucoumaric acid, oleanderoic acid, oleanderen , 8α-Methoxy Helicobacter-18-Acid, 12-Usene, Mannoside (kaneroside), Neriumoside, 3β-O-(D-Geoside)-2α-Hydroxy-8,14β-Epoxy -5β-Cardiosta-16:17,20:22-Dienolide, 3β-O-(D-Digenoside) -2α,14β-Dihydroxy-5β-cardiosta-16:17,20:22-dienolide, 3β,27-dihydroxy-urso-18-ene-13,28-lactone, 3β,22α ,28-trihydroxy-25-nor-lupine-1(10),20(29)-dien-2-one, cis -karenin(3β-hydroxy-28-Z-p-coumarin) Ethyloxy-ursop-12-en-27-acid), trans -Carinine (3-β-hydroxy-28-E-p-coumaroloxy-ursop-12-en-27-acid) , 3β-hydroxy-5α-cardiosta-14(15), 20(22)-dienolide (β-anhydrousaglycin), 3β-O-(D-digoxigenin)-21-hydroxy -5β-cardiosta-8,14,16,20(22)-tetraenolide (neriumogenin-A-3β-D-digitaloside), bovine horn Citrullin (proceragenin), Ouerdienone A, 3β,27-dihydroxy-12-ursene-28-acid, 3β,13β-dihydroxyursop-11-en-28-acid, 3β- Hydroxyursop-12-en-28-al, 28-methylursop-12-en-3β-ol, ursop-12-en-3β-ol, ursop-12-en-3β,28-di Alcohol, 3β,27-dihydroxy-12-oleanene-28-acid, (20S,24R)-epoxidamarane-3β,25-diol, 20β,28-epoxy-28α-methan Oxydandelion st-3β-ol, 20β,28-epoxydandelion st-21-en-3β-ol, 28-nor-urso-12-ene-3β,17β-diol, 3β-hydroxyuranyl So-12-en-28-aldehyde, α-neriursate, β-neriursate, 3α-acetylphenoxy-ursop-12-en-28-acid, oleandroic acid, kanerodione, 3β- p-Hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursene-28-acid, 28-hydroxy-20(29)-lupine-3,7-dione, kanerocin, 3α-hydroxy - Ursol-18,20-dien-28-acid, D-salmonose, D-digestinose, ganglioside, neridin, isoricinoleic acid, gentiostanoside (gentiobioside neuron gentioside), gentiobiosylbeaumontoside, gentiobiosyloleandrin, folinerin, 12β-hydroxy-5β-carda-8,14,16,20(22 )-tetraenolactone, 8β-hydroxy-digoxigenin, △16-8β-hydroxy-digoxigenin, △16-neriagenin, black Fanol, ursaldehyde, 27(p-coumaryloxy) ursolic acid, oleander alcohol, 16-anhydro-deacetyl-ganglioside, 9-D-hydroxy-cis-12-octadecanoic acid, adipic acid Adigoside (adigoside), oleandrin B, α-scentrin, β-sitosterol, campesterol, rubber (caoutchouc), capric acid, caprylic acid, choline, cornerin, cortenerin, deacetylene oleandrin C, Diacetyl-ganglioside, oleandrin C, pseudocuramine, quercetin, quercetin-3-rhamnoglucoside, quercetin, rosaginin, rutin, stearic acid, Stigmasterol, digitalis glycosides, urehitoxin and usa aglycone. Additional components present in the extract are disclosed by 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 extracts derived from suspension cultures of Agrobacterium tumefaciens transformed callus. Hot water, organic solvents, aqueous organic solvents or supercritical fluid extracts of Agrobacterium can be used according to the invention.

Oleandrin can also be obtained from extracts of oleander in vitro microcultures, thereby Stem section culture can be initiated from seedlings and/or shoot tips of oleander species such as Splendens Giganteum, Revanche or Alsace or others. According to the present invention, hot water, organic solvent, aqueous organic solvent or supercritical fluid extract of micro-cultured oleander can be used.

Extracts may also be obtained by extraction of cell mass (eg, present in cell culture) of any of the aforementioned plant species.

The present invention also provides the use of cardiac glycosides in the preparation of a medicament for treating viral infection in a subject. In some embodiments, the aforementioned medicaments are prepared comprising: providing one or more antiviral compounds of the invention; comprising a dose of the antiviral compound(s) in a pharmaceutical dosage form; and packaging the pharmaceutical dosage form. In some embodiments, the preparation can be performed as described in PCT International Application No. PCT/US06/29061. The preparation may also include one or more additional steps, such as: delivering the packaged dosage form to suppliers (retailers, wholesalers and/or distributors); selling or otherwise providing the packaged dosage form to subjects suffering from a viral infection ; contains drug labels and package inserts that provide instructions for use of the dosage form, dosing schedule, administration, content, and toxicological profile. In some embodiments, treatment of a viral infection comprises: determining that the subject has a viral infection; administering to the subject a pharmaceutical dosage form according to the dosing regimen; administering to the subject one or more pharmaceutical dosage forms, wherein according to The dosing regimen administers one or more of the aforementioned pharmaceutical dosage forms.

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

The solubilizer is at least a single surfactant, but it can also be a combination of materials, such as a combination of: a) surfactant and water-miscible solvent; b) surfactant and water-immiscible solvent; c) surface Active agents, antioxidants; d) surfactants, antioxidants, and water-immiscible solvents; e) surfactants, antioxidants, and water-immiscible solvents; f) surfactants, water-miscible solvents, and water-immiscible solvents Solvents; or g) surfactants, antioxidants, water-miscible solvents and water Immiscible solvent.

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

In some embodiments, the water-miscible solvent is a low molecular weight (less than 6000) PEG, ethylene glycol, or ethanol. In some embodiments, the surfactant is a pegylated surfactant, meaning that the surfactant contains poly(ethylene glycol) functional groups.

The invention includes all combinations of aspects, embodiments, and sub-embodiments of the invention disclosed in this specification.

The following drawings form a part of this specification and describe exemplary embodiments of the claimed invention. From these drawings and the description of this specification, one of ordinary skill in the art can implement the present invention without undue experimentation.

Figures 1-2 are graphs depicting summarizing the in vitro dose-response antiviral activity of various compositions against Ebola virus.

Figures 3-4 are graphs depicting summarizing the in vitro dose-response antiviral activity of various compositions against Marburg virus.

Figure 5 is a graph depicting a summary of the in vitro dose-response antiviral activity of oleandrin against Zika virus (SIKV PRVABC59 strain) in Vero E6 cells.

Figure 6 is a graph depicting summarizing the in vitro dose-response antiviral activity of digitonin against Zika virus (SIKV PRVABC59 strain) in Vero E6 cells.

Figure 7 is a graph depicting a summary of the in vitro dose-response antiviral activity of various compositions (oleandrin, digitonin, and PBI-05204) against Ebola virus in Vero E6 cells surface.

Figure 8 is a graph depicting a summary of the in vitro dose-response antiviral activity of various compositions (oleandrin, digitonin, and PBI-05204) against Marburg virus in Vero E6 cells.

Figure 9 is a graph depicting a summary of the in vitro cell viability of Vero E6 cells in the presence of various compositions (oleandrin, digitonin, and PBI-05204).

Figures 10A and 10B are graphs depicting summarizing the ability of the compositions (oleandrin and PBI-05204) to inhibit Ebola virus in Vero E6 cells shortly after exposure to the virus: Figure 10A - 2 hours post infection; Figure 10B - post infection 24 hours.

Figures 11A and 11B are graphs depicting summarizing the ability of the compositions (oleandrin and PBI-05204) to inhibit Marburg virus in Vero E6 cells shortly after exposure to the virus: Figure 11A - 2 hours post infection; Figure 11B - post infection 24 hours.

Figures 12A and 12B are graphs depicting the ability of the summary compositions (oleandrin and PBI-05204) to inhibit infectious progeny products by virus-infected Vero E6 cells that had been exposed to oleandrin: Figure 12A - Ebola virus; Figure 12B - Marburg virus.

Figures 13A and 13B are depictions summarizing the effect of various compositions (oleandrin, digitoxin, and PBI-05204) on Venezuelan equine encephalomyelitis virus (Figure 13A) and western equine encephalomyelitis virus (Figure 13B) in Vero E6 cells ) of the in vitro dose-response antiviral activity graph.

Figure 14 is a graph summarizing the effects of vehicle control, oleandrin or extracts of oleander on HTLV-1 replication or the release of newly synthesized virus particles as determined by quantification of HTLV-1 p19 ^Gag (see Examples). Examples 19 and 20). Untreated (UT) cells are shown for comparison. Any data is representative of at least three independent experiments. Data represent mean ± standard deviation of experiments (error bars).

Figure 15 is a graph depicting a summary of the relative cytotoxicity of vehicle control, oleandrin, and oleander extracts against the HTLV-1+SLB1 lymphoma T cell line. any data generation Table at least three independent experiments. Data represent mean ± standard deviation of experiments (error bars).

Figures 16A-16F are representative micrographs depicting the results of phospholipid binding protein V-FITC (green) and PI (red) staining shown by DIC phase contrast in merged images. Separate phospholipid binding protein V-FITC and PI fluorescence channel images are also provided. Scale bar, 20 μm.

Figure 17 is a graph depicting a summary of the effects of vehicle control, oleandrin or extracts of European oleander on HTLV-1 replication or release of newly synthesized viral particles from oleandrin-treated HTLV-1+ lymphoma T cells.

Figure 18 is a graph depicting a summary of the relative cytotoxicity of vehicle control, oleandrin or oleander extract on treated huPBMCs.

Figure 19 is a graph depicting a summary of the relative inhibition of HTLV-1 transmission in a huPBMC co-culture assay with a vehicle control, oleandrin-European oleander extract containing huPBMC.

Figure 20 is a representative micrograph depicting the GFP-expressing HTLV-1+SLB1 T cell line: fluorescence microscopy (upper panel) and immunoblotting (lower panel).

Figure 21 is a representative microscopic image depicting viral synapses between huPBMC and mitomycin C-treated HTLV-1+SLB1/pLenti-GFP lymphoblasts (green cells).

FIG. 22 is a graph of mean data with standard error (error bars) depicting quantification of the microscopic images from FIG. 21 .

Figures 23A-23D depict 24 hours and 48 hours after "treatment" for VERO E6 cells infected with SARS-CoV-2 virus and treated with oleandrin (red bars) or control vehicle (medium) (black bars). Graph of log SARS-CoV-2 viral titer (PFU/mL) versus time (hours) (Example 28). Cells were pretreated with oleandrin prior to infection. After the initial 2-hour incubation post-infection, the infected cells were washed to remove extracellular virus and oleandrin. Next, the recovered infected cells were treated as described below. Infected cells were treated with oleandrin (FIG. 23A: 1 μg/mL as aqueous component in 0.1% DMSO in water with RPMI 1640 medium; FIG. 23C: 0.1 μg/mL in 0.01% DMSO in water with RPMI 1640) or Control vehicle only (FIG. 23B: 0.1% DMSO in water with RPMI 1640; FIG. 23D: 0.01% DMSO in water with RPMI 1640), and virus titers were determined.

Figure 24A is a graph depicting viral replication (Y1, left axis) and Vero-E6 cell counts (Y2, right axis: expression of the potential cytotoxicity of oleandrin against the aforementioned cells) in culture medium at 24 hours post infection versus oleandrin concentration Dual y-axis plot of percent inhibition (μg/mL) (Example 29). Figure 24B is the culture used in Figure 24A, but obtained 48 hours post infection.

Figure 25 is a graph depicting the percentage of Vero-E6 cells (cellular titers) in medium relative to the concentration of oleandrin (μg/mL) 24 hours after the cells were continuously exposed to the indicated concentrations of oleandrin (Example 30) .

Figures 26A-26B depict 24 hours (Figure 26A) and 48 hours (Figure 26B) after "treatment" for infection with SARS-CoV-2 virus followed by oleandrin (blue circles) or control vehicle (medium) (red squares) VERO CCL-81 cells (halophilic limpet kidney normal cells; https://www.atcc.org/products/all/CCL-81.aspx) treated with SARS-CoV- 2 Graph of log viral titer (PFU/mL) versus oleandrin concentration (Example 31).

For the samples of Figures 26A and 26B, the fold reduction in viral titer was determined at 24 hours (Figure 26C) and 48 hours (Figure 26D).

Figures 27A-27D depict 24 hours and 48 hours after "treatment" for VERO CCL-24 and 48 hours after infection with SARS-CoV-2 virus and treated with oleandrin (blue circles) or control vehicle (medium) (red squares). 81 cells, graph of log SARS-CoV-2 viral titer (PFU/mL) versus time (hours) (Example 28). Pretreatment of fines with oleandrin prior to infection cell. After the initial 2-hour incubation post-infection, the infected cells were washed to remove extracellular virus and oleandrin. Next, the recovered infected cells were processed as follows. Infected cells were treated with oleandrin ( FIG. 27A : 0.05 μg/mL in DMSO (0.005%) in water with RPMI 1640 medium as the aqueous component; FIG. 27B : 0.1 in DMSO (0.01%) in water with RPMI 1640 μg/mL); FIG. 27C: 0.5 μg/mL in DMSO (0.05%) in water with RPMI 1640; FIG. 27D: 1 μg/mL in DMSO (0.1%) in water with RPMI 1640) and assay for virus Power price.

Figures 28A and 28B are graphs depicting "treatment" at 24 hours (Figure 28A) and 48 hours (Figure 28B) for infection with SARS-CoV-2 virus, followed by oleandrin (dark blue circles (Example 2) and VERO CCL-81 cells treated with light blue circles (Example 3)) or control vehicle (medium) (dark red squares (Example 2) and light red squares (Example 3)), SARS- Graph of the logarithm of CoV-2 viral titer (PFU/mL) versus the concentration of oleandrin. Examples 2 and 3 are merely repetitions of this assay.

Figures 29A and 29B are bar graphs depicting viral titers relative to oleandrin concentration in culture medium where viral titers were determined at 24 hours (Figure 29A) and 48 hours (Figure 29B) post-infection. For some samples, cells were treated with oleandrin (blue solid bars) or DMSO-only control vehicle (red solid bars) before and after infection (2 hours). For other samples, oleandrin (dotted blue line: 12 hours post-infection; open blue bars: 24 hours post-infection) or DMSO-only control vehicle (dashed red line: 12 hours post-infection; open red bars: infection 24 hours later) to treat the cells.

Figures 30A and 30B are graphs depicting the anti-COVID-19 activity used to evaluate the dual extract composition composition (PBI-A). In Figure 30B, the text description of μg/ml (oleandrin concentration) means that PBI-A was provided as a 1 μg/ml (oleandrin concentration) solution. Viral titers (Log ₁₀ (PFU/mL)) were determined relative to the Log ₁₀ dilution index (Figure 30A) or relative to the Log ₁₀ concentration of oleandrin (Figure 30B). FIG. 30A is for the data processing of the pre-infection assay of Example 31, and FIG. 30B is for the processing of the post-infection assay of Example 34.

The present invention provides treatment in a subject by chronically or acutely administering to the subject one or more effective doses of an antiviral composition (or a pharmaceutical composition comprising an antiviral composition and at least one pharmaceutical excipient). Methods of viral infection. The composition is administered according to the dosing regimen most suitable for the subject, and the suitability of the clinical dosage and dosing regimen is determined according to routine clinical trials and clinical treatment endpoints of viral infections.

As used herein, the term "subject" means warm-blooded animals such as mammals, eg, cats, dogs, mice, guinea pigs, horses, cattle, sheep, and humans.

As used in this specification, subjects at risk of viral infection are: a) Mosquitoes living in particular Aedes species ( Aedes egypti , Aedes albopictus ), etc. Subjects living in a geographic area; b) Subjects living with or near a person or group of people with a viral infection; c) Subjects in a sexual relationship with a person with a viral infection; d) Living in In particular, subjects of the genus Ixodes species (species: Ixodes marx , Ixodes scapularis or Ixodes cooke ) in geographic areas where ticks live; e) living in Subjects in geographic areas where fruit bats live; f) subjects living in tropical regions; g) subjects living in Africa; h) subjects exposed to bodily fluids of other subjects with viral infections; i) Children; or j) Subjects with a weakened immune system. In some embodiments, the subject is a female, a capable female, or a pregnant female.

Subjects treated in accordance with the present invention will exhibit a therapeutic response. "Therapeutic response" means that a subject suffering from viral infection will enjoy at least one of the following clinical benefits as a result of treatment with cardiac glycosides: a reduction in the active viral titer in the subject's blood or plasma, the subject's blood or Eradication of active virus in plasma, amelioration of infection, reduction in the occurrence of symptoms associated with infection, partial or complete remission of infection or increased time to infection progression, and/or reduction in the infectivity of the viruses responsible for the aforementioned viral infections. The therapeutic response can be a total or partial therapeutic response.

As used in this specification, "time to progression" is the period, length, or duration of time after diagnosis (or treatment) of a viral infection until the infection begins to worsen. The term refers to the period when the infection level is flat without further deterioration, and that period ends when the infection begins to progress again. Disease progression is determined by "staging" infected subjects before or at the start of treatment. For example, the health of the subject is determined before or at the start of treatment. The subject is then treated with an antiviral composition and the viral titer is monitored periodically. At a later point in time, symptoms of the infection may worsen, thereby marking the progression of the infection and the end of the "time to progression". The period of time during which the infection does not progress or the level or severity of the infection does not worsen is the "progression time".

