JP2022522074A - Methods and Compositions for Treating Coronavirus Infections - Google Patents

Abstract

Methods are provided for treating viral infections, such as viral infections caused by viruses of the Coronaviridae family. Compositions with at least oleandrin are used to treat viral infections.

Description

Incorporation by Reference 37 In accordance with CFR 1.52 (e) (5), the present application includes a sequence listing approved in electronic format via EFS, which is incorporated herein by reference. The sequence information is PBI22PCT9_SEQ_ST25. Included as a 1KB sized electronic file named txt, created on July 10, 2020 using Patent-in 3.5.1 and Checker 4.4.6, in its entirety by reference. Is incorporated herein by.
The present invention is an antiviral composition and use thereof for treating arenavirus infection, bunyavirus infection, flavivirus infection, togavirus infection, paramyxovirus infection, retrovirus infection, coronavirus infection or phyllovirus infection in mammals. Regarding. Some embodiments relate to the treatment of hemorrhagic viral infections.

A member of the Nerium species, the Nerium oleander is an ornamental plant that is widely distributed in subtropical Asia, the southwestern United States, and the Mediterranean Sea. Its medical and toxicological properties have long been recognized. For example, it has been proposed for use in the treatment of hemorrhoids, ulcers, leprosy, snakebites, cancers, tumors, neuropathy, vulgaris and cell proliferation diseases. Zibbu et al. (J. Chem. Pharma. Res. (2010), 2 (6), 351-358) briefly touch on the chemical and pharmacological activity of the Nerium oleander.
Extraction of constituents from plants of the Nerium species has traditionally been carried out using boiling water, cold water, supercritical fluids or organic solvents.

ANVIRZEL ™ (US 5,135,745 of Ozel) comprises a concentrated or powdered form of a hot water extract of Nerium oleander. Muller et al. (Pharmazier. (1991) Sept. 46 (9), 657-663) disclose the results of the analysis of the water extract of Nerium oleander. They report that the polysaccharides present are predominantly galacturonic acid. Other sugars include rhamnose, arabinose, and galactose. The polysaccharide content in the hot water extract of Nerium oleander and the individual sugar compositions of the polysaccharides have also been reported by Newman et al. (J. Herbal Pharmacotherapy, (2001) vol1, pp. 1-16). The composition analysis of the hot water extract ANVIRZEL ™ has been described by Newman et al. (Anal. Chem. (2000), 72 (15), 3547-3552). US Pat. No. 5,869,060 of Selvaraj et al. Concerns the extraction and production method of Nerium species. To prepare the extract, put the plant material in water and bring to a boil. The crude extract is then separated from the plant and sterilized by filtration. The resulting extract can then be lyophilized to produce a powder. U.S. Pat. No. 6,565,897 (Pre-Registration No. 2002114852 and PCT International Publication No. 2000/016793 of Selvaraj et al.) Provides a hot water extraction process for the preparation of substantially sterilized water extracts. It is disclosed. Ishikawa et al. (J. Nutr. Sci. Vitaminol. (2007), 53, 166-173) used a hot water extract of Nerium oleander and its fraction by liquid chromatography using a mixture of chloroform, methanol and water. It is disclosed. These are also N. It is reported that leaf extract of oleander is used for the treatment of type II diabetes. Panyosan's US20060188585, published August 24, 2006, discloses a hydrothermal extract of Nerium oleander. US10323055 of Mothers, published June 18, 2019, discloses a method of extracting plant material with aloe and water to obtain an extract containing aloe and cardiac glycosides. Rashan et al., US 20070154573, published July 5, 2007, discloses a cold water extract of Nerium oleander and its use.

Erdemoglu et al. (J. Ethnopharmacol. (2003) Nov. 89 (1), 123-129) compared aqueous and ethanol extracts of plants containing Nerium oleander based on anti-nociceptive and anti-inflammatory effects. The results are disclosed. Fartyal et al. (J. Sci. Innov. Res. (2014), 3 (4), 426-432) show comparative results of Nerium oleander's methanol extract, aqueous extract and petroleum ether extract based on their antibacterial activity. Is disclosed.
Organic solvent extracts of Nerium oleander were also found in Adobe et al. (Afr. Health Sci. (2003) Aug. 3 (2), 77-86; ethanolic extract), el-Shazly et al. (J. Egypt SEC. Parasitol. ), August 26 (2), 461-473; ethanolic extract), Vegas et al. (Phytochemistry (1999) Feb. 50 (3), 435-438; metanolic extract), Zia et al. (J. Ethnolpharmacol. .49 (1), 33-39; mechanical extract), and Vlasenko et al. (Farmatsia. (1972) Sept.-Oct. 21 (5), 46-47; alcoholic extract). Turkmen et al. (J. Planar Chroma. (2013), 26 (3), 279-283) disclose an aqueous ethanol extract of the leaves and stems of the Nerium oleander. Yamauchi's US3833472, published September 3, 1974, discloses the extraction of Nerium chloroform SOL (Nerium chloroformer Linn) leaves with water, organic or aqueous organic solvents, where the leaves are squeezed from 60 ° C. Heated to 170 ° C. and then extracted, the organic solvent is methanol, ethanol, propyl ether or chloroform.