The dosing regimen comprises a therapeutically relevant dose (or effective dose) of one or more cardiac glycosides and/or triterpenoid(s) administered according to the dosing schedule. Thus, a therapeutically relevant dose is the therapeutic response observed with administration of an antiviral composition to treat a viral infection, and a therapeutic dose of the antiviral composition can be administered to a subject without excess unwanted, or deleterious side effects. The therapeutically relevant dose is non-lethal to the subject, although it may cause some side effects in the patient. The level of clinical benefit to a subject administered the antiviral composition from administration of a therapeutically relevant dose will outweigh the level of adverse side effects experienced by the subject as a result of administration of the antiviral composition or its component(s). Treatment-relevant doses will vary in different subjects according to various established pharmacological, pharmacodynamic, and pharmacokinetic principles. However, therapeutically relevant doses (eg relative to oleandrin) are typically about 25 μg, about 100 μg, about 250 μg, about 500 μg or about 750 μg of cardiac glycosides per day, or it may be in the range of about 25-750 μg of cardiac glycosides per dose, Alternatively, there may be no more than about 25 μg, about 100 μg, about 250 μg, about 500 μg, or about 750 μg of cardiac glycosides per day. Another example of a therapeutically relevant dose (eg, relative to the triterpenes alone or together) is typically about 0.1 μg to 100 μg, about 0.1 mg to about 500 mg, about 100 to about 1000 mg per kg body weight, about 15 to about 25 mg/kg, about 25 to about 50 mg/kg, about 50 to about 100 mg/kg, about 100 to about 200 mg/kg, about 200 to A range of about 500 mg/kg, about 10 to about 750 mg/kg, about 16 to about 640 mg/kg, about 15 to about 750 mg/kg, about 15 to about 700 mg/kg, or about 15 to about 650 mg/kg of body weight. From the basic principles of pharmacy, it is known in the art that the actual amount of an antiviral composition required to provide a target therapeutic outcome in a subject will vary from subject to subject.

Digoxigenin treatment can be performed using two or more administration phases: an implementation phase and a maintenance phase. The dosing regimen described below may be used in the implementation phase until steady-state plasma levels of digoxigenin are achieved, and after the implementation phase is completed, the maintenance phase may be used in the following dosing regimen.

A therapeutically relevant dose can be administered according to any dosing regimen commonly used in the treatment of viral infections. A therapeutically relevant dose can be administered once, twice, three times or more per day. It can be every other day, every three days, every four days, every five days, every half week, every week, every two weeks, every three weeks, every four weeks, every month, every two months (bimonthly), every half month, every Administer every three months, every four months, every six months, every year, or according to any combination of the above to achieve a suitable dosing schedule. For example, therapeutically relevant doses may be administered one or more times per day (up to 10 times per day or more) for one or more weeks to the highest dose).

Example 15 provides a detailed description of the in vitro assays used to evaluate oleandrin (as the only active), Anvirzel ^™ (hot water extract of oleander) and PBI-05204 (supercritical fluid (SCF) extraction of oleander) Ebola virus (Figure 1~2) and Marburg virus (Figure 3~4) infection treatment efficacy of the composition), Ebola virus and Marburg virus are both filoviruses.

The hot water extract can be administered orally, sublingually, subcutaneously and intramuscularly. One embodiment is available as a liquid dosage form under the tradename ANVIRZEL ^™ (Nerium Biotechnology, Inc., San Antonio, TX; Salud Integral Medical Clinic, Tegucigalpa, Honduras; www.saludintegral.com; www.anvirzel.com). For sublingual administration, a typical dosing regimen is 1.5 mL per day, or 0.5 mL doses three times a day. For administration by injection, a typical dosing regimen is about 1 to about ² mL/day; or about 0.1 to about 0.4 ml/m2/day for about 1 week to about 6 months or longer; or about 0.4 to about 0.8 ml / ^m2 /day for about 1 week to about 6 months or longer; or about 0.8 to about 1.2 ml/ ^m2 /day for about 1 week to about 6 months or longer. Because the maximum tolerated dose of ANVIRZEL ^™ is higher, higher doses may be used. ANVIRZEL ^™ contains oleandrin, oleandrin, polysaccharide extracted from oleander (hot water extraction). A commercially available vial contains about 150 mg of oleander extract as a lyophilized powder (before reconstitution with water prior to administration) containing about 200 to about 900 μg of oleandrin, about 500 to about 700 μg of oleandrin extracted from oleander, and polysaccharides. The aforementioned vials may also contain pharmaceutical excipients such as at least one osmotic agent, such as mannitol, sodium chloride, at least one buffer, such as sodium ascorbate and ascorbic acid, at least one preservative, such as propylparaben, paraben Methyl formate.

Experiments were set up by adding the composition to cells at 40 μg/mL, followed by virus and incubating for 1 hour. When virus was added to cells, the final concentration of the composition was 20 μg/mL. The composition containing different amounts of oleandrin can be adjusted according to the concentration of oleandrin contained therein, and converted into a molar concentration. Figures 1-4 depict the efficacy of the extract based on the oleandrin content. OL itself is valid. PBI-05204, an SCF extract of oleander containing OL, OA, UA and BA, was substantially more effective than OL itself. Anvirzel ^TM , which is a hot water extract of oleander, is more effective than OL itself. Both extracts clearly demonstrated efficacy in the nanomolar range. The percentage of oleandrin in PBI-05204 extract (1.74%) was higher than that in Anvirzel (0.459%, 4.59 μg/mg). At the highest dose of PBI-05204, it completely inhibited EBOV and MARV infection, however Anvirzel ^™ did not exhibit complete inhibition because of toxicity observed at doses above 20 μg/mL of Anvirzel ^™ . The data indicated that PBI-05204 had the highest antiviral activity against Ebola virus and Marburg virus. The combination of triterpenes in PBI-05204 increases the antiviral activity of oleandrin.

Example 6 provides a detailed description of the in vitro assays used to evaluate the efficacy of cardiac glycosides in the treatment of Zika virus (a flavivirus) infection. Vero E6 cells were infected with Zika virus (ZIKV PRVABC59 strain) at MOI of 0.2 in the presence of oleandrin (FIG. 5) or digitonin (FIG. 6). Cells were incubated with virus and cardiac glycosides for 1 hour, followed by removal of inoculum and unabsorbed cardiac glycosides (if present). Cells were immersed in fresh medium and cultured for 48 hours, then cells were fixed with formalin and stained for ZIKV infection. The data demonstrate that both cardiac glycosides have antiviral activity against Zika virus; however, oleandrin exhibited higher (almost 8-fold greater) antiviral activity than digitonin.

Example 14 provides a detailed description of the experiments used to evaluate the antiviral activity of test compositions against Zika virus and Dengue virus. The data demonstrate that oleandrin shows efficacy against Zika virus and Dengue virus.

Figure 7 is a graph summarizing the in vitro dose-response antiviral activity of various compositions (oleandrin, digitoxin, and PBI-05204) against Ebola virus (EBOV) in Vero E6 cells. Figure 8 is a summary of the various compositions (oleandrin, digitoxin and PBI-05204) Graph of in vitro dose-response antiviral activity of Marburg virus (MARV) in Vero E6 cells. Figure 9 is a graph depicting a summary of the in vitro cell viability of Vero E6 cells in the presence of various compositions (oleandrin, digitonin, and PBI-05204). For Figures 7-8, host cells were exposed to the composition prior to infection with virus. EBOV/Kik (Fig. 7, MOI=1) or MARV/Ci67 (Fig. 8, MOI) in the presence of oleandrin, digitonin or PBI-05204 (a plant extract containing oleandrin) =1) Infection of Vero E6 cells. 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 counted using Operetta.

To ensure that no false positives were observed for antiviral activity, cell viability in the presence of the composition was tested. For the data in Figure 9, Vero E6 cells were treated with the above compounds. ATP levels were determined by CellTiter-Glo as a measure of cell viability. It has been determined that oleandrin, digitoxin and PBI-05204 do not reduce cell viability, meaning that the antiviral activities detailed in other figures in this specification are not caused by false positives due to the cytotoxicity of a single compound .

Accordingly, the present invention provides a method of treating a viral infection in a mammal or host cell, the aforementioned method comprising: prior to contracting the aforementioned viral infection, administering to the mammal or host cell an antiviral composition, whereby the aforementioned mammal or host is infected with an antiviral composition When cells are infected with a virus, the antiviral composition reduces viral titer and ameliorates, reduces or eliminates the viral infection.

The antiviral compositions and methods of the present invention are also useful in treating viral infections that occur prior to administration of the antiviral compositions. Vero E6 cells were infected with EBOV (FIGS. 10A, 10B) or MARV (FIGS. 11A, 11B). At 2 hours post-infection (Figures 10A, 11A) or 24 hours post-infection (Figures 10B, 11B), oleandrin or PBI-05204 were added to cells for 1 hour, then discarded, and cells were grown into culture.

Figures 10A and 10B are graphs depicting summarizing the ability of the compositions (oleandrin and PBI-05204) to inhibit Ebola virus in Vero E6 cells shortly after exposure to the virus: Figure 10A - 2 hours post infection; Figure 10B - post infection 24 hours. Viral titer antiviral compositions provide effective treatment and reduce EBOV viral titers when administered within 2 hours (or up to 12 hours) after viral infection. The viral composition was effective even after 24 hours; however, its efficacy decreased with time after initial viral infection. Do the same for MARV. Figures 11A and 11B are graphs depicting summarizing the ability of the compositions (oleandrin and PBI-05204) to inhibit Marburg virus in Vero E6 cells shortly after exposure to the virus: Figure 11A - 2 hours post infection; Figure 11B - post infection 24 hours. Viral titer antiviral compositions provide effective treatment and reduce MARV viral titers when administered within 2 hours (or up to 12 hours) after viral infection. The viral composition was effective even after 24 hours; however, its efficacy decreased with time after initial viral infection.

Considering that the antiviral activity of the composition of the present specification is reduced for a single passage of virus-infected cells, for example, within 24 hours after infection, we assessed whether the antiviral composition was able to inhibit viral reproduction, meaning whether it inhibited the production of infectious progeny. . Vero E6 cells were infected with EBOV or MARV in the presence of oleandrin or PBI-05204 and cultured for 48 hours. Supernatants from infected cell cultures were transferred to fresh Vero E6 cells for 1 hour and then discarded. Cells containing the transferred supernatant were cultured for 48 hours. Cells infected with EBOV (B) or MARV (C) were assessed as described in this specification. The control infection rate was 66% for EBOV and 67% for MARV. The antiviral compositions of the present invention inhibit the production of infectious progeny.

Thus, the antiviral compositions of the present invention: a) may be administered prophylactically prior to viral infection to inhibit viral infection following exposure to the virus; b) may be administered post viral infection to inhibit or reduce viral replication and the rate of infectious progeny produce; or c) a combination of a) and b).

VEE virus and WEE virus in Vero E6 cells were used to evaluate the antiviral activity of antiviral compositions against Togaviridae Alphavirus. Figures 13A and 13B are depictions summarizing the effects of various compositions (oleandrin, digitoxin, and PBI-05204) on Venezuelan equine encephalomyelitis virus (Figure 13A) and western equine encephalomyelitis virus (Figure 13B) in Vero E6 cells ) of the in vitro dose-response antiviral activity graph. 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) in the presence or absence of the indicated compounds for 18 hours. Infected cells were detected and counted on the Operetta as previously described. The antiviral compositions of the present invention have been found to be effective.

Accordingly, the present invention provides a method of treating a viral infection in a subject or host cell caused by: Arenaviridae, Filoviridae, Flaviviridae (Flaviridae), Retroviridae A virus, delta retrovirus, coronavirus, Paramyxoviridae, or Togaviridae virus, the foregoing method comprising administering an effective amount of an antiviral composition, thereby exposing the virus to the antiviral composition and treating the foregoing Viral infection.

We evaluated the use of oleandrin and extracts described in this specification for the treatment of HTLV-1 (human T-lymphotropic virus type 1; enveloped retrovirus; delta retrovirus) infection. To determine whether purified oleandrin compounds or oleander extracts could inhibit HTLV-1 provirus replication and/or the production and release of p19 ^Ga -containing viral particles, increased concentrations of oleandrin or oleander extracts, or sterile The virus-producing HTLV-1-transformed SLB1 lymphoma T cell line was treated with a control vector (MilliQ treated with 20% DMSO in ddH ₂ O), followed by incubation at 37° C., 10% CO ₂ for 72 hours. The cells were then pelleted by centrifugation and the relative amount of extracellular p19 ^Gag -containing viral particles released into the culture supernatant was determined by performing an anti-HTLV-1 p19 ^Gag ELISA (Zeptometrix).

Figure 14 is a graph depicting the use of control vehicle (1.5 μL, 7.5 μL or 15 μL), or increasing concentrations (10 μg/mL, 50 μg/mL and 100 μg/mL) of oleandrin compounds or extracts of European oleander (Examples 19 and 20). ) quantification data of HTLV-1 p19 ^Gag expressed by HTLV-1+SLB1 lymphoma T cell line treated for 72 hours. Viral replication and extracellular particles released into culture supernatants were quantified by performing an anti-HTLV-1 p19 ^Gag ELISA (Zeptometrix). Oleandrin did not significantly inhibit HTLV-1 replication or the release of newly synthesized viral particles. We determined that neither the extract nor oleandrin alone significantly inhibited viral replication or the release of p19 ^Gag -containing particles into the culture supernatant. Therefore, we expected no further antiviral activity; however, we unexpectedly found that viral particles collected from treated cells exhibited reduced infectivity to primary human peripheral blood mononuclear cells (huPBMC). Unlike HIV-1, extracellular HTLV-1 particles are less infective, and viral transmission typically occurs through direct cell-to-cell interactions across viral synapses.

The present invention thus provides a method of producing HTLV-1 virions with reduced infectivity, the aforementioned method comprising treating HTLV-1 virions with an antiviral composition of the invention to provide HTLV-1 virions with reduced infectivity.

To ensure that the observed antiviral activity was not an artifact due to the potential cytotoxicity of the antiviral composition on HTLV-1+SLB1 lymphoblastoid cells, we evaluated the Cytotoxicity of different dilutions of purified oleandrin compounds and European oleander extracts (Example 21). SLB1 T cells were treated with increasing concentrations (10 μg /ml, 50 and 100 μg/ml) of oleandrin or oleander extract for 72 hours as described in this specification. As a negative control, cells were further treated with increments (1.5 μl, 7.5 μl and 15 μl) of carrier solution corresponding to the volumes used in the drug-treated cultures. Cyclophosphamide (50 μM; Sigma-Aldrich) treated cells were included as a positive control for apoptosis. Next, the samples were washed and stained with Annexin V-FITC and propidium iodide (PI) and analyzed by conjugated focus fluorescence microscopy. The relative percentage of AnnexinV-FITC and/or PI positive cells was quantified by fluorescence microscopy and three replicate fields were counted using a 20x objective.

The results (FIG. 15 and FIGS. 16A-16F) demonstrate that the lowest concentration (10 μg/ml) of oleandrin and European oleander extract did not induce significant cytotoxicity/apoptosis. However, higher concentrations (about 50 and about 100 μg/ml) of the crude plant extract induced significantly more apoptosis than the oleandrin compound. This is consistent with the fact that oleandrin represents about 1.23% of the European oleander extract. Cytotoxicity caused by oleandrin was not significantly higher than that of the control vehicle in treated TLV-1+SLB1 cells.

We next investigated whether oleandrin or oleander extract could inhibit viral transmission from green fluorescent protein (GFP)-expressing HTLV-1+ lymphoma T cell lines to huPBMCs in a co-culture assay (Example 20). For these studies, HTLV-1+SLB1 lymphoma T cells were treated with increasing concentrations of oleandrin compounds or oleander extract, or control vehicle in 96-well microtiter plates for 72 hours, followed by collection of virus-containing supernatants And directly used to infect primary cultured in vitro human peripheral blood mononuclear cells (huPBMC). After 72 hours, an anti-HTLV-1 p19 ^Gag ELISA was performed to quantify the relative levels of extracellular p19 ^Gag -containing viral particles released into the culture supernatant as a result of direct infection.

The HTLV-1+SLB1 lymphoma T cell line was treated with control vehicle, increasing concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml) of European oleander extract or oleandrin compound for 72 hours, followed by collection of virus-containing supernatants solution and directly used to infect primary huPBMC. Control vehicle, European oleander extract or oleandrin were also included in the medium of huPBMC. After 72 hours, culture supernatants were collected and the relative amount of extracellular virus particles produced was quantified by performing an anti-HTLV-1 p19 ^Gag ELISA.

The data (FIG. 17) show that both oleandrin and oleander extract inhibit release to the culture supernatant of treated cells, even at the lowest concentration (10 μg/ml), compared to an equivalent amount of control vehicle Infectivity of newly synthesized virus particles containing p19 ^Gag in liquid. Both oleandrin and the crude extract inhibited the formation of viral synapses and the spread of HTLV-1 in vitro. Extracellular virus particles produced by oleandrin-treated HTLV-1+ lymphoma T cells exhibited reduced infectivity to primary huPBMCs. Importantly, oleandrin exhibits antiviral activity against enveloped viruses by reducing the incorporation of envelope glycoproteins into mature particles, which represents a unique phase of the retroviral infection cycle.

To ensure that the observed antiviral activity was not an artifact due to the potential cytotoxicity of the antiviral composition on the treated huPBMCs, we also investigated (Example 21), in the treated huPBMCs compared to the vehicle negative control Cytotoxicity of purified oleandrin and European oleander extracts. Primary buffy coat huPBMCs were isolated, stimulated with phytohemagglutinin (PHA), and cultured in the presence of recombinant human interleukin-2 (hIL-2). Cells were then treated with increasing concentrations of oleandrin or European oleander extract, or with increasing volumes of vehicle for 72 hours. The samples were then stained with Annexin V-FITC and PI and counted by conjugated focus fluorescence microscopy in triplicate to quantify the relative percentage of apoptotic (ie Annexin V-FITC and/or PI positive) cells per field.

The cytotoxic effects of control vehicle, European oleander extract and oleandrin compounds were assessed by treating primary huPBMCs for 72 hours, followed by staining of cultures with AnnexinV-FITC and PI. The relative percentage of apoptotic (ie AnnexinV-FITC and/or PI positive) cells was quantified by fluorescence microscopy and counting three replicate fields using a 2Ox objective. The total number of cells 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 high toxicity.

The data (FIG. 18) indicated that oleandrin showed moderate cytotoxicity in huPBMCs (eg, 35-37% at the lowest concentrations) compared to the control vehicle. In contrast, oleander extract was significantly cytotoxic and induced high levels of apoptosis even at the lowest concentrations. Compared with HTLV-1+SLB1 lymphoblastoid cells, huPBMCs were slightly more sensitive to purified oleandrin. However, huPBMC was more sensitive to crude oleander extract, which The extract also contains other cytotoxic compounds, such as the triterpenes described in this specification.

We further investigated (Example 22) whether oleandrin or European oleander extract could interfere with the dissemination of HTLV-1 particles to target huPBMCs in co-culture experiments. For these studies, virus-producing HTLV-1+SLB1 T cell lines were treated with mitomycin C, followed by increasing amounts of oleandrin, oleander extract, or control vehicle for 15 minutes or 3 hours. SLB1 cells were washed 2 times with serum-free medium, then an equal amount of huPBMC was added to each well, and the samples were co-cultured in complete medium for 72 hours in a humidified incubator at 37°C, 10% _CO . . Relative cell-to-cell spread 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 with control vehicle, increasing concentrations (10 μg/mL, 50 μg/mL, and 100 μg/mL) of European oleander extract or oleander HTLV-1+SLB1 lymphoma T cells were pretreated with glycoside compounds for 15 minutes or 3 hours. Control vehicles, extracts and compounds were also present in the co-culture medium. After 72 hours, the supernatant was collected and the amount of extracellular viral particles released was quantified by performing an anti-HTLV-1 p19 ^Gag ELISA.

The results depicted in Figure 19 demonstrate that both oleandrin and oleander extract inhibited the spread of HTLV-1 compared to the control vehicle; however, nothing was observed between the 15-minute and 3-hour pretreatment of HTLV-1+SLB1 cells difference.

We also investigated whether oleandrin inhibits viral synapse formation and transmission of HTLV-1 in a co-culture assay (Example 22). HTLV-1 expressing GFP was generated by transduction of SLB1 lymphoma T cells with the pLenti-6.2/V5-DEST-GFP vector and two weeks of selection with blasticidin (5 μg/mL; Life Technologies) +SLB1 T cell line. GFP positive colonies were screened by fluorescence microscopy (Fig. 20 upper panel) and immunoblotting (Fig. 20 lower panel), expanded and subcultured repeatedly. DIC phase contrast images are provided for comparison.

Viral synapse formation between huPBMC and mitomycin C-treated HTLV-1+SLB1/pLenti-GFP lymphoblasts (green cells) visualized by fluorescence microscopy Blast cells had been pretreated with control vehicle or increasing amounts (10 μg/mL, 50 μg/mL and 100 μg/mL) of European oleander extract or oleandrin compound for 3 hours ( FIG. 21 ). Virus transmission was assessed by quantifying the relative percentage of infected (ie, 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 the control vector image). Data obtained by quantitative fluorescence microscopy (FIG. 22). It can be confirmed from the data that the antiviral composition inhibited viral synapse formation and transmission of HTLV-1 in co-culture assays.

Accordingly, the present invention also provides a method for inhibiting (reducing) the infectivity of HTLV-1 particles released into treated cell culture supernatants and for reducing HTLV-1 cell-to-cell transmission by inhibiting Env-dependent formation of viral synapses. A method, the foregoing method comprising treating virus-infected cells (in vitro or in vivo) with an effective amount of an antiviral composition.

The antiviral activity of the compositions described in this specification was evaluated against rhinovirus infection. Rhinoviruses belong to the Picornaviridae family and the Enterovirus genus. It is a non-enveloped, (+) polar ss-RNA virus. At the concentrations and tests employed in this specification, oleandrin has been found to be inactive against rhinoviruses because it does not inhibit viral replication.

As detailed in Example 26, CoV infection can be treated in vivo, wherein the antiviral composition is administered to a subject as a monotherapy or combination therapy. The efficacy of oleandrin against CoV was established in vivo according to Example 27. Infants recovered from COVID-19 infection by administering 0.25 ml of recombinant ANVIRZEL ^™ to children in a small portion of orange juice, followed by 0.5 ml of recombinant ANVIRZEL ^™ to children every 12 hours for approximately 2-3 days.

Further demonstration of the efficacy of oleandrin (a composition containing oleandrin) against coronaviruses such as SARS-CoV-2 (COVID-19) was obtained by in vitro evaluation in accordance with Example 28, wherein Vero cells were pretreated with oleandrin, followed by After infection with SARS-CoV-2, after cell infection, extracellular virus and oleandrin were washed away, followed by treatment of infected cells with oleandrin (Fig. 23A: oleandrin at 1 μg/mL in 0.1% v/v aqueous DMSO; Fig. 23C: Oleandrin at 0.1 μg/mL in 0.01% v/v aqueous DMSO) or only aqueous DMSO as control vehicle (Figure 23B: 0.1% v/v aqueous DMSO; Figure 23D: 0.01% v/v aqueous DMSO ). The results showed that a) oleandrin pretreatment caused a 1368-fold reduction in viral load at 24 hours and a 369-fold reduction at the 48 hour time point; b) oleandrin was effective over the entire concentration range of about 0.1 to about 1.0 μg/mL , where high doses are slightly better than low doses, so oleandrin is likely to be effective at even lower concentrations, such as 0.01 to 0.1 μg/mL; c) oleandrin should be administered repeatedly, as a single dose is not sufficient to completely stop viral replication; and d) pre-incubating Vero cells with oleandrin alone for 30 minutes was only slightly effective in reducing initial viral infection and did not appear to affect the infectivity of progeny virions. The results also showed that oleandrin was not overly toxic to Vero cells at concentrations of 0.1 and 1.0 μg/mL. The results further demonstrate that oleandrin inhibits the infectivity of the progeny virus by: a) about 1 log ₁₀ without continuous drug treatment; and b) about >3 log ₁₀ with continuous drug treatment (no toxicity).

To determine whether oleandrin directly inhibits viral replication, Vero-E6 cells were infected with SARS-CoV-2 virus according to Example 29 and treated with varying concentrations of oleandrin. The results are depicted in Figures 24A and 24B. At the 24 hour time point (FIG. 24A), in wells treated with oleandrin alone, antiviral activity was observed only during the uptake phase (pre-processing data), with an approximate _IC50 of 0.625 μg/mL. In wells treated with oleandrin, for the duration of the test (duration data), oleandrin significantly restricted viral entry and/or viral replication, even in the presence of large inoculated virus. At the 48 hour time point (Figure 24B), in wells treated with oleandrin only during the uptake phase (pre-processing data), the lowest antiviral activity was observed at the end of the time period. In wells treated with oleandrin for the duration of the test (duration data), oleandrin significantly inhibited viral infection. Possible methods of action include inhibition of viral replication, assembly and/or release.

To ensure that the observed antiviral activity of oleandrin against SARS-CoV-2 was not due to the extracellular toxicity of oleandrin against Vero-E6 cells, it was determined at the 24 h (Fig. 24A) and 48 h (Fig. 24B) time points Cell force. At concentrations of oleandrin above 1.0 μg/mL, cytotoxicity appeared and potentially interfered with the assay; however, at concentrations of oleandrin below 0.625 μg/mL, the interference of cytotoxicity was significantly reduced, confirming that even at very low The strong antiviral activity of oleandrin at the concentration of . Additional evidence for the degree of toxicity of oleandrin against Vero-E6 cells was observed in the assay of Example 30 (Figure 25). At a concentration of 0.625 μg/mL oleandrin, approximately 80% of Vero cells were viable at the 24-hour time point, and even lower toxicity was observed at lower concentrations. It should be understood that the toxicity of oleandrin to Vero-E6 cells does not indicate that oleandrin is toxic to humans. When measuring antiviral activity, this toxicity assay is used only to determine the potential effect of background cell death.

Therefore, oleandrin has at least a dual mechanism (pathway) for the treatment of viral infections, especially coronavirus infections, such as SARS-CoV-2 infection: a) direct inhibition of viral replication; and b) reduced infectivity of progeny viruses.

Furthermore, oleandrin has antiviral activity even at very low doses, and oleandrin exhibits substantial preventive effects. This is illustrated according to Example 31, where VERO CCL-81 cells were infected with SARS-CoV-2. Cells were pretreated with oleandrin prior to infection. After an initial culture of 2 hours after infection, the infected cells were washed to remove extracellular virus and oleandrin. Next, the recovered infected cells were processed as follows. Infected cells were treated with oleandrin (in various concentrations of oleandrin in aqueous DMSO with RPMI 1640 broth as an aqueous component) or with control vehicle only (DMSO in water with RPMI 1640), and after "treatment" Viral titers were determined at 24 hours (FIG. 26A) and 48 hours (FIG. 26B). In the absence of oleandrin, SARS-CoV-2 reached a high (approximately 6 log ₁₀ plaque forming units (pfu)/mL) titer at the 24-hour time point and maintained that titer at subsequent time points: It was consistently maintained at or below the detection limit of the assay. Oleandrin at concentrations ranging from 1 μg/mL to 0.05 μg/mL provided greatly reduced viral titers even at just 24 hours. The two higher doses substantially reduced viral titers at or below the detection limit, and no cytotoxicity was observed at any of the oleandrin concentrations tested. The calculated viral titers of these samples decreased by multiples. Fold reductions in viral titers (Figures 26C and 26D) ranging from about 1,000-fold to about 40,000-fold were observed at the 48 hour time point, and about 1,000-fold to about 20,000-fold were observed at the 24 hour time point. Although the 10 ng/mL dose had no significant effect compared to its DMSO control group at 24 hours post-infection, it resulted in a significant reduction in force titers at 48 hours post-infection. It is worth noting that the highest value of the fold reduction in viral titer caused by oleandrin appeared not at the 24th hour, but at the 48th hour. The increase in prophylactic efficacy of oleandrin over time (24 hours and 48 hours) was reflected in its _EC50 values, which were calculated as 11.98 ng/ml at 24 hours post-infection and 7.07 ng/ml at 48 hours post-infection.

Genomic analysis of the above-mentioned Vero CCL-81 cells was performed to determine whether inhibition of SARS-CoV-2 was global or only at the level of its infectious particle production. RNA was extracted from the cell culture supernatants of the prophylactic studies and the genomic equivalents were quantified by qRT-PCR (Example 39). The preventive effect of oleandrin initially observed by the infectivity assay was confirmed at the level of the genomic equivalent. At 24 hours post-infection, oleandrin significantly reduced the SARS-CoV-2 genome in the supernatant at the four highest doses. The preventive effect of oleandrin initially observed by the infectivity assay was confirmed at the level of the genomic equivalent. At 24 hours post-infection, oleandrin significantly reduced the SARS-CoV-2 genome in the supernatant at the four highest doses.

Additional studies were conducted to determine the dose-response of oleandrin to COVID-19 infection at 24 and 48 hours post-infection (Figures 27A-27B). A dose-response was observed, whereby increasing the concentration of oleandrin in the culture medium significantly reduced viral titers. even detecting The lowest concentration in the medium (0.05 μg/mL) also caused a decrease in titer 24 hours after infection, and an even greater decrease in titer at 48 hours after infection. The highest dose resulted in a more than 1,000-fold reduction in infectious SARS-CoV-2 titer, with the 0.5 μg/mL and 100 ng/mL doses resulting in a greater than 100-fold reduction and the 50 ng/ml dose resulting in a 78-fold reduction.

Figures 28A and 28B depict the results of replicate studies, each conducted in triplicate, to determine the dose-response of COVID-19 to treatment with varying concentrations of oleandrin in the medium (0.005 to 1 μg/mL). At concentrations greater than 0.01 μg/mL, substantial antiviral activity was observed even at 24 and 48 hours post-infection in Vero CCL-81 cells. Even at a very low concentration of 0.05 μg/mL, a substantial reduction in virus titer was observed.

In order to determine the antiviral efficacy of oleandrin after infection, a study according to Example 34 was performed. Vero CCL-81 cells were not pretreated with oleandrin before infection. Instead, cells were infected with the COVID-19 virus, followed by treatment with oleandrin (at the indicated concentrations) at 12 and 24 hours post-infection. Viral titers were then determined at 24 hours (FIG. 29A) and 48 hours (FIG. 29B) post-infection. The data demonstrate that even with only a single treatment, oleandrin can exhibit antiviral activity at least 12 hours, at least 24 hours, or at least 36 hours after infection. Notably, the assay is temporally compressed compared to the course of viral infection in humans, with the 24-hour time point corresponding to approximately 5 to 7 days post-human infection and the 48-hour time point corresponding to approximately 10 to 7 days post-human infection. 14 days.

A dual extraction composition was used (PBI-A, containing 1 wt% ethanol extract dissolved in DMSO (98 wt%), 1 wt% Example 36). 30A details the results of evaluating the composition of the dual extract composition according to the assay of Example 31, and FIG. 30B details the results of evaluating the dual extract (1 wt %) according to the assay of Example 34. The data in Figure 30A illustrate the relative antiviral (anti-COVID-19) efficacy of PBI-A based on relative dilution of the original stock solution. The data in Figure 30B are based on the relative concentration (μg/mL) of oleandrin in the assay solution. The dotted line in each graph depicts the lowest concentration of virus that can be detected using the CFU (colony forming unit) assay.

Based on the results of Figures 30A and 30B, the dual extraction composition composition is effective as an antiviral agent against COVID-19 at concentrations comprising 0.05 to 1.0 μg/mL, which is the same range as observed in pure oleandrin .

Equally important, the concentrations of oleandrin assessed in the assay were observed to be clinically relevant in terms of dose and plasma concentration.

Evidence for the safety of compositions containing oleandrin is further provided by in vitro cellular assays used to determine the release of lactate dehydrogenase following exposure of the aforementioned cells to solutions containing varying concentrations of oleandrin. It was determined that at concentrations up to 1 μg/mL, there was no additional toxicity compared to the control vehicle.

The efficacy of oleandrin (the composition containing oleandrin, the extract containing oleandrin) in the treatment of COVID-19 virus infection was further obtained according to Example 35 under the Expanded Access program of the FDA, by applying to recipients Subjects administered sublingual dosage forms containing oleandrin (Examples 32 or 37) to establish. Four 15 μg doses of oleandrin (as a dual extract composition) per day at about 6 hour intervals or three 15 mg doses per day at about 8 hour intervals were administered daily to subjects aged 18 to 78 years. Subjects were observed for clinical status and/or viral titer prior to initiation of treatment. Some subjects received palliative care or hospice care. Clinical status and/or viral titers were determined periodically over a one to two week, ten to fourteen day treatment period. The following results were observed after starting the treatment.

An additional in vivo study was conducted under the FDA's Expanded Access program with a second cohort of subjects displaying varying degrees of COVID-19-related symptoms. Before initiating treatment, determine the subject's clinical status and/or viral titer to confirm COVID-19 infection. Some subjects exhibited moderate to severe symptoms. Four groups of 15 μg doses of oleandrin (as a dual extract composition) were administered daily to subjects of varying ages at approximately 6 hour intervals. Clinical status and/or viral titers were determined periodically over a one to two week, ten to fourteen day treatment period. All subjects fully recovered from COVID-19 infection within five to twelve days of starting treatment.

Oleandrin has been shown to produce a strong anti-inflammatory response, which may help prevent excessive inflammation caused by SARS-CoV-2 infection.

Accordingly, the present invention provides a method of treating a COVID-19 viral infection, the aforementioned method comprising administering multiple doses of cardiac glycosides (a cardiac glycoside-containing composition, or a cardiac glycoside-containing extract) to a subject having the aforementioned infection. Multiple doses may be administered at the following frequency: one dose per day The above doses are administered more than two days per week; optionally, more than one week per month; and optionally, more than one month per year. The ideal cardiac glycoside is oleandrin.

Accordingly, the present invention provides a method of treating a coronavirus infection, particularly an infection by a coronavirus pathogenic to humans such as SARS-CoV-2, the aforementioned method comprising chronically administering to a subject having the aforementioned infection a therapeutically effective dose of a cardiotonic glycosides (compositions containing cardiac glycosides). Chronic administration can be achieved by repeated administration of one or more (plurality) therapeutically effective doses of cardiac glycosides (cardiac glycoside-containing compositions). More than one dose per day may be administered more than one day per week, optionally more than one week per month, and optionally more than one month per year.

Accordingly, the present invention provides a method of treating a virus, such as a CoV infection, in a subject (particularly a subject) in need thereof, the aforementioned method comprising administering to the subject one or more doses of an antiviral composition, the aforementioned The antiviral composition comprises a) oleandrin; or b) oleandrin extracted from Oleander species and one or more other compounds. Oleandrin can be present as part of an extract of Oleander sp., wherein the extract can be a) a supercritical fluid extract; b) a hot water extract; c) an organic solvent extract; d) an aqueous organic solvent extract; e) using a supercritical fluid, optionally, plus an extract of at least one organic solvent (extraction modifier); f) using a supercritical fluid, optionally adding an extract of at least one organic solvent (extraction modifier) ; or g) any combination of any two or more of the foregoing extracts.