Supercritical fluid extracts of the Nerium species are known (US8394434, US8187644, US7402325), neuropathy (US8481086, US92020778, US93582993, US201601243143A1, US9877979, US10383886) and cell proliferation disorders (US83767363, US9494589). It has also been shown to be effective in treating several viral infections (US10596186, WO2018053123A1, WO201905511A1).

Triterpenes are known to have a wide variety of therapeutic activities. Some of the known triterpenes include oleanolic acid, ursolic acid, bethulinic acid, baldoxolone, maslinic acid and the like. The therapeutic activity of triterpenes is primarily assessed individually, not as a combination of triterpenes.
Oleanolic acid belongs to the class of triterpenoids represented by compounds such as valdoxolone, which naturally leads to increased transcription of the program of downstream antioxidant genes, including the antioxidant transcription response element (ARE), when activation of the transcription factor Nrf2. It has been shown to be a potent activator of the cell phase 2 detoxification pathway. Valdoxolone itself has been extensively investigated in clinical trials of inflammatory conditions, however, due to adverse events that may be associated with the known cytotoxicity of certain triterpenoids, including elevated concentrations of baldoxolone. Phase 3 clinical trials for chronic kidney disease have been completed.

Compositions containing triterpenes in combination with other therapeutic ingredients are found as plant extracts. Fumico et al. (Biol. Pharma. Bull (2002), 25 (11), 1485-1487) described Rosmarimus officelis L. et al. For the treatment of trypanosomasis. Discloses the evaluation of the methanol extract of. Addington et al. (US8481086, US9220778, US9358293, US2016 / 0243143A1) disclose a supercritical fluid extract (SCF; PBI-05204) of Nerium oleander containing oleandrin and triterpenes for the treatment of neurological conditions. There is. Addington et al. (US9011937, US2015 / 0283191A1) disclose a triterpene-containing fraction (PBI-04711) of a Nerium oleander SCF extract containing oleandrin and triterpenes for the treatment of neurological conditions. Jager et al. (Molecules (2009), 14, 2016-2031) disclose various botanical extracts, including mixtures of oleanolic acid, ursolic acid, bethulinic acid, and other constituents. Mishra et al. (PLoS One 2016 25; 11 (7): e015430.Epub 2016 Jul 25) disclosed an extract of Betula utilis bark containing a mixture of oleanolic acid, ursolic acid, bethulinic acid, and other constituents. There is. Wang et al. (Molecules (2016), 21, 139) disclose an extract of Alstonia scholaris containing a mixture of oleanolic acid, ursolic acid, bethulinic acid, and other constituents. L. e Silva et al. (Molecules (2012), 17, 12197) disclose an extract of Eriope blanchetti containing a mixture of oleanolic acid, ursolic acid, bethulinic acid, and other constituents. Rui et al. (Int. J. Mol. Sci. (2012), 13, 7648-7662) disclosed an extract of Eucaplyptus globulus containing a mixture of oleanolic acid, ursolic acid, bethulinic acid, and other constituents. There is. Ayatorlahi et al. (Iran. J. Pharma. Res. (2011), 10 (2), 287-294) have extracted an extract of Euphorbia microsciadia containing a mixture of oleanolic acid, ursolic acid, betulinic acid, and other constituents. It is disclosed. Wu et al. (Molecules (2011), 16, 1-15) disclose an extract of Ligusturum species containing a mixture of oleanolic acid, ursolic acid, bethulinic acid, and other constituents. Lee et al. (Biol. Pharma. Bull (2010), 33 (2), 330) disclose an extract of Forsythia viridisshima containing a mixture of oleanolic acid, ursolic acid, bethulinic acid, and other constituents.

Oleanolic acid (O or OA), ursolic acid (U or UA), and bethulinic acid (B or BA) are PBI-05204 (PBI-23; supercritical fluid extract of Nerium oleander) and PBI-04711 (PBI-). It is the three major triterpene constituents found in the triterpene-containing fractions 0-4) of 05204. The inventors (two of us) described the contribution of triterpenes to efficacy by comparing their neuroprotective activity in a brain slice oxygen glucose deficiency (OGD) model assay at similar concentrations. Previously reported (Van Kanegan et al. Nature Scientific Reports (May 2016), 6: 25626. Doi: 10.1038 / rep25626). PBI-05204 (PBI) and PBI-04711 (fractions 0-4) have been found to provide neuroprotective activity.