PBI-05204 (such as this specification and US 8187644 B2 of Addington (published on May 29, 2012), US 7402325 B2 of Addington (published on July 22, 2008), US 8394434 B2 of Addington et al. (published on March 2013) 12 Announcement), the entire disclosure of which is incorporated into this specification by reference) includes cardiac glycosides (oleandrin, OL) and triterpenes (oleanolic acid (OA), ursolic acid (UA) and betulinic acid. (BA)) as the main pharmacologically active component. The molar ratio of OL to all triterpenes was 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 oleander when compared based on equimolar OL Antiviral activity of peach glycosides. PBI-04711 is a fraction of PBI-05204, but it 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. Therefore, the antiviral composition containing OL, OA, UA and BA is more effective than the composition containing OL as the only active ingredient based on OL at equimolar content. In some embodiments, the molar ratio of the individual triterpenes to oleandrin 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 to 0.7 (BA): about 0.9 to 1.1 (OL); or about 4.6 (OA): about 4.4 (UA): about 0.6 (BA): about 1 (OL).

Antiviral compositions containing oleandrin as the sole antiviral agent are within the scope of the present invention. Antiviral compositions containing digitonin as the sole antiviral agent are within the scope of the present invention.

Antiviral compositions containing oleandrin and various triterpenes as antiviral agents are within the scope of the present invention. In some embodiments, the antiviral composition comprises oleandrin, oleanolic acid (free acid, salt, derivative or prodrug thereof), ursolic acid (free acid, salt, derivative or prodrug thereof) and birch Acids (free acids, salts, derivatives or prodrugs thereof). The molar ratio of the compound is described in this specification.

Antiviral compositions containing multiple triterpenes as the main active ingredient (meaning no steroids, cardiac glycosides and pharmacologically active components) are also within the scope of the present invention. As described above, PBI-04711 contains OA, UA and BA as main active ingredients, and it 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 thereof.

PBI-01011 is a triterpenoid-based modified antiviral composition containing 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 at least comprises oleanolic acid (including its free acid, salt, derivative or prodrug) and ursolic acid (including its free acid, salt, derivative or prodrug) in the molar ratio of OA to UA described in this specification. including its free acid, salt, derivative or prodrug). The molar amount of OA is larger than that of UA.

In some embodiments, the antiviral composition comprises at least oleanolic acid (including its free acid, salt, derivative or prodrug) and betulinic acid (including its free acid, salt, derivative or prodrug) in the molar ratio of OA to BA described in this specification. its free acid, salt, derivative or prodrug). The molar amount of OA is greater than that of BA.

In some embodiments, the antiviral composition at least comprises oleanolic acid (including its free acid, salt, derivative or prodrug) in the molar ratio of OA to UA and OA to BA described in this specification, Ursolic acid (including its free acid, salt, derivative or prodrug) and betulinic acid (including its free acid, salt, derivative or prodrug). The molar amount of OA is larger than that of UA and BA.

In some embodiments, the triterpene-based antiviral composition does not comprise cardiac glycosides.

In general, the following methods are used to treat infections with arenaviridae, arteriviridae, filoviridae, flaviviridae (flaviridae), delta retroviridae, coronaviridae, paramyxoviridae, Orthomyxoviridae or Togaviridae infected subjects. The subject is assessed to determine whether the aforementioned subject is infected with the aforementioned virus, administration of the antiviral composition is indicated, and the subject is administered an initial dose of the antiviral composition for a period of time (one treatment period) according to the indicated dosing regimen. The subject's clinical response and therapeutic response level are determined periodically, and if the therapeutic response level is too low at a dose, the dose is escalated according to a predetermined dose escalation plan until When the desired level of therapeutic response is achieved in the subject, the subject continues to be treated with the antiviral composition as needed. The dose or dosing regimen can be adjusted as needed until the patient achieves the desired clinical endpoint or endpoints, such as cessation of the infection itself, reduction in symptoms associated with the infection, and/or reduction in the progression of the infection.

If a clinician intends to treat a subject with a viral infection with a combination of an antiviral composition and one or more other therapeutic agents, and the subject is known to have a viral infection that is at least partially therapeutic response, the method invention comprises administering to a subject in need thereof a therapeutically relevant dose of an antiviral composition and a therapeutically relevant dose of one or more of the foregoing other therapeutic agents, wherein the antiviral is administered as in the first dosing regimen composition and one or more other therapeutic agents are administered as a second dosing regimen. In some embodiments, the first and second dosing regimens are the same. In some embodiments, the first and second dosing regimens are different.

The antiviral composition(s) of the present invention can be administered as primary antiviral therapy, adjunctive antiviral therapy, or combined antiviral therapy. The methods of the present invention comprise separate administration or co-administration of the antiviral composition with at least one other known antiviral composition, meaning that the antiviral composition of the present invention can be used in a known antiviral composition (one or more compounds) or before, during or after administration of the composition for the treatment of symptoms associated with viral infection. For example, a drug for the treatment of inflammation, vomiting, nausea, headache, fever, diarrhea, urticaria, conjunctivitis, malaise, muscle pain, arthralgia, epilepsy or paralysis can be administered together with or separately from the antiviral composition of the present invention .

The other therapeutic agent(s) may be administered at a dose and baseline dosing regimen recognized by a clinician as therapeutically effective, or at a dose recognized by a clinician that is less than a therapeutically effective dose. The clinical benefit and/or therapeutic effect provided by the administration of the antiviral composition in combination with one or more other therapeutic agents may be additive or synergistic, and the extent of such benefit or effect may vary. Determined by comparing administration in combination with administration of the antiviral composition component(s) alone, and one or more other therapeutic agents. FDA, World Health Organization, European Medicines Agency (EMEA), Drug Administration (TGA, Australia), Pan American Health Organization (PAHO), Medicines and Medical Device Safety Authority (Medsafe, New Zealand) blue) or the dosages and baseline dosing regimens recommended or described by various world health authorities to administer one or more other therapeutic agents.

Exemplary other therapeutic agents included in the antiviral compositions of the present invention can be used to treat viral infections, including antiretroviral agents, alpha-interferon (IFN-a), zidovudine, lamivudine lamivudine, cyclosporine A, CHOP with arsenic trioxide, sodium valproate, methotrexate, azathioprine, one or more symptom reliever drugs, steroid sparing drugs, Corticosteroids, cyclophosphamide, immunosuppressants, anti-inflammatory agents, Janus kinase inhibitors, tofacitinib, calcineurin inhibitors, tacrolimus, mTOR inhibitors, sirolimus sirolimus, everolimus, IMDH inhibitors, azathioprine, leflunomide, mycophenolate, biologics, abatacept, adalim Mab (adalimumab), anakinra (anakinra), certolizumab (certolizumab), etanercept (etanercept), golimumab (golimumab), infliximab (infliximab), ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, monoclonal antibodies, basiliximab, daclizumab, polyclonal antibodies, nucleoside analogs, reverse transcriptase inhibitors, emtricitabine ( emtricitabine), telbivudine (telbivudine), abacavir (abacavir), adefovir (adefovir), didanosine (didanosine), emtricitabine (emtricitabine), entecavir (entecavir), Stavudine, tenofovir, azithromycin (azithromycin), macrolide-type antibiotic (macrolide-type antibiotic), protease inhibitor, interferon, immune response modifier, mRNA synthesis inhibitor, protein synthesis, inhibitor, thiazolide, CYP3A4 inhibitor, Heterocyclic biguanides, CCR5 receptor inhibitors, and combinations thereof. The treatments studied also included plasma exchange and/or radiation. Antibodies to specific viruses can also be administered to subjects treated with the antiviral compositions of the present invention. Plasma obtained from the blood of a survivor of the first viral infection can be administered to other subjects with the same type of viral infection who are also administered the antiviral composition of the present invention. For example, plasma from a survivor of a COVID-19 infection can be administered to another subject suffering from a COVID-19 infection who is also administered an antiviral composition of the invention.

Example 5 provides exemplary treatment procedures for Zika virus infection in mammals. Example 12 provides exemplary treatment procedures for filovirus infections (Ebola virus, Marburg virus) in mammals. Figure 13 provides a graph of flavivirus infections in mammals (yellow fever, dengue, Japanese encephalitis, West Nile virus, Zika virus, tick-borne encephalitis, Kejeldahl disease, Alkhurma disease, Omsk hemorrhagic fever, Powa Exemplary treatment steps for viral infections). Example 25 provides exemplary treatment procedures for delta retrovirus (HTLV-1) infection.

The antiviral compounds (eg, one or more triterpenes, one or more cardiac glycosides, etc. . . . ) present in the pharmaceutical composition may be present in their unmodified forms, salt forms, derivative forms, or combinations thereof. As used in this specification, the term "derivative" refers to: a) a chemical substance that is structurally related to, and theoretically derivable from, a first chemical substance; b) a compound that results from a similar first compound; or conceivably because One atom of the first compound is substituted by another atom or group of atoms, thereby producing a compound; c) a compound derived or obtained from a parent compound and containing the essential elements of the parent compound; or d) similar in one or more steps The chemical compound produced by the first compound of the structure. For example, derivatives may comprise their deuterated, oxidized, dehydrated, unsaturated, polymer-conjugated, or glycosylated forms, or may comprise their esters, amides, lactones, Homologs, ethers, thioethers, cyano, amines, alkylamines, sulfhydryls, heterocycles, fused heterocycles, polymeric, pegylated, benzylidene, triazolyl, piperazinyl or Deuterated form.

As used in this specification, unless otherwise indicated, the term "oleandrin" refers to all known forms of oleandrin. Oleandrin can exist in racemic, optically pure or optically enriched form. Oleander plant material is available, for example, from commercial plant suppliers such as Aldridge Nursery (Atascosa, Texas).

Supercritical fluid (SCF) extracts can be prepared as described 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 with supercritical carbon dioxide can be performed in the presence or absence of modifiers (organic solvents) such as ethanol.

Other extracts containing cardiac glycosides, especially oleandrin, can be prepared by various different processes. The extract can be prepared by a process developed by Dr. Huseyin Ziya Ozel (US Patent No. 5,135,745), which describes the procedure for preparing a hot water extract. The aqueous extract was reported to contain various polysaccharides ranging in molecular weight from 2KD to 30KD, oleandrin, oleandrin, odonoside and oleandroside. The polysaccharide is reported to contain acidic homogalacturonic acid or arabinogalacturonic acid. US Patent No. 5,869,060 to Selvaraj et al. discloses hot water extracts of Oleander sp. and methods for their production, eg, Example 2. The resulting extract is then lyophilized to yield a powder. US Patent No. 6,565,897 (US Pre-Grant Publication No. 20020114852 to Selvaraj et al. and PCT International Publication No. WO 2000/016793) discloses a hot water extraction process for preparing substantially sterile extracts. Erdemoglu et al. ( J. Ethnopharmacol. (2003) Nov. 89(1), 123-129) disclosed comparative results of aqueous and ethanolic extracts of oleander-containing plants based on their analgesic and anti-inflammatory activities. Adome et al. ( Afr. Health Sci. (2003) Aug. 3(2), 77-86; ethanolic extract), el-Shazly et al. ( J. Egypt Soc. Parasitol. (1996) Aug. 26 (2), 461-473; ethanolic extract), Begum et al. ( Phytochemistry (1999) Feb. 50(3), 435-438; methanolic extract), Zia et al. ( J. Ethnolpharmacol. (1995) ten Jan. 49(1), 33-39; methanol extract) and Vlasenko et al. ( Farmatsiia. (1972) Sep-Oct. 21(5), 46-47; Organic solvent extract. US Pregrant Patent Application Publication No. 20040247660 to Singh et al. discloses the preparation of protein stabilized liposomal formulations of oleandrin for use in cancer therapy. US Pre-Grant Patent Application Publication No. 20050026849 to Singh et al. discloses water-soluble formulations of oleandrin containing cyclodextrins. US Pre-Grant Patent Application Publication No. 20040082521 to Singh et al. discloses the preparation of protein-stabilized nanoparticle formulations of oleandrin from hot water extracts.

Oleandrin can also be obtained from extracts derived from supernatant cultures of Agrobacterium tumefaciens transformed callus (Ibrahim et al., "Stimulation of oleandrin production by combined Agrobacterium tumefaciens mediated transformation and fungal elicitation in Nerium oleander cell cultures"). "in Enz. Microbial Techno. (2007), 41(3), 331-336, the entire disclosure of which is incorporated herein by reference). According to the present invention, hot water, organic solvent, aqueous organic solvent or supercritical fluid extracts of Agrobacterium can be used.

Oleandrins can also be obtained as extracts from in vitro microcultures of oleander, whereby stem segment cultures can be initiated from seedlings and/or shoot tips of oleander species such as Splendens Giganteum, Revanche, Alsace or others (Vila et al., "Micropropagation of Oleander"). (Nerium oleander L.)" in Hort Science (2010), 45(1), 98-102, the entire disclosure of which is incorporated herein by reference). Hot water, organic solvents, aqueous organic solvents or supercritical fluid extracts of microcultured oleander can be used according to the present invention.

The polysaccharide and carbohydrate content of the extracts also varied. The hot water extract contained 407.3 dextrose equivalent units of carbohydrates relative to a standard curve made with dextrose, while analysis of the SCF _CO2 extract found the presence of carbohydrates well below the minimum level required for quantification. However, the amount of carbohydrates in the hot water extract of oleander was at least 100 times higher than the amount of carbohydrates in the SCF _CO2 extract. The polysaccharide content of the SCF extract may be 0 wt%, <0.5 wt%, <0.1 wt%, <0.05 wt%, or <0.01 wt%. In some embodiments, the SCF extract does not contain polysaccharides obtained during extraction of the plant material.

The SCF _CO2 extract and Part of the hot water extract.

The SCF extract of Oleander sp. or Oleander sp. is a mixture of pharmacologically active compounds such as oleandrin and triterpenes. The extract obtained by the SCF process is a viscous semi-solid that is substantially insoluble in water (after removal of the solvent) at ambient temperature. SCF extracts contain many distinct components with various ranges of water solubility. The extract from the supercritical fluid process contains a theoretical range of 0.9% to 2.5% oleandrin by weight, or 1.7% to 2.1% oleandrin, or 1.7% to 2.0% oleandrin by weight theoretically. SCF extracts containing varying amounts of oleandrin have been obtained. In one embodiment, the SCF extract comprises about 2 wt% oleandrin. Compared with hot water extract, SCF extract contains 3-10 times higher concentration of oleandrin. This was confirmed by both HPLC and LC/MS/MS (tandem mass spectrometry) analysis.

The SCF extract contains oleandrin and the triterpenes oleanolic acid, betulinic acid and ursolic acid and any other components described in this specification. The content of oleandrin and triterpenes can be found in batches differences; however, the degree of variation should not be too great. For example, these four components were analyzed against a batch of SCF extract (PBI-05204) and each was found to contain the following approximate amounts.

WRT表示「相對於」。

WRT stands for "relative to".

The content of each component may vary between ±25%, ±20%, ±15%, ±10% or ±5% relative to the indicated value. Therefore, the content of oleandrin in the SCF extract may range from 20 mg ± 5 mg per mg of SCF extract (which is ± 25% of 20 mg).

Oleandrin, oleanolic acid, ursolic acid, betulinic acid and derivatives thereof can also be purchased from Sigma-Aldrich ( www.sigmaaldrich.com ; St. Louis, MO, USA). Digoxigenin 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, 0.25mg/mL), West-Ward Pharmaceuticals International LTD (NDA A083391, injection, 0.25mg/mL), Covis Pharma BV (NDA N009330, 0.1mg/mL, 0.25mg/ 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, 0.25mg), Sun Pharmaceutical Industries Inc. (NDA A076363, tablet, 0.125mg, 0.25mg), Concordia Pharmaceuticals Inc. (NDA A020405, tablet, 0.0625, 0.125mg, 0.1875 mg, 0.25mg, 0.375mg, 0.5 mg, LANOXIN), GlaxoSmithKline LLC (NDA 018118, capsules, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, LANOXICAPS).

As used herein, an individually named triterpenoid may be independently selected at each occurrence from its native (unmodified, free acid) form, its salt form, derivative form, prodrug form, or a combination thereof. Compositions containing triterpenes in deuterated form and methods of using triterpenes in deuterated form are also within the scope of the invention.

Oleanolic acid derivatives, prodrugs and salts are disclosed in US 20150011627 A1 to Gribble et al. (published Jan. 8, 2015), US 20140343108 A1 to Rong et al. (published Nov. 20, 2014) ), US 20140343064 A1 by Xu et al. (published on November 20, 2014), US 20140179928 A1 by Anderson et al. (published on June 26, 2014), US 20140100227 A1 by Bender et al. (published on April 10, 2014) published), US 20140088188 A1 by Jiang et al. (published on March 27, 2014), US 20140088163 A1 by Jiang et al. (published on March 27, 2014), US 20140066408 A1 by Jiang et al. (published on March 6, 2014) published on November 28, 2013), US 20130317007 A1 by Anderson et al. (published on November 28, 2013), US 20130303607 A1 by Gribble et al. (published on November 14, 2013), US 20120245374 by Anderson et al. published on September 20, 2012), US 20120238767 A1 by Jiang et al. (published on September 20, 2012), US 20120237629 A1 by Shode et al. (published on September 20, 2012), US 20120214814 A1 by Anderson et al. (published on August 2012) 23), US 20120165279 A1 by Lee et al. (published on June 28, 2012), US 20110294752 A1 by Arntzen et al. (published on December 1, 2011), US 20110091398 A1 by Majeed et al. published on July 21), US 20100189824 A1 by Arntzen et al. (published on July 29, 2010), US 20100048911 A1 by Jiang et al. (published on February 25, 2010), and US 20060073222 A1 by Arntzen et al. (2006 published April 6, 2004), the entire disclosure of which is incorporated herein by reference.

Ursolic acid derivatives, prodrugs and salts are disclosed in the following documents: US 20150011627 A1 to Gribble et al. (published on Jan. 8, 2015), US 20130303607 A1 to Gribble et al. (published on Nov. 14, 2013) , US 20150218206 A1 of Yoon et al. (published on August 6, 2015), US 6824811 of Fritsche et al. (authorized on November 30, 2004), US 7718635 of Ochiai et al. (authorized on May 8, 2010), US 8729055 to Lin et al. (issued May 20, 2014), and US 9120839 to Yoon et al. (issued September 1, 2015), the entire disclosures of which are incorporated herein by reference.

Betulinic acid derivatives, prodrugs and salts are disclosed in the following documents: US 20150011627 A1 by Gribble et al. (published on Jan. 8, 2015), US 20130303607 A1 by Gribble et al. (published on Nov. 14, 2013), US 20120237629 A1 to Shode et al. (published September 20, 2012), US 20170204133 A1 to Regueiro-Ren et al. (published July 20, 2017), US 20170096446 A1 to Nitz et al. (April 6, 2017) published), 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), US 20140296546 A1 by Yan et al. published on Aug. 2), US 20140243298 A1 by Swidorski et al. (published on Aug. 28, 2014), US 20140221328 A1 by Parthasaradhi Reddy et al. (published on Aug. 7, 2014), and US 20140066416 A1 by Leunis et al. (2014 published on March 6, 2013), US 20130065868 A1 by Durst et al. (published on March 14, 2013), US 20130029954 A1 by Regueiro-Ren et al. (published on January 31, 2013), US 20120302530 by Zhang et al. A1 (published on November 29, 2012), US 20120214775 A1 by Power et al. (published on August 23, 2012), US 20120101149 A1 by Honda et al. (published on April 26, 2012), US Patent by Bullock et al. 20110224182 (published on Sept. 15, 2011), US 20110313191 A1 by Hemp et al. (published on Dec. 22, 2011), US 20110224159 A1 by Pichette et al. (published on Sept. 15, 2011) Published), US 20110218204 to Parthasaradhi Reddy et al. (published September 8, 2011), US 20090203661 A1 to Safe et al. (published August 13, 2009), US 20090131714 A1 to Krasutsky et al. Published on March 1), US 20090076290 to Krasutsky et al. (published March 19, 2009), US 20090068257 A1 to Leunis et al. (published March 12, 2009), US 20080293682 to Mukherjee et al. (November 27, 2008) published), US 20070072835 A1 to Pezzuto et al. (published March 29, 2007), US 20060252733 A1 to Jansen et al. (published November 9, 2006), and US 2006025274 A1 to O'Neill et al. published on November 9), the entire disclosure of which is incorporated herein by reference.

Antiviral compositions can be formulated in any suitable, pharmaceutically acceptable dosage form. Parenteral, otic, ocular, nasal, inhalable, buccal, sublingual, enteral, topical, oral, oral, and injectable dosage forms are particularly useful. Certain dosage forms include solid or liquid dosage forms. Exemplary suitable dosage forms include tablets, capsules, pills, caplets, lozenges, granules, solutions, suspensions, dispersions, vials, bags, bottles, injectable liquids, iv (intravenous), im (intramuscular). ) or ip (intraperitoneal) administrable fluids, and other such dosage forms known to those of ordinary skill in the art of pharmacy.

Because viral infection can affect multiple organs simultaneously and cause multiple organ failure, it is 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, compositions containing cardiac glycosides can be advantageously administered as inhalable compositions and oral compositions; sublingual compositions and oral compositions; inhalable compositions and sublingual compositions; inhalable compositions and non-digestible compositions Canal compositions; sublingual compositions and parenteral compositions; oral compositions and parenteral compositions, or other such combinations.

Suitable dosage forms containing the antiviral composition can be prepared by mixing the antiviral composition with a pharmaceutically acceptable excipient, as described in this specification or in the following documents: Pi et al. ("Ursolic acid nanocrystals for dissolution rate and bioavailability enhancement: influence of different particle size” in Curr. Drug Deliv. (Mar 2016), 13(8), 1358-1366), Yang et al. (“Self-microemulsifying drug delivery system for improved oral bioavailability of oleanolic acid: design and (2013), 8(1), 2917-2926), Li et al. (Development and evaluation of optimized sucrose ester stabilized oleanolic acid nanosuspensions prepared by wet ball milling with design of experiments” in Biol .Pharm.Bull.(2014), 37(6), 926-937), Zhang et al. (“Enhancement of oral bioavailability of triterpene through lipid nanospheres: preparation, characterization, and absorption evaluation” in J.Pharm.Sci.( June 2014), 103(6), 1711-1719), Godugu et al (“Approaches to improve the oral bioavailability and effects of novel anticancer drugs berberine and betulinic acid” in PLoS One (Mar 2014), 9(3): e89919 ), Zhao et al. (“Preparation and characterization of betulin nanoparticles for oral hypoglycemic drug by antisolvent precipitation” in Drug Deliv. (Sep 2014), 21(6), 467-479), Yang et al. (“Physicochemical properties and oral bioavailability of ursolic acid nanoparticles using supercritical anti-solvent (SAS) process” in Food Chem. (May 2012), 132(1), 319-325), Cao et al. (“Ethylene glycol-linked amino acid diester prodrugs of oleanolic acid for PEPT1-mediated transport: synthesis, testinal permeability and pharmacokinetics” in Mol. Pharm. (Aug. 2012), 9(8), 2127-2135), Li et al. (“Formulation, biological and pharmacokinetic studies of sucrose ester” -stabilized nanosuspensions of oleanolic acid” in Pharm. Res. (Aug 2011), 28(8), 2020-2033), Tong et al. (“Spray freeze drying with polyvinylpyrrolidone and sodium caprate for improved dissolution and oral bioavailablity of oleanolic acid, a BCS Class IV compound”in Int.J.Pharm.(Feb 2011), 404(1-2), 148-158), Xi et al. (Formulation development and bioavailability evaluation of a self-nanoemulsified drug delivery system of oleanolic acid” in AAPS PharmSciTech (2009), 10(1), 172-182), Chen et al. (“Oleanolic acid nanosuspensions: preparation, in-vitro characterization and enhanced hepatoprotective effect" in J. Pharm. Pharmacol. (Feb 2005), 57(2), 259-264), the entire disclosure of which is incorporated herein by reference.

Also according to US 8187644 B2 of Addington (issued May 29, 2012), US 7402325 B2 of Addington (issued July 22, 2008), US 8394434 B2 of Addington et al. (issued March 12, 2013) Suitable dosage forms are prepared, the entire contents of which are incorporated herein by reference. Suitable dosage forms can also be prepared as described in Examples 13-15.

Of particular note is the effective or therapeutically relevant amount of the antiviral compound (cardiac glycosides, triterpenes or combinations thereof). The term "effective amount" is to be understood as the intended pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount or quantity of active ingredient sufficient to achieve a desired or desired therapeutic response; or in other words, an amount sufficient to produce an appreciable biological response when administered to a patient. Perceivable biological responses can occur as a result of administration of single or multiple doses of the active substance. A dose may contain one or more dosage forms. It is to be understood that the particular dosage level for any patient will depend on a variety of factors, including the indication for treatment, the severity of the indication, the patient's health, age, sex, weight, diet, pharmacological response, the particular dosage form employed, and other such factors. class factor.

The desired dose for oral administration is up to 5 dosage forms, although as few as 1 and as many as 10 dosage forms may be administered as a single dose. Exemplary dosage forms may contain 0.01 to 100 mg or 0.01 to 100 μg of the antiviral composition per dosage form, 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 can be predetermined and/or tailored to achieve a particular therapeutic response or clinical benefit in a subject.

Cardiac glycosides can be provided in an amount sufficient to provide the subject, from about 20 to about 100 μg, An initial dose of about 12 μg to about 300 μg or about 12 μg to about 120 μg of oleandrin is present in the dosage form. The dosage form may comprise about 20 to about 100 μg of oleandrin, about 0.01 μg to about 100 mg, or about 0.01 μg to about 100 μg of oleandrin, an oleandrin extract, or an extract of European oleander containing oleandrin.

Antiviral agents can be included in oral dosage forms. Some embodiments of the dosage form are not enteric-coated and release their entrained antiviral composition in 0.5 to 1 hour or less. Some embodiments of dosage forms are enteric-coated and release their entrained antiviral compositions, eg, downstream from the stomach, such as the jejunum, ileum, small intestine, and/or large intestine (colon). The enteric-coated dosage form will release the antiviral composition into the systemic circulation within 1-10 hours after oral administration.

The antiviral composition can be contained in a fast-release, immediate-release, controlled-release, sustained-release, prolonged-release, delayed-release, burst-release, continuous-release, slow-release, or pulsed-release dosage form, or in both forms that exhibit these types of release. in one or more dosage forms. The release profile of the antiviral composition from the dosage form can be zero-order, pseudo-zero-order, first-order, pseudo-first-order, or sigmoidal. The plasma concentration profile of triterpenes in subjects to which the antiviral composition is administered may exhibit one or more maxima.

Based on human clinical data, it is expected that 50% to 75% of the administered dose of oleandrin will be orally available, thus providing about 10 to about 20 μg, about 20 to about 40 μg, about 30 to about 50 μg, about 40 to about 40 μg per dosage form 60 μg, about 50 to about 75 μg, or about 75 to about 100 μg of oleandrin. Considering that the average blood volume of an adult is 5 liters, the expected plasma concentrations of oleandrin will be in the range of about 0.05 to about 2 ng/ml, about 0.005 to about 10 ng/ml, about 0.005 to about 8 ng/ml, about 0.01 to about 7 ng /ml, in the range of about 0.02 to about 7 ng/ml, about 0.03 to about 6 ng/ml, about 0.04 to about 5 ng/ml, or about 0.05 to about 2.5 ng/ml. The recommended daily dose of oleandrin present in the SCF extract is generally from about 0.2 μg to about 4.5 μg/kg body weight twice a day. The dosage of oleandrin can be about 0.2 μg to about 1 μg/kg body weight/day, about 0.5 to about 1.0 μg/kg body weight/day, about 0.75 to about 1.5 μg/kg body weight/day, about 1.5 to about 2.52 μg/kg body weight/day, about 2.5 to about 3.0 μg/kg body weight/day, about 3.0 to about 4.0 μg/kg body weight/day, or about 3.5 to 4.5 μg oleandrin/kg body weight/day. The maximum tolerated dose of oleandrin can be from about 3.5 μg/kg body weight/day to about 4.0 μg/kg body weight/day. The minimum effective dose can be 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.

Because there are different combinations of triterpenes and different molar ratios therein, the antiviral composition can be administered from low to high doses. A therapeutically effective dose for humans is about 100-1000 mg or about 100-1000 μg of the antiviral composition/Kg body weight. The above dose can be administered up to 10 times in 24 hours. Other suitable dosage ranges are specified below.

All numerical values are approximate, meaning "approximately equal" to the above-mentioned numerical values.

It should be noted that the compounds of this specification may have one or more functions in the compositions or formulations of the invention. For example, a compound can be used as both a surfactant and a water-miscible solvent or as both a surfactant and a water-immiscible solvent.

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

As used in this specification, the terms "water-soluble solvent" or "water-miscible solvent" are used interchangeably to refer to an organic liquid that does not form a biphasic mixture with water, or is sufficiently soluble in water to provide a solvent containing at least 5% and no Liquid-phase separated organic liquids of aqueous solvent mixtures. Solvents are suitable for administration to humans or animals. Exemplary water-soluble solvents include - such as, but not limited to - PEG (poly(ethylene glycol)), PEG 400 (poly(ethylene glycol) having a molecular weight of about 400), ethanol, acetone, alkanols, alcohols, diethyl ether, propylene glycol , glycerol, triacetin, poly(propylene glycol), PVP (poly(vinylpyrrolidone)), dimethylsulfoxide, N,N-dimethylformamide, formamide, N,N- Dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

As used in this specification, the terms "water-insoluble solvent" or "water-immiscible solvent" are used interchangeably to refer to organic liquids that form a biphasic mixture with water, or form phase separation when the concentration of solvent in water exceeds 5% organic liquid. Solvents are suitable for administration to humans or animals. Exemplary water insoluble solvents include - such as but not limited to - medium/long chain triglycerides, oils, castor oil, corn oil, vitamin E, vitamin E derivatives, oleic acid, fatty acids, olive oil, softisan 645 (diglycerol caprylic acid) Acetate/Caprate/Stearate/Hydroxystearate Adipate), miglyol, captex (Captex 350: Tricaprylate/Caprate/Trilaurate; Captex 355: Glycerin Tricaprylate/Capric Triglyceride; Captex 355 EP/NF: Tricaprylate/Capric Medium Chain Triglyceride).

Listed in the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance for industry Q3C Impurities: Residual Solvents (1997) A suitable solvent was developed, which suggested what amount of residual solvent in the drug should be considered safe. 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-butyl methyl ether, cumene, ethanol, diethyl ether, ethyl acetate, ethyl formate Ester, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propane alcohol, pentane, 1-pentanol, 1-propanol, 2-propanol or propyl acetate.

Other materials that can be used as water immiscible solvents in the present invention include: Captex 100: Propylene Glycol Dicaprate; Captex 200: Propylene Glycol Dicaprylate/Dicaprate; Captex 200P: Propylene Glycol Dicaprylate/Dicaprate Caprate; Propylene Glycol Dicaprylocaprate ; Captex 300: Caprylic Triglyceride/Caprate; Captex 300 EP/NF: Caprylic Triglyceride/Capric Medium Chain Triglyceride; Captex 350: Caprylic Triglyceride/Capric Acid/Laureate; Captex 355: Caprylic Triglyceride/Capric Acid; Captex 355 EP/NF: Caprylic Triglyceride/Capric Medium Chain Triglyceride; Captex 500: Triacetin; Captex 500 P: Triacetin (Pharmaceutical Grade); Captex 800: Propylene Glycol Di(2-ethylhexanoate); Captex 810 D: Tricaprylin/Caprate/Sub Oleate; Captex 1000: Tricaprin; Captex CA: Medium Chain Triglyceride; Captex MCT-170: Medium Chain Triglyceride; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP/NF: Glycerin Monooleate; Capmul MCM: Medium Chain Mono- &Diglycerides; Capmul MCM C8: Glycerol Monocaprylate; Capmul MCM C10: Glycerol Monocaprate; Capmul PG-8: Propylene Glycol Monocaprylate; Capmul PG-12: Propylene Glycol Monolaurate; Caprol 10G10O: Decaoleate; Caprol 3GO: Triglycerol Monooleate; Caprol ET: Polyglycerol Mixed Fatty Acids Glycerides; Caprol MPGO: Hexaglycerol Dioleate; Caprol PGE 860: Decaglycerol Mono-, Dioleate.

As used in this specification, "surfactant" refers to a compound that contains a polar or charged hydrophilic moiety and a non-polar hydrophobic (lipophilic) moiety; that is, a surfactant is amphiphilic. The term surfactant can refer to a compound or a mixture of compounds, a surfactant can be a solubilizer, emulsifier or dispersant, and a surfactant can be hydrophilic or hydrophobic.

The hydrophilic surfactant can be any hydrophilic surfactant suitable for use in pharmaceutical compositions, which can be anionic, cationic, zwitterionic or nonionic, although nonionic hydrophilic surfactants are currently used. agent is better. As noted above, such nonionic hydrophilic surfactants will typically have HLB values greater than about 10. Mixtures of hydrophilic surfactants are also within the scope of the present invention.

Similarly, the hydrophobic surfactant can 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 other suitable solubilizers include: alcohols and polyols such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butylene glycol and its isomers, glycerol, pentaerythritol, sorbitol , mannitol, carbitol (transcutol), isosorbide dimethyl ether, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methyl cellulose and other cellulose derivatives, cyclodextrin and cyclodextrin Dextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol polyethylene glycol ether (tetraethylene glycol, commercially available from BASF under the trade name Tetraglycol) Or methoxypolyethylene glycol (Union Carbide); amides such as 2-pyrrolidone, 2-piperidone, caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkyl Piperidones, N-alkylcaprolactamides, dimethylacetamides, and polyvinylpyrrolidones; esters such as ethyl propionate, tributyl citrate, acetonitrile triethyl citrate, acetonitrile Tributyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, glycerol triacetate, propylene glycol monoethyl acid esters, propylene glycol diacetate, caprolactone and its isomers, valerolactone and its isomers, butyrolactone and its isomers; and other solubilizers known in the art, such as dimethyl Acetamide, dimethyl isosorbide (Arlasolve DMI (ICI)), N-methylpyrrolidone (Pharmasolve (ISP)), glycerol monocaprylate, diethylene glycol monoethyl ether (available from Gattefosse under the trade name Transcutol ) and water. Mixtures of solubilizers are also within the scope of the present invention.

Unless otherwise indicated, the compounds mentioned in this specification are readily available from standard commercial sources.

Although not required, the composition or formulation may further comprise one or more chelating agents, one or more preservatives, one or more antioxidants, one or more adsorbents, one or more acidifying agents, one or more alkalizing agents, one or more one or more defoamers, one or more buffers, one or more colorants, one or more electrolytes, one or more salts, one or more stabilizers, one or more tonicity modifiers, one or more diluents , or a combination thereof.

The compositions of the present invention may also include oils, such as fixed oils, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil; fatty acids, such as oleic acid, stearic acid, and isostearic acid; and fatty acid esters, such as Ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. The composition may also include alcohols such as ethanol, isopropanol, cetyl 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 is to be understood that compounds used in the field of pharmaceutical formulation often have multiple functions or purposes. Thus, if a compound named in this specification is mentioned only once or used to define more than one term in this specification, its purpose or function should not be construed as being limited to the stated purpose or functions, or function or functions. .