Extracts of the Nerium species are known to contain a number of different classes of compounds such as cardiac glycosides, glycosides, steroids, triterpenes and polysaccharides. Specific compounds include oleandrin; neritaroside; odroside; oleanolic acid; ursolic acid; beturic acid; oleandrigenin; oleacid A; bethulin (urus-12-en-3-β, 28-diol); 28-Norulus-12-en-3-β-ol; Urs-12-en-3β-ol; 3β, 3β-hydroxy-12-oleanen-28-acid; 3β, 20α-dihydroxyurus-21-en-28 -Acid; 3β, 27-dihydroxy-12-ursen-28-acid; 3β, 13β-dihydroxyurs-11-ene-28-acid; 3β, 12α-dihydroxyoleanan-28,13β-oride; 3β, 27- Dihydroxy-12-oleanan-28-acid; and other constituents.

Viral hemorrhagic fever (VHF) can be caused by five different viral families: arenavirus, bunyavirus, filoviridae, flaviviridae, and paramyxoviridae. Filoviruses, such as Ebola virus (EBOV) and Marburg virus (MARV), are the most pathogenic viruses known to humans and are the causative agents of the development of viral hemorrhagic fever with a case fatality rate of up to 90%. be. Each virion contains one molecule of single-stranded negative sense RNA. Other than supportive or symptomatic therapy, there are no over-the-counter therapeutic agents and prophylactics available for the treatment of EBOV (Ebola virus) and MARV (Marburg virus) infections, ie filovirus infections. Five species of Ebola virus have been identified: Thai Forest (formerly Côte d'Ivoire), Sudan, Zaire, Reston, and Bundibugyo.

Negative sense single-stranded envelope RNA virus ((-)-(ss) -envRNAV) is found in Arenavirus, Bunyavirales (Bunyavirales), Phyllovirus, Orthomixovirus, Paramyxovirus and Rabdovirus. Contains viruses. Negative viral RNA is complementary to mRNA and must be converted to plus RNA by RNA polymerase prior to translation; therefore, purified RNA of minus sense virus is converted to plus sense RNA for replication. It is not infectious in itself as it needs to be. Exemplary viruses and infectious diseases from the arenavirus family include lassavirus, aseptic meningitis, ganaritovirus, funinevirus, lujovirus, machupovirus, saviavirus and whitewater aroyovirus. Exemplary viruses and infectious diseases from the Bunyaviridae family include orthohantavirus, Crimean-Congo hemorrhagic fever orthonyrovirus. Exemplary viruses and infectious diseases from the Paramyxoviridae family include mumps virus, nipavirus, hendravirus, respiratory polynuclear virus (RSV), human parainfluenza virus (HPIV) and NDV. Exemplary viruses and infectious diseases from the Orthomyxoviridae are influenza virus (A to C), isavirus, togotovirus, quaranjavirus, H1N1, H2N2, H3N2, H1N2, Spanish cold, Asian cold, Hong Kong cold. , Including Soviet cold. Exemplary viruses and infectious diseases from the rhabdoviridae include rabies virus, becyclovirus, lyssavirus, cytorabdovirus.

The flavivirus is a plus sense single-stranded envelope RNA virus ((+)-(ss) -envRNAV). They are found in arthropods, primarily mites and mosquitoes, and cause widespread morbidity and mortality worldwide. Some mosquito-borne viruses include yellow fever, dengue fever, Japanese encephalitis, West Nile virus, and Zika virus. Tick-borne viral infections include tick-borne encephalitis, Kyasanur forest 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 Alterviridae (animal pathogens). ..

Coronavirus (CoV) is a general name for the family Coronaviridae. In humans, CoV is typically mild, but can be fatal in rare forms such as SARS (Severe Acute Respiratory Syndrome) -CoV, MERS (Middle East Respiratory Syndrome) -CoV and COVID-19. Causes respiratory infections. CoV has a helically symmetric nucleocapsid and has a genomic size ranging from about 26 to about 32 kilobases. Other exemplary human CoVs include CoV 229E, CoV NL63, CoV OC43, CoV HKU 1 and CoV HKU 20. The CoV envelope has three glycoproteins: S-spike protein: receptor binding, cell fusion, major antigen; E-envelope protein: small protein with envelope; and M-membrane protein: transmembrane-germinate & envelope Formation. In some types of CoV, a fourth glycoprotein is present: HE-hemagglutinin-esterase. The genome has a 5'methylation cap and 3'poly-A and functions directly as mRNA. Invasion of CoV into human cells occurs via endocytosis and membrane fusion; replication occurs in the cytoplasm of the cell. CoV is transmitted by the aerosol of respiratory secretions, by the fecal-oral route, and by mechanical transmission. Most viral growth occurs in epithelial cells. Occasionally, liver, kidney, heart or eye, and other cell types such as macrophages can be infected. In cold-like respiratory infections, growth is localized to the epithelium of the upper respiratory tract. Coronavirus infections are extremely common and occur worldwide. The incidence of infectious diseases is highly seasonal and is highest in children in winter. Infection in adults is less common. The number of coronavirus serotypes and the extent of antigenic variation are unknown. Reinfection appears to occur throughout life, which means multiple serotypes (at least 4 known) and / or antigenic variation, thus immunization of all serotypes with a single vaccine. There is little chance of. SARS is a type of viral pneumonia with symptoms including fever, dry cough, dyspnea (shortness of breath), headache and hypoxemia (low blood oxygen concentration). Findings of typical laboratory findings include lymphopenia (decreased lymphocyte count) and moderately elevated aminotransferase levels (indicating liver damage). Death can result from progressive respiratory failure due to alveolar damage. The typical clinical course of SARS is accompanied by improvement of symptoms during the first week of infection, followed by exacerbation during the second week. There is still considerable need for effective antiviral treatments for human CoV (compositions and methods).