One or more components of the formulation may be present in their free base, free acid, or pharmaceutically or analytically acceptable salt form. As used herein, a "pharmaceutically or analytically acceptable salt" refers to a compound that has been modified by reacting it with an acid as desired to form an ionic bond pair. Examples of acceptable salts include conventional non-toxic salts formed from, for example, non-toxic inorganic or organic acids. Suitable non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfonic, sulfamic, phosphoric, nitric, and the like, and others known to those of ordinary skill in the art. Salts prepared from organic acids such as amino acids, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, and others known to those of ordinary skill in the art , tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid , Fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid. On the other hand, where the pharmaceutically active ingredient has an acidic functional group, a pharmaceutically acceptable base is added to form a pharmaceutically acceptable salt. A list of other suitable salts is found in Remington 's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, 1985, p. 1418, the relevant disclosure of which is incorporated herein by reference.

The term "pharmaceutically acceptable" as used in this specification means, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or any other problems or complications, and with reasonable benefit/risk ratio of such compounds, materials, compositions and/or dosage forms.

Dosage forms can be manufactured by any conventional means known in the pharmaceutical industry. Liquid dosage forms can be prepared by providing at least one liquid carrier and an antiviral composition in a container. One or more other excipients may be included in the liquid dosage form. Solid dosage forms can be prepared by providing at least one solid carrier and an antiviral composition. One or more other excipients may be included in the solid dosage form.

The dosage form can be packaged using conventional packaging equipment and materials. It may be contained in a package, bottle, vial, bag, syringe, envelope, packet, blister pack, box, ampule, or other similar container.

The compositions of the present invention may be included in any dosage form. Certain dosage forms include solid or liquid dosage forms. Exemplary suitable dosage forms include tablets, capsules, pills, caplets, lozenges, granules, and other congener dosage forms known to those of ordinary skill in the art of pharmacy.

In view of the foregoing description and the following examples, one of ordinary skill in the art will be able to practice the claimed invention without undue experimentation. The foregoing will be better understood with reference to the following examples, which detail specific procedures for making embodiments of the present invention. All references in the following examples are for illustrative purposes. The following examples should not be considered exhaustive, but merely illustrate a few of the many embodiments contemplated by the present invention.

Vero CCL81 cells were used in prophylactic and therapeutic trials (ATCC, Manassas, VA). Plaque assays were performed in Vero E6 cells, a gift from Vineet Menachery (UTMB, Galveston, TX). Cells were maintained in a 37 °C incubator with 5% _CO . Cells were grown in Dulbecco's Modified Eagle Medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin/streptomycin (Gibco, NY). Maintenance medium reduced FBS to 2%, but was otherwise the same. SARS-CoV-2, strain USA_WA1/2020 (Genbank accession number MT020880) was provided by the World Reference Center for Emerging Viruses and Arboviruses. All studies used NextGen-sequenced Vero Generation 4 stocks of SARS-CoV-2.

【Example】

Example 1

Supercritical fluid extraction of powdered oleander leaves

Method A. Using carbon dioxide

The oleander leaf material is harvested, washed, and dried, followed by the preparation of powdered oleander leaves by such crushing and dewatering equipment as described in US Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. The starting material used had a weight of 3.94 kg.

The starting material was combined with pure CO ₂ at a pressure of 300 bar (30 MPa, 4351 psi) and a temperature of 50 °C (122 °F) in an extractor device. A total of 197 kg of _CO2 was used to achieve a solvent to raw material ratio of 50:1. Next, the mixture of CO ₂ and raw materials is sent to a separation device, and the extract and carbon dioxide are separated by changing the pressure and temperature of the mixture.

A brown, sticky, viscous extract (65 g) was obtained with good fragrance. The color may be due to chlorophyll and other residual chromophoric compounds. To determine the exact yield, the tube and separator were flushed with acetone, and the acetone was evaporated to give an additional 9 g of extract. The total extracted amount was 74 g. The yield of the extract was 1.88% based on the weight of the starting material. The content of oleandrin in the extract was calculated using high pressure liquid chromatography and mass spectrometry to be 560.1 mg, or 0.76% yield.

Method B. Using a mixture of carbon dioxide and ethanol

The oleander leaf material is harvested, washed, and dried, followed by the preparation of powdered oleander leaves by such crushing and dehydration equipment as described in US Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. The weight of the starting material used was 3.85 kg.

In an extractor setup, the starting material was combined 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). A total of 160 kg of _CO2 and 8 kg of ethanol were used to achieve a solvent to raw material ratio of 43.6:1. Next, the mixture of CO ₂ , ethanol and raw materials is sent to a separation device, and the extract and carbon dioxide are separated by changing the pressure and temperature of the mixture.

After removal of the ethanol, a dark green, sticky, viscous blocky extract (207 g) was obtained, which was It obviously contains some chlorophyll. The yield of the extract was 5.38% based on the weight of the starting material. The content of oleandrin in the extract calculated using high pressure liquid chromatography and mass spectrometry was 1.89 g, or 0.91% yield.

Example 2

Hot water extract of powdered oleander leaves.

(Comparative example)

Hot water extraction is commonly used to extract oleandrin and other active components 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. Add 10 volumes of boiling water (by weight of the oleander starting material) to the powdered oleander leaves and continue stirring the mixture for 6 hours. The mixture was then filtered and the leaf residue was collected and extracted once more 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 material was dissolved in water and analyzed for oleandrin content using high pressure liquid chromatography and mass spectrometry. The amount of oleandrin was determined to be 3.68 mg. The yield of oleandrin was calculated to be 0.26% based on the amount of the extract.

Example 3

Preparation of pharmaceutical compositions.

Method A. Cremophor-Based Drug Delivery System

The following ingredients are provided in the amounts indicated.

Carrier #1 (LN2005-055-035)

The excipients were dispensed into jars and shaken in a New Brunswick Scientific C24KC Refrigerated Incubator shaker at 60°C for 24 hours to ensure homogeneity. The sample was then removed and visually inspected for dissolution. After 24 hours, the excipients and antiviral compositions were all dissolved in all formulations.

Method B. GMO/Cremophor-Based Drug Delivery System

The following ingredients are provided in the amounts indicated.

Carrier #2 (LN2005-055-045)

Follow the steps of Method A.

Method C. Labrasol-Based Drug Delivery System

The following ingredients are provided in the amounts indicated.

Carrier #3 (LN2005-055-047)

Follow the steps of Method A.

Method D. Vitamin E-TPGS-based micelle-forming system

The following ingredients are provided in the amounts indicated.

Follow the steps of Method A.

Method E. Multicomponent Drug Delivery System

The following ingredients are provided in the amounts indicated.

Follow the steps of Method A.

Method F. Multicomponent Drug Delivery System

The following ingredients are provided in the amounts indicated for inclusion in the capsule.

Follow the steps of Method A.

Example 4

Preparation of enteric-coated capsules

Step 1: Preparation of Liquid-Filled Capsules

Hard gelatin capsules (50, 00 size) were filled with the liquid composition of Example 3. The capsules were manually filled with 800 mg of the formulation, followed by hand sealing with 50% ethanol/50% aqueous solution. The capsules were then manually strapped with the indicated amounts of a 22% gelatin solution containing the following ingredients.

Mix the gelatin solution well and swell for 1-2 hours. After the swelling period, the solution was capped tightly and placed in a 55°C oven to liquefy. Once the entire gelatin solution became liquid, banding was performed.

Apply the sol solution to the capsule using a pointed 3/0 paint brush. Use the strapping tool provided by Shionogi. After strapping, the capsules were held at ambient conditions for 12 hours to cure the strapping.

Step II: Coating of Liquid-Filled Capsules

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

[Table 13]

If using capsules strapped as in Step I, the dispersion was applied to the capsules at a coating level of 20.0 ^mg /cm. The following conditions were used to carry out the coating of capsules.

Example 5

Treatment of Zika virus in subjects

Method A. Antiviral Composition Therapy

The antiviral composition is administered to a subject exhibiting a Zika virus infection, and the subject is administered a therapeutically relevant dose for a period of time as specified in the dosing schedule. The subject's level of treatment response was determined periodically. Treatment response can be determined by determining the Zika virus titer in the blood or plasma of the subject level. If the level of therapeutic response at a dose is too low, the dose is escalated according to a predetermined dose escalation plan until the desired level of therapeutic response is achieved in the subject. Treatment of the subject with the antiviral composition is continued as needed, and the dose or dosing regimen can be adjusted as needed until the patient achieves the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Compositions and Other Agents

Method A above is followed except that the subject is instructed and administered one or more other therapeutic agents for the treatment of Zika virus infection or symptoms thereof. Thus, one or more other therapeutic agents can be administered before, after, or with the antiviral composition. Dose escalation (or decrementation) of one or more other therapeutic agents may also be performed.

Example 6

In vitro assessment of antiviral activity against Zika virus infection

Method A. Pure Compounds

In the presence of cardiac glycosides, Zika virus (ZIKV; PRVABC59 strain; ATCC VR-1843; https://www.atcc.org/Products/All/VR-1843.aspx was administered at a viral infectious dose (MOI) of 0.2 ) infected Vero E6 cells (also known as Vero C1008 cells, ATTC No. CRL-1586; https://www.atcc.org/Products/All/CRL-1586.aspx). Cells were incubated with virus and compound for 1 hour, after which the inoculum and compound were discarded. Cells were given fresh medium and incubated for 48 hours, after which they were fixed with formalin and stained for ZIKV infection. Representative infection rates for oleandrin (FIG. 1A) and digitonin (FIG. 1B) determined by scintigraphy are depicted. Other compounds were evaluated under the same conditions, and they showed very different degrees of antiviral activity against Zika virus.

Method B. Compounds in the form of extracts

Extracts containing target compounds to be detected were evaluated as detailed in Method A, except that the amount of extract was normalized to the amount of target compound in the extract. For example, an extract containing 2% by weight of oleandrin contains 20 μg of oleandrin per 1 mg of extract. Therefore, if the The expected amount of oleandrin is 20 μg, then 1 mg of the extract will be used for the assay.

Example 7

Preparation of tablets containing antiviral compositions

An initial tableting mix of 3% Syloid 244FP and 97% microcrystalline cellulose (MCC) was mixed. Next, the existing batch of the composition prepared according to Example 3 was mixed into the Syloid/MCC mixture by wet granulation. This mixture is labeled "Initial Tablet Mix" in the table below. Additional MCC was added extragranularly to increase compressibility. This addition to the initial tableting mix is labeled "extragranular addition". The resulting mixture from extragranular addition was of the same composition as the "Final Tablet Mix".

Extra-granular addition

Final Tablet Mix: Abbreviated

[Table 17]

Final Tablet Mix: Detailed

Syloid 244FP is a colloidal silica manufactured by Grace Davison. Colloidal silica is commonly used to provide various functions such as adsorbents, glidants and tablet disintegrants. Syloid 244FP was chosen for its ability to absorb 3 times its weight in oil and has a 5.5 micron particle size.

Example 8

HPLC analysis of solutions containing oleandrin

Samples (oleandrin standard, SCF extract and hot water extract) were analyzed in HPLC (Waters) using the following conditions: Symmetry C18 column (5.0 μm, 150×4.6 mm I.D.; Waters); MeOH:water=54:46 (v/v) mobile phase and flow rate of 1.0 ml/min. 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 approximately the target concentration of oleandrin. The retention time of oleandrin was determined by using an internal standard. The concentration of oleandrin can be determined/corrected by forming a signal response curve using an internal standard.

Example 9

Preparation of pharmaceutical compositions

The pharmaceutical compositions of the present invention can be prepared by any of the following methods. Mixing can be done in wet or dry conditions. During preparation, the pharmaceutical composition can be compressed, dried, or compressed and dried. Pharmaceutical compositions can be dispensed into dosage forms.

method a.

At least one pharmaceutical excipient is mixed with at least one antiviral compound disclosed in this specification.

method b.

At least one pharmaceutical excipient is mixed with at least two antiviral compounds disclosed in this specification.

method c.

At least one pharmaceutical excipient is mixed with at least one cardiac glycoside disclosed in this specification.

method D.

At least one pharmaceutical excipient is mixed with at least two triterpenes disclosed in this specification.

method E.

At least one pharmaceutical excipient is mixed with at least one cardiac glycoside disclosed in this specification and at least two triterpenes disclosed in this specification.

method F.

At least one pharmaceutical excipient is mixed with at least three triterpenes disclosed in this specification.

Example 10

Preparation of Triterpene Mixtures

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

For each composition, three distinct respective solutions were prepared such that 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 combining the individual triterpene components to form a mixture. The triterpenoid mixtures prepared as described above that provide acceptable antiviral activity can be formulated into antiviral compositions.

Antiviral composition with oleanolic acid and ursolic acid

Known amounts of oleanolic acid and ursolic acid are mixed according to predetermined molar ratios of the components as defined in this specification. The components are mixed in solid form or in the following solvent(s): e.g. methanol, ethanol, chloroform, acetone, propanol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethy Methylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains components in relative molar ratios as described in this specification.

For a pharmaceutically acceptable antiviral composition, at least one pharmaceutically acceptable excipient is mixed with a pharmacologically active agent. Antiviral compositions are formulated for administration to mammals.

Antiviral composition with oleanolic acid and betulinic acid

Known amounts of oleanolic acid and betulinic acid are mixed according to predetermined molar ratios of the components as defined in this specification. The components are mixed in solid form or in the following solvent(s): e.g. methanol, ethanol, chloroform, acetone, propanol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethy Methylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains the components in relative molar ratios as described in this specification.

For a pharmaceutically acceptable antiviral composition, at least one pharmaceutically acceptable excipient is mixed with a pharmacologically active agent. Antiviral compositions are formulated for administration to mammals.

Antiviral composition with oleanolic acid, ursolic acid and betulinic acid

Known amounts of oleanolic acid, ursolic acid and betulinic acid are mixed according to predetermined molar ratios of the components defined in this specification. The components are mixed in solid form or in the following solvent(s): e.g. methanol, ethanol, chloroform, acetone, propanol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethy Methylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains the components in relative molar ratios as described in this specification.

For a pharmaceutically acceptable antiviral composition, at least one pharmaceutically acceptable excipient is mixed with a pharmacologically active agent. Antiviral compositions are formulated for administration to mammals.

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

Known amounts of oleandrin, oleanolic acid, ursolic acid and betulinic acid are mixed according to predetermined molar ratios of the components as defined in this specification. The components are mixed in solid form or in the following solvent(s): e.g. methanol, ethanol, chloroform, acetone, propanol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethy Methylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains the components in relative molar ratios as described in this specification.

For a pharmaceutically acceptable antiviral composition, at least one pharmaceutically acceptable excipient is mixed with a pharmacologically active agent. Antiviral compositions are formulated for administration to mammals.

Example 12

Treatment of Filovirus Infections in Subjects

Exemplary filovirus infections include Ebola virus and Marburg virus.

Method A. Antiviral Composition Therapy

The antiviral composition is administered to a subject exhibiting a filovirus infection, and the subject is additionally administered a therapeutically relevant dose for a period of time as specified in the dosing schedule. The subject's level of treatment response was determined periodically. The level of therapeutic response can be determined by determining the filovirus titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose is escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition is continued as needed, and the dose or dosing regimen may be adjusted as needed until the patient achieves the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Compositions and Other Agents

Method A above is followed except that the subject is instructed and administered one or more additional therapeutic agents for the treatment of filovirus infection or symptoms thereof. Thus, one or more other therapeutic agents can be administered before, after, or with the antiviral composition. Dose escalation (or decrementation) of one or more other therapeutic agents may also be performed.

Example 13

Treatment of Flavivirus Infections in Subjects

Typical flavivirus infections include yellow fever, dengue fever, Japanese encephalitis, West Nile virus, Zika virus, tick-borne encephalitis, Kejeldahl disease, Alkhurma disease, Chikungunya virus, Omsk hemorrhagic fever, and Powassan Viral infection.

Method A. Antiviral Composition Therapy

The antiviral composition is administered to a subject exhibiting a flavivirus infection, and the subject is additionally administered a therapeutically relevant dose for a period of time as specified in the dosing schedule. Periodically determine the subject's response to treatment level. The level of therapeutic response can be determined by determining the flavivirus titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose is escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition is continued as needed, and the dose or dosing regimen may be adjusted as needed until the patient achieves the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Compositions and Other Agents

Method A above is followed except that the subject is instructed and administered one or more other therapeutic agents for the treatment of flavivirus infection or symptoms thereof. Thus, one or more other therapeutic agents can be administered before, after, or with the antiviral composition. Dose escalation (or decrementation) of one or more other therapeutic agents may also be performed.

Example 14

Evaluation of antiviral activity against Zika virus and Dengue virus

Target cells were infected over a range of concentrations and CPE-based antiviral assays were performed in the presence or absence of test compositions. Infection of target cells results in cytopathic effects and cell death. In this type of assay, the decrease in CPE and the corresponding increase in cell viability in the presence of the test composition are used as indicators of antiviral activity. For CPE-based assays, cell viability was determined with neutral red readings. Living cells bind neutral red in their lysosomes. Neutral red uptake is dependent on the ability of living cells to maintain the pH within their lysosomes below that in the cytoplasm, an active process that requires ATP. Once inside the lysosome, the neutral red dye becomes charged and remains within the cell. After incubation with neutral red (0.033%) for 3 hours, the extracellular dye was removed, the cells were washed with PBS, and the intracellular neutral red was lysed with a solution of 50% ethanol + 1% acetic acid. The amount of neutral red in solution was quantified by reading the absorbance (optical density) of each well at 490 nm.

Adherent cell lines were used to assess the antiviral activity of the compositions against a panel of viruses. The composition was preincubated with target cells for 30 minutes prior to adding virus to the cells. composition during the incubation period of infection present in the cell culture medium. For each infection assay, viability assays were set up in parallel using the same concentrations of the compositions (in duplicate) to determine the cytotoxic effects of the compositions in the absence of virus.

The antiviral activity of the test composition is determined by comparing the level of infection (immunostain-based assay) or survival (CPE-based assay) of cells under the test conditions to the level of infection or survival of uninfected cells. Cytotoxic effects were assessed in uninfected cells by comparing survival in the presence of inhibitor to that of sham-treated cells. Cytotoxicity was determined by XTT viability assays performed at the same time points as the readings of the corresponding infection assays.

The test compositions were dissolved in 100% methanol. Eight concentrations of the composition (in duplicate) were generated by making 8-fold dilutions, starting with 50 [mu]M as the highest concentration tested. The highest tested concentration of the composition (50 μM) resulted in a final concentration of methanol in the culture broth of 0.25% (v/v%). Each test plate contained an 8-fold dilution series of methanol carrier at concentrations that reflected the final concentration of methanol in the test conditions for each composition. Where possible, the EC50 and CC50 of the compositions of each assay were determined using GraphPad Prism software.

Antiviral activity was assessed by the degree of protection against virus-induced cytopathic effect (CPE). Cells were challenged with virus in the presence of varying concentrations of controls or compositions. After 6 days of infection (ZIKV, Zika virus) or 7 days (DENV, Dengue virus), by quantifying cell viability under different experimental conditions and comparing the values with untreated cells and vehicle alone (infection medium ) treated cells were compared to monitor the degree of protection against CPE.

Quality control of neutralization assays was performed on each plate to determine: i) signal versus background (S/B) values; ii) inhibition by known inhibitors; and iii) assay variation, which was determined by all data points The coefficient of variation (CV) was determined. The overall change in the infection test ranged from 3.4% to 9.5%, and in the survival test, the overall change ranged from 1.4% to 3.2%, calculated as the mean of all C.V. values. Infect The signal-to-background (S/B) range for the assay was 2.9 to 11.0, while the signal-to-background (S/B) range for the survival assay was 6.5 to 29.9.

DENV2-induced cytopathic effect (CPE) protected with neutral red readout: For DENV2 antiviral assays, the 08-10381 Montserrat strain was used. Virus stocks were produced in C6/36 insect cells. Vero cells (epithelial kidney cells derived from green monkey ( Cercopithecus aethiops )) were cultured in MEM with 5% FBS (MEM5). For both infection and viability assays, cells were seeded at 10,000 cells/well in 96-well clear plates and maintained in MEM5 for 24 hours at 37°C. On the day of infection, samples were diluted 8-fold in a U-chassis using MEM with 1% bovine serum albumin (BSA). Test material dilutions were prepared at a final concentration of 1.25X and 40 μl were incubated with target cells for 30 minutes at 37°C. After pre-incubation of the test material, 10 μl of virus dilution prepared in MEM with 1% BSA was added to each well (50 μl final volume/well) and the plates were placed in a humidified incubator with 5% CO ₂ . Incubate at 37°C for 3 hours. The volume of virus used in the assay was predetermined to generate a signal in the linear range of inhibition by Ribavirin and Compound A3, a known inhibitor of DENV2. After infection culture, cells were washed with PBS followed by MEM (MEM2) containing 2% FBS to remove unbound virus. Thereafter, 50 μl of a culture medium containing inhibitor dilutions prepared at a concentration of 1X in MEM2 was added to each well. 96-well clear plates were cultured at 37°C for 7 days in an incubator (5% _CO2 ). The assay plates contained a virus-free control ("sham infection"), infected cells incubated with broth only, infected cells incubated with vehicle (methanol) only, and wells without cells (for background determination). Control wells containing 50 [mu]M ribavirin and 0.5 [mu]M Compound A3 were also included on the assay plate. Seven days after infection, cells were stained with neutral red to monitor cell viability. Test materials were evaluated in duplicate using serial 8-fold dilutions in infection medium. Controls included cells cultured without virus ("pseudo-infection"), infected cells cultured with medium alone, or infected cells in the presence of ribavirin (0.5 μM) or A3 (0.5 μM). Complete replicate inhibition curves with methanol vehicle only were included on the same assay plate.

ZIKV-induced cytopathic effect (CPE) protected with neutral red readout: For ZIKV antiviral assays, the PLCal_ZV strain was used. Vero cells (epithelial kidney cells derived from green monkeys) were cultured in MEM with 5% FBS (MEM5). For both infection and viability assays, cells were seeded at 10,000 cells/well in 96-well clear plates and maintained in MEM5 for 24 hours at 37°C. On the day of infection, samples were diluted 8-fold in a U-chassis using MEM with 1% bovine serum albumin (BSA). Test material dilutions were prepared at a final concentration of 1.25X and 40 μl were incubated with target cells for 30 minutes at 37°C. After pre-incubation of the test material, 10 μl of virus dilutions prepared in MEM with 1% BSA were added to each well (50 μl final volume per well) and the plates were placed in a humidified incubator with 5% _CO . Incubate at 37°C for 3 hours. After infection culture, cells were washed first with PBS followed by MEM (MEM2) containing 2% FBS to remove unbound virus. Thereafter, 50 μl of a culture medium containing inhibitor dilutions prepared at a concentration of 1X in MEM2 was added to each well. Plates were incubated at 37°C for 6 days in an incubator (5% _CO2 ). A virus-free control group ("sham infection"), infected cells incubated with broth only, infected cells incubated with vehicle (methanol) only, and cell-free wells (to determine background) were included in the assay plate. Six days after infection, cells were stained with neutral red to monitor cell viability. Test material was evaluated in duplicate using serial 8-fold dilutions in infection medium. Controls included cells cultured without virus ("pseudo-infection"), infected cells cultured with medium only, or infected cells in the presence of A3 (0.5 μM). Complete replicate inhibition curves with 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 the absorbance at 490 nm. The average signal obtained in cell-free wells was subtracted from all samples. Next, all data points were calculated as a percentage of the average signal observed in 8 wells of sham (uninfected) cells on the same assay plate. Infected cells treated with media alone reduced the signal to an average of 4.2% (for HRV), 26.9% (for DENV) and 5.1% (for ZIKV) of the signal observed in uninfected cells. The signal-to-background (S/B) for this assay was 2.9 (for DENV) and 7.2 (for ZIKV), which was determined as the percent survival in "pseudo-infected" cells compared to the percent survival in infected cells treated with vehicle alone.

Viability Assay (XTT) to Assess Compound-Induced Cytotoxicity: Mock-infected cells were incubated with inhibitor dilution (or medium only) using the same experimental setup and inhibitor concentration as used in the corresponding infection assay. The incubation temperature and duration of the incubation period were the same as the conditions for 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 yellow tetrazolium salt (XTT), which forms an orange formazan dye. The aforementioned reactions occur only in living cells with active mitochondria. Formazan dye was directly quantified using a scanning multiwell spectrophotometer. Background levels obtained from cell-free wells were subtracted from all data points. The viability assay plate contained a control vehicle with methanol only (7 concentrations, the same as the final percentage of methanol for each oleandrin assay well). Viability was monitored by measuring absorbance at 490 nm.

Analysis of cytotoxicity data: For XTT experiments, cell viability was determined by monitoring absorbance at 490 nm. The average signal obtained in cell-free wells was subtracted from all samples. Next, all data points were calculated as a percentage of the average signal observed in 8 wells of sham (uninfected) cells on the same assay plate. The signal-to-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 determined to be compared to the signal observed in cell-free wells Percent survival in "pseudo-infected" cells.

Example 15

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

method a.

EBOV/Kik (A, MOI=1) or MARV/Ci67 (B, MOI=1) in the presence of oleandrin, digitonin or PBI-05204 (a plant extract containing oleandrin) Vero E6 cells were infected. 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 counted using Operetta. C) Vero E6 cells were treated with the above compounds. ATP levels were determined 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 hours post infection (A, C) or 24 hours post infection (B, D), oleandrin or PBI-05204 were added to cells for 1 hour, then discarded and cells were grown back into culture. 48 hours after infection, the analysis of infected cells is shown in Figure 1.

method c.

Vero E6 cells were infected with EBOV or MARV in the presence of oleandrin or PBI-05204 and cultured for 48 hours. Supernatants from infected cell cultures were transferred to fresh Vero E6 cells, incubated for 1 hour, and then discarded (as described in A). Cells containing the transferred supernatant were cultured for 48 hours. Cells infected with EBOV (B) or MARV (C) were detected as previously described. The control infection rate was 66% for EBOV and 67% for MARV.

Example 16

Assessment of antiviral activity against Togaviridae viruses

(Alphavirus: 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 hours in the presence or absence of the indicated compounds. Infected cells were detected and counted on the Operetta as described in this specification.

Example 17

Treatment of Paramyxoviridae Infections in Subjects

Exemplary Paramyxoviridae viral infections include Henivirus infections, Nipah virus infections, or Hendra virus infections.

Method A. Antiviral Composition Therapy

The antiviral composition is administered to a subject exhibiting a Paramyxoviridae infection, in addition to which the subject is administered a therapeutically relevant dose for a period of time as indicated by the dosing regimen. The subject's level of treatment response was determined periodically. The level of therapeutic response can be determined by determining the viral titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose is escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition is continued as needed, and the dose or dosing regimen may be adjusted as needed until the patient achieves the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Compositions and Other Agents

Method A above is followed except that the subject is instructed and administered one or more additional therapeutic agents for the treatment of Paramyxoviridae infections or symptoms thereof. Thus, one or more other therapeutic agents can be administered before, after, or with the antiviral composition. Dose escalation (or decrementation) of one or more other therapeutic agents may also be performed.

Example 18

Cell line and isolation of primary huPBMC

Supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biowest), 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 20 μg/mL cyanine sulfate in a humidified incubator at 37 °C, 10% _CO Viral-producing HTLV-1-transformed (HTLV-1+) SLB1 lymphoma T cell line (Arnold et al. , 2008; kindly provided by P. Green, The Ohio State University-Comprehensive Cancer Center).

Primary human peripheral blood mononuclear cells (huPBMC) were isolated from whole blood samples provided by SMU Memorial Health Center under a protocol approved by the SMU Institutional Review Board, A sample without an identifier and in compliance with the Declaration of Helsinki principles. 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 samples were then gently plated in 3 ml of lymphocyte separation. culture medium (MP Biomedicals). Samples were centrifuged at 400 x g for 30 min in a swing rotor at room temperature, then the buffy coat huPBMCs were aspirated, washed twice in RPMI-1640 medium (ATCC No. 30-2001), and washed at 260 x The pellet was centrifuged at g for 7 min. Cells were resuspended in supplemented 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) and stimulated with 10 ng/ml phytohemagglutinin (PHA; Sigma-Aldrich) for 24 h and grown in a humidified incubator at 37 °C, 10% _CO . The next day, cells were pelleted by centrifugation at 260 x g for 7 min, washed twice with RPMI-1640 medium to remove PHA, and then resuspended in complete medium supplemented with antibiotics and 50 U/ml hu-IL-2 and cultivate.

Example 19

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

To generate HTLV-1+SLB1 T cell clones expressing GFP, ^2x106 SLB1 cells were plated in 60 ^mm2 tissue culture dishes (Corning) in IMDM supplemented with 10% heat-inactivated FBS and antibiotics, followed by pLenti -6.2/V5-DEST green fluorescent protein expression vector, and lentiviral particles carrying blasticidin resistance gene for transduction. 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 then resuspended in complete medium (Life Technologies) supplemented with 5 μg/mL blasticidin. ) and aliquoted into 96-well microtiter plates (Corning). Cultures were maintained for two weeks with blasticidin screening in a humidified incubator at 37 °C and 10% _CO . GFP-expressing lymphoblasts were screened by fluorescence microscopy, followed by GFP-expressing homogenous cell colonies by limiting dilution plating in 96-well microtiter plates. The obtained HTLV-1+SLB1/pLenti-GFP T lymphocyte strain was expanded and passaged repeatedly; GFP expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the use of rabbit antibodies. Confirmation by immunoblotting (Santa Cruz Biotechnology) of GFP(FL) polyclonal antibody.

Example 20

Quantification of virus production and particle infectivity by anti-HTLV-1 p19 _Gag ELISA

To determine the effect of oleandrin or oleander extract on HTLV-1 provirus replication and release of newly synthesized extracellular viral particles, the HTLV- ¹ +SLB1 lymphoma T cell line was plated at 2x10 cells/well in 96 wells 300 μl of complete medium supplemented with antibiotics in microtiter plates and incubated at 37 °C, 10% CO ₂ . Purified oleandrin compounds and European oleander extract (Phoenix Biotechnology; see Singh et al., 2013) were resuspended in carrier solution ( ₂₀ % v/v dimethylsulfite, DMSO, distilled in MilliQ/deionized H 0) at a stock concentration of 2 mg/ml, followed by sterilization using a luer-lock 0.2 μm syringe filter (Millipore). HTLV-1+SLB1 cells were treated with oleandrin or oleander extract at concentrations of 10, 50 and 100 μg/ml, or with increasing (1.5, 7.5 and 15 μl) vehicle controls for 72 hours. The 96-well microtiter plates were then centrifuged at 260 x g for 7 min at room temperature in a swing rotor using an Eppendorf A-2-DWP to pellet the cells and assayed by colorimetric anti-p19 ^Gag enzyme-linked immunosorbent assay (ELISA). ; Zeptometrix) to quantify the levels of extracellular p19 ^Gag -containing HTLV-1 particles released into the culture supernatant relative to the p19 ^Gag protein standard. Samples were analyzed in triplicate in absorbance mode at 450 nm on a Berthold Tristar LB 941 multimode microplate reader.

To assess the infectivity of newly synthesized extracellular HTLV-1 particles collected from oleandrin-treated cells, 2x10 HTLV- ¹ +SLB1 T lymphoblastoid cells were plated in 300 μl of complete culture medium supplemented with antibiotics to Cultures were treated with increasing concentrations (10, 50 and 100 μg/ml) of oleandrin or European oleander extract, or control vehicle (1.5, 7.5 and 15 μl) for 72 hours. Next, 50 μl of the virus-containing supernatant was used to directly infect huPBMCs plated at a density of 2×10 ⁴ cells/well on complete medium supplemented with antibiotics and hu-IL-2 in 96-well microtiter plates. Oleandrin compounds, European oleander extract, or control vehicle were maintained in huPBMC medium to control reinfection events that may be caused by newly produced particles. After 72 hours, the relative levels of extracellular release of p19 ^Gag -containing HTLV-1 virions into culture supernatants of infected huPBMCs were quantified by anti-HTLV-1 p19 ^Gag ELISA.

Example 21

Determination of apoptosis

To assess the relative cytotoxicity of oleandrin compounds, extracts of European oleander, or control vehicle in treated cell cultures, 2x10 HTLV- ¹ +SLB1 lymphoma T cells or activated/cultured huPBMCs were plated in 300 μl in complete medium supplemented with antibiotics and maintained in a humidified incubator at 37 °C, 10% _CO . Oleandrin or Oleander extract at increasing concentrations (10, 50 and 100 μg/ml), or control vehicle (1.5, 7.5 and 15 ml) were incubated for 72 hours. Cyclophosphamide (50 μM; Sigma-Aldrich)-treated cells were included as a positive control for apoptosis. Cells were then aspirated and plated on Permanox 8-chamber tissue culture slides (Nalge), which had been treated with 0.01% Poly- _L -Lysine and Concanavalin A (1 mg/mL; Sigma-Aldrich) pretreatment with sterile solution. The samples were then stained using a microscopic apoptosis detection kit, phospholipid-binding protein V was conjugated to fluorescein isothiocyanate (phospholipid-binding protein V-FITC) and propidium iodide (PI; BD-Pharmingen), and The relative percentage of apoptotic (ie, phospholipid binding protein V-FITC and/or PI-positive) cells per field was quantified in triplicate by conjugated fluorescence microscopy using a 20x objective. The total number of cells per field was quantified by microscopy using a DIC phase-contrast filter.

Example 22

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

Because transmission of HTLV-1 typically occurs by direct contact across viral synapses between infected cells and uninfected target cells (Igakura et al., 2003; Pais-Correia et al., 2010; Gross et al., 2016 ; Omsland et al., 2018; Majorovits et al., 2008), we tested whether oleandrin, European oleander extract, or a control vehicle could affect viral synapse formation and/or the in vitro passage of infectious HTLV-1 particles between cells Spread of interaction. For these experiments, 2x10 ⁴ virus-producing HTLV-1+SLB1 T cells were plated in 96-well microtiter plates and incubated with mitomycin in 300 μl of complete medium at 37°C, 10% _CO C (100 μg/mL) was treated for 2 hours (Bryja et al., 2006). The culture medium was then removed, cells were washed twice with serum-free IMDM, and cells were treated with increasing (10, 50, and 100 μg/ml) oleandrin or European oleander extract, or control vehicle (1.5, 7.5, and 15 μl) 15 minutes or 3 hours. Alternatively, 2x10 GFP ^- expressing HTLV-1+SLB1/pLenti-GFP T cells were plated on 8-chamber tissue culture slides in 300 μl of complete medium and treated with mitomycin C and treated with serum-free IMDM washed 2 times, followed by treatment with oleandrin, European oleander extract, or control vehicle as described in the conjugate focus microscopy experiments. Next, we aspirated the culture medium, washed HTLV-1+SLB1 cells twice with serum-free medium, and added 2x10 ^huPBMCs to 300 μl of huPBMC supplemented with 20% FBS, antibiotics, and 50 U/ml hu-IL-2 The cells were then co-cultured in each well of RPMI-1640 medium in a humidified incubator at 37°C, 10% _CO for an additional 72 hours (cells were co-cultured for 6 hours using SLB1/pLenti-GFP lymphoblastoid cells). Viral synapse formation and virus transmission were observed by conjugation focus microscopy). As a negative control, huPBMCs were cultured alone in the absence of virus-producing cells. Oleandrin, European oleander extract and vehicle were maintained in the co-culture medium. Relative levels of extracellular p19 ^Gag -containing HTLV-1 particles released into co-culture supernatants due to cell-to-cell virus transmission were quantified by performing an anti-HTLV-1 p19 ^Gag ELISA. GFP-positive HTLV-1+SLB/pLenti-GFP cells and huPBMCs were visualized using immunofluorescence conjugate focus microscopy by staining fixed samples with anti-HTLV-1 gp21 ^Env primary antibody and rhodamine red-conjugated secondary antibody Viral synapses formed. Diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes) nuclear staining was included for comparison and visualization of uninfected (ie, HTLV-1 negative) cells. Cell-to-cell spread of HTLV-1 to huPBMCs in co-culture assays was quantified by counting the relative percentages of HTLV-1 gp21 ^Env -positive (and GFP-negative) huPBMCs in 20 fields using a 20x objective.