Extracts of oleandrin and Nerium oleander have been shown to prevent HIV-1 gp120 envelope glycosides from incorporating into mature viral particles and inhibit viral infectivity in vitro (Singh). Et al., Fitoterapia (2013) 84, 32-39, "Nerium oranger dried cardiac glycoside olivelandrin is a novel inhibitor of HIV infection").

Oleandrin has shown anti-HIV activity but has not been evaluated against many viruses. Triterpenes oleanolic acid, bethulinic acid, and ursolic acid have been reported to exhibit different levels of antiviral activity, but have not been evaluated for many viruses. Betulinic acid has shown some antiviral activity against HSV-1 strain 1C, influenza A H7N1, ECHO 6, and HIV-1. Oleanolic acid has shown some antiviral activity against HIV-1, HEPC, and the HCV H strain NS5B. Some ursoleic acid is associated with HIV-1, HEP C, HCV H strain NS5B, HSV-1, HSV-2, ADV-3, ADV-8, ADV-11, HEP B, ENTV CVB1 and ENTV EV71. Shows antiviral activity. The antiviral activity of oleandrin, oleanolic acid, ursolic acid, and bethulinic acid is unpredictable with respect to their effectiveness against certain viruses. There are viruses in which oleandrin, oleanolic acid, ursolic acid, and / or bethulinic acid have little or no antiviral activity, which is specific to oleandrin, oleanolic acid, ursolic acid, and / or bethulinic acid. It means that it is not possible to predict a priori whether it shows antiviral activity against a genus virus.

Barrows et al. (Cell Host Microbe (2016), 20, 259-270 "A screen of FDA-approved drugs for inhibitors of Zikavirus infection") show that digoxin is too active against the virus, antiviral to Zika virus. It reports that it is likely to be toxic. Cheung et al. (“Antival activity of lanatoside C against dengue virus infection” in Experimental Res. (2014) 111, 93-99) report that lanatoside C exhibits antiviral activity against dengue virus.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus belonging to the family Retrovirus family and Deltaretrovirus genus. It has a plus sense RNA genome that is reverse transcribed into DNA and then integrated into cellular DNA. Once integrated, HTLV-1 remains present only as a provirus that can spread cell-to-cell via viral synapses. If present, some acellular virions are produced, usually the detectable virus is not present in plasma, but the virus is present in genital secretions. HTLV-1 mainly infects CD4 + T-lymphocytes, among other autoimmune / inflammatory conditions including infectious dermatitis, rheumatoid arthritis, vegetative inflammation, keratoconjunctivitis, dryness syndrome, Sjogren's syndrome and HAM / TSP. In addition, it rarely causes adult T-cell leukemia / lymphoma (ATLL), as well as invasive hematological malignancies with a high probability of refractory, and generally has poor clinical outcomes. HAM / TSP is clinically characterized by chronic progressive spastic paraplegia, urinary incontinence and mild sensory deficits. ATLL is pathologically involved in viral latency, carcinogenic transformation and clonal development of HTLV-1-infected cells, but inflammation such as HTLV-1-related myelopathy / tropical spastic paraparesis (HAM / TSP). Sexual disorders are caused by autoimmune and / or immunopathological responses to proviral replication and expression of viral antigens. HAM / TSP is a progressive neuroinflammatory disease that causes damage to the lower spinal cord and demyelination. Circulating T-cells infected with HTLV-1 infiltrate the central nervous system (CNS) and provoke an immunopathogenic response to the virus and possible components of the CNS. Nerve damage and subsequent degeneration can cause severe disability in patients with HAM / TSP. Persistence of provirus replication and HTLV-1-infected cell proliferation in CNS leads to a response of targeted cytotoxic T-cells to viral antigens, which respond