Example 23

microscope

Phospholipid-binding protein V-conjugated fluorescence microscopy was visualized using a Plan-Apochromat 20x/0.8 objective and Zeiss ZEN system software (Carl Zeiss Microscopy) on a Zeiss LSM800 instrument equipped with an Airyscan detector and staged _CO incubator. FITC/PI stained samples to quantify apoptosis and cytotoxicity. Viral synapse formation between mitomycin C-treated HTLV-1+SLB1/pLenti-GFP lymphoblasts and cultured huPBMCs was observed by immunofluorescence conjugate focus microscopy using a Plan-Apochromat 20x/0.8 objective. Viral spread (ie, determined by quantifying the relative percentage of anti-HTLV-1 gp21 ^Env -positive huPBMCs). Relative fluorescence intensity of DAPI, anti-HTLV-1 gp21 ^Env specificity (positive for Rhodamine Red) and GFP signal was quantified graphically using Zen 2.5D analysis tool (Carl Zeiss Microscopy). Plan Fluor 10x/0.30 objective and DIC retardation filter by conjugation fluorescence microscope on Nikon Eclipse TE2000-U inverted microscope equipped with 633nm and 543nm He/Ne and 488nm Ar lasers and D-Eclipse Conjugate Imaging System Light slides (Nikon Instruments) were used to screen for GFP-expressing HTLV-1+SLB1/pLenti-GFP T cell colonization.

Example 24

Statistical Analysis

Statistical significance of the experimental dataset was determined using an unpaired two-tailed Student's t-test (α=0.05), and P-values were calculated using the 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 (****). Error bars represent SEM from at least three independent experiments unless otherwise stated.

Example 25

Treatment of delta retroviral infection in subjects

Exemplary delta retroviral infection containing HTLV-1

Method A. Antiviral Composition Therapy

The antiviral composition is administered to a subject exhibiting HTLV-1 infection, and the subject is additionally administered a therapeutically relevant dose for a period of time as specified in the dosing schedule. The subject's level of treatment response was determined periodically. The level of therapeutic response can be determined by determining the HTLV-1 viral titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose is escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition is continued as needed, and the dose or dosing regimen may be adjusted as needed until the patient achieves the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Compositions and Other Agents

Method A above is followed except that the subject is instructed and administered one or more additional therapeutic agents for the treatment of HTLV-1 infection or a symptom thereof. Thus, one or more other therapeutic agents can be administered before, after, or with the antiviral composition. Dose escalation (or decrementation) of one or more other therapeutic agents may also be performed. Exemplary other therapeutic agents are described in this specification.

Example 26

Treatment of CoV infection in subjects

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

The antiviral composition is administered to a subject exhibiting a CoV infection, and the subject is additionally administered a therapeutically relevant dose for a period of time as specified in the dosing schedule. Periodically determine the subject's therapeutic response to water flat. The level of therapeutic response can be determined by determining the CoV viral titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose is escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition is continued as needed, and the dose or dosing regimen may be adjusted as needed until the patient achieves the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Compositions and Other Agents

Method A above is followed except that the subject is instructed and administered one or more other therapeutic agents for the treatment of CoV infection or its symptoms. Thus, one or more other therapeutic agents can be administered before, after, or with the antiviral composition. Dose escalation (or decrementation) of one or more other therapeutic agents may also be performed. Exemplary other therapeutic agents are described in this specification.

Example 27

Treatment of COVID-19 Infections in Subjects Using ANVIRZEL ^TM

To children (infants) presenting with COVID-19, ANVIRZEL ^™ is administered as follows to treat symptoms associated with COVID-19. The subject's viral infection worsened prior to administration of ANVIRZEL ^™ . Subjects were assigned and administered ANVIRZEL ^™ according to the following dosing schedule: an initial dose of 0.25 mL of recombinant ANVIRZEL ^™ , followed by 0.5 mL of recombinant ANVIRZEL ^™ every twelve hours for two to three days, the subject's COVID-19 The infection resolved and no drug-related toxicity was observed.

Example 28

In vitro evaluation of oleandrin against COVID-19 infection

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

A stock solution of oleandrin in methanol (10 mg oleandrin/mL) was prepared for the preparation of a stock solution containing DMSO (in aqueous medium RPMI 1640 and oleandrin (20 μg/mL, 10 μg/mL, 1.0 μg/mL or 0.1 μg/mL). μg/mL) is 0.1% or 0.01% v/v) culture medium, culture medium such as shown below.

Uninfected VERO E6 cells in culture (target initial cell count of 1 x ¹⁰⁶ ) were incubated for 30 minutes at 37°C in each vial of the indicated medium. A viral inoculum of SARS-CoV-2 was then added to each vial to achieve the target initial viral titer (approximately PFU/mL ^1x104 ). The target MOI (viral infectious dose) was about 0.1. The solution was incubated at 37°C for an additional 2 hours to achieve infection of VERO E6 cells. Infected VERO E6 cells were then washed with control vehicle to remove extracellular virus and oleandrin. A new aliquot of each medium was added to each corresponding vial of infected VERO E6 cells. Those in the second aliquot that received oleandrin were denoted as "+ post-infection treatment", while those in the second aliquot that did not receive oleandrin were denoted as "- post-infection treatment" (Fig. 23A). ~23D). Viral titers were determined for each vial at about 24 hours and about 48 hours after infection.

As a means to determine the potential toxicity of oleandrin against VERO E6 cells, parallel cultures were prepared against uninfected VERO E6 cells based on the above broth.

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

Example 29

In vitro evaluation of oleandrin against COVID-19 virus

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

Growth medium was removed from confluent monolayers of approximately 106 Vero-E6 cells in ⁶ -well dishes. Oleandrin was serially diluted in culture broth and then added to Vero-E6 cells seeded in 96-well plates. Replace the growth medium with 200 μl of maintenance medium containing 10 μg/mL, 5 μg/mL, 1 μg/mL, 0.5 μg/mL, 100 ng/mL, or 50 ng/mL oleandrin, or a matched DMSO-only control. Plates were incubated at 37°C for approximately 30 minutes before virus addition.

SARS-CoV-2 virus was added to oleandrin-treated and untreated cells at an MOI (viral infectious dose) of 0.4 (entry assay) or 0.02 (replication assay). After 1 hour incubation at 37°C, oleandrin was retained in the wells.

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

Medium alone (no oleandrin) was added back to the oleandrin-treated wells on the data slide designated "pre-treatment". The medium with the indicated concentrations of oleandrin was added back to the wells on the data slides designated "Duration".

Disks were fixed 24 hours (entry assay) or 48 hours (replication assay) post-infection and immunostained with virus-specific antibodies and fluorescently labeled secondary antibodies.

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

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

Example 30

In vitro assessment of the toxicity of oleandrin against Vero-E6 cells

The purpose of this experiment was to determine the relative toxicity potential of oleandrin against Vero-E6 cells.

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

The results are shown in FIG. 25 .

Example 31

In vitro evaluation of oleandrin against COVID-19 virus

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

The procedure of Example 28 was repeated except that the following lower concentrations of oleandrin were used: 1 μg/mL, 0.5 μg/mL, 0.1 μg/mL, 0.05 μg/mL, 0.01 μg/mL and 0.005 μg/mL. In addition, VERO CCL-81 cells were used in place of VERO E6 cells.

Viral titers were determined according to Example 28, and the fold reduction in viral titers was calculated by comparing with control samples. The results are shown in Figures 26A-26D, 27A-27D, and 28A and 28B.

Example 32

Sublingual liquid dosage form

The sublingual liquid dosage form comprises oleandrin or a composition containing oleandrin (e.g. oleandrin-containing extract; 2 wt%) with medium chain triglycerides (95 wt%) and flavoring (3 wt%) Oleandrin made by mixing. The oleandrin content in the dosage form was about 25 μg/mL.

Example 33

Preparation of Oleander Subcritical Fluid Extract

An improved method for the preparation of oleandrin-containing extracts was developed by extracting oleander biomass using subcritical fluid extraction rather than supercritical liquid.

The dried and powdered biomass is placed in the extraction chamber, which is then sealed. Carbon dioxide (about 95 wt%) and alcohol (about 5 wt%; methanol or ethanol) were injected into the chamber. The internal temperature and pressure of the chamber maintain the extraction medium in a subcritical fluid phase rather than a supercritical fluid phase for most or substantially all of the extraction time period: temperatures in the range of about 2°C to about 16°C (about 7°C to about 8°C). °C), the pressure is in the range of about 115 to about 135 bar (about 124 bar). Extraction time is about 4 hours to about 12 hours (about 6 hours to about 10 hours). The extraction environment was then filtered and the supernatant was collected. Carbon dioxide was vented from the supernatant and the resulting crude extract was diluted into ethanol (about 9 parts ethanol:about 1 part extract) and frozen at about -50°C for at least 12 hours. The solution was thawed and filtered (100 micron pore size filter). The filtrate was concentrated to about 10% of its original volume, followed by sterile filtration (0.2 micron pore size filter). Next, 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 fluid (SbCL) extract comprises oleandrin extractable from oleander and one or more other compounds as defined herein.

Example 34

In vitro evaluation of oleandrin against COVID-19 virus

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

The procedure of Example 28 was repeated except that no pretreatment with oleandrin was performed prior to infection. Instead, infected cells were treated with oleandrin or control vehicle at 12 and 24 hours post infection. In addition, VERO CCL-81 was used in place of VERO E6 cells, and lower concentrations of oleandrin were used: 1 μg/mL, 0.5 μg/mL, 0.1 μg/mL and 0.05 μg/mL. The data are shown in Figures 29A and 29B.

Example 35

In vivo evaluation of oleandrin against the COVID-19 virus

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

Subjects sufficiently representative of a broad population distribution and exhibiting COVID-19 infection were assessed to determine their clinical status prior to sublingual, buccal or oral administration of an OCE prepared according to the dosage form of Example 32. The composition is safely administered to the subject by dripping the liquid into the subject's mouth By. The dosing regimen is about 0.5 mL per dose, four doses per day (one dose about every six hours), about 50 micrograms of oleandrin per day. Alternatively, only half of the total daily dose may be administered. All subjects recovered fully.

Example 36

Preparation of ethanolic extract of oleander

The purpose of this study was to prepare ethanolic extracts by extracting oleander biomass with an aqueous ethanolic solution.

Ground dried leaves were repeatedly treated with an aqueous ethanol solution (90% v/v ethanol; 10% v/v water). The combined ethanolic supernatants were combined and filtered, followed by concentration by vacuum evaporation to reduce the amount of ethanol and water therein to provide a crude ethanolic extract (with about 50% v/mL of extract containing about 25 mg of oleandrin/mL). v ethanol content).

Example 37

Preparation of a dosage form comprising a combination of extracts of oleander

The purpose of this study was to prepare a dosage form as in Example 32, except that the fraction (1 wt %) of the ethanol extract of Example 36 was compared with the fraction (1 wt %) of the SbCL extract of Example 33, the middle Chain triglycerides (95% by weight) and flavoring (3% by weight) were combined.

Example 38

In vivo evaluation of digitonin against COVID-19 virus

The purpose of this study was to determine the efficacy of a digitonin-containing composition (DCC) in the treatment of subjects already infected with the COVID-19 virus. A commercially available dosage form containing digitonin was used.

Subjects presenting with COVID-19 infection were assessed to determine clinical status prior to oral or systemic administration of DCC. Commercially available compositions are as described in this specification. Each safe dosing regimen is described in the respective NDA and package insert. The composition is safely applied to each subjects. Perform clinical monitoring to determine therapeutic response and titrate dose accordingly.

Example 39

Determination of genome to infectious particle ratio in SARS-CoV-2 infection treated with oleandrin

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

To quantify the genomic copies of the samples, 200 μl samples were extracted with TRIzol LS (Ambion, Carlsbad, CA) in a 5:1 volume ratio using standard manufacturer’s protocols and resuspended in 11 μl water. Extracted RNA was detected for SARS-CoV-2 by qRT-PCR according to a previously disclosed 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); probe [[5'-FAM-GCAAATTGTGCAATTTGCGG-TAMRA-3'; (SEQ ID NO. 3)]. Using the iTaq Universal Probe One-Step kit (BioRad, Hercules, CA), prepare a 20 μl reaction mix according to the manufacturer’s instructions: reaction mix (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 thermal cycler StepOnePlus ^™ real-time PCR system (Applied Biosystems). The reaction was carried out at 50°C for 5 minutes, 95°C for 20 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The positive control RNA sequence (nucleotides 26,044–29,883 of the COVID-2019 genome) was used to estimate the RNA copy number of the N gene in the assessed samples.

As used in this specification, the terms "about" or "approximately" refer to ±10%, ±5%, ±2.5%, or ±1% of a specified value. As used in this specification, the term "substantially" means "substantially" or "at least most of" or "more than 50%".

The foregoing are detailed descriptions of specific embodiments of the present invention. It should be understood that, although specific embodiments of the invention have been described in this specification for illustrative purposes, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the present invention is not limited except within the scope of the appended claims. In accordance with the present disclosure, all embodiments disclosed and claimed in this specification can be made and carried out without undue experimentation.

Pursuant to 37 CFR 1.52(e)(5), this application contains a Sequence Listing, which has been submitted in electronic format through EFS, which is hereby incorporated by reference. Using Patent-in 3.5.1 and Checker 4.4.6, created on July 10, 2020, named PBI22PCT9_SEQ_ST25.txt, the sequence information contained in the 1KB electronic file is incorporated herein by reference in its entirety.

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Claims (15)

A use of an antiviral composition comprising oleandrin in the preparation of a drug for treating COVID-19 or SARS-CoV-2 coronavirus infection, characterized by administering one or multiple doses to a subject in need during the treatment period, which is effective in treatment dose of the antiviral composition. The use according to claim 1, wherein the treatment period is at least 5 days to one or more months. The use according to claim 1, wherein the administration frequency during the treatment period is more than two days per week. The use of any one of claims 1 to 2, wherein the total daily dose of oleandrin is 0.01 μg to 750 μg per occurrence. The use according to any one of claims 1 to 3, wherein the dose of the antiviral composition is 0.05-0.5 mg/kg/day or 0.05-0.5 μg/kg/day, which is based on a daily subject The unit amount of oleandrin per kg of body weight. The use according to any one of claims 1 to 2, wherein a) the maximum daily dose of oleandrin is: 100 μg/day; and/or b) the minimum daily dose of oleandrin is: 0.5 μg/day . The use of any one of claims 1 to 2, wherein the antiviral composition is administered to a subject in need thereof in two doses per day or one dose every 12 hours in a therapeutically effective dose for the treatment period, and the The amount of oleandrin in the agent is 0.25 μg to 50 μg. The use according to any one of claims 1 to 2, wherein the one or more doses are 2 to 10 therapeutically effective doses of the antiviral composition. The use according to any one of claims 1 to 2, wherein oleandrin is present in A composition comprising at least one extract obtained from biomass containing oleandrin; wherein the extract is independently selected at each occurrence from the group consisting of hot water extract, solvent extract and supercritical A fluid extract; wherein the extract comprises a combination of oleandrin and one or more compounds extracted from the oleandrin-containing biomass; wherein the one or more compounds extracted from the oleandrin-containing biomass comprise One or more cardiac glycoside precursors, one or more glycoside components of cardiac glycosides, or a combination thereof. The use of any one of claims 1 to 2, wherein, after administration of the one or more doses, the subject's plasma concentration of oleandrin is in the range of 0.005 to 10 ng/mL, which is per Amount of oleandrin in mL of plasma. The use according to any one of claims 1 to 2, wherein the administration is continued for one or more weeks per month, or for one or more months per year. The use of any one of claims 1 to 2, wherein the administration is systemic. The use of any one of claims 1 to 2, wherein the antiviral composition is administered after infection, or within 1 to 5 days after infection, or after diagnosis of viral infection apply. The use of any one of claims 1 to 2, wherein the antiviral composition is administered as primary antiviral therapy, adjunctive antiviral therapy or combined antiviral therapy, and the administering comprises: combining the composition with The at least one other antiviral composition is administered separately or co-administered with at least one other composition for the treatment of symptoms associated with the viral infection. The use according to claim 9, wherein the extract comprises oleandrin and one or more compounds selected from the group consisting of cardiac glycosides, aglycones, steroids, carbohydrates, alkaloids, fats, proteins .

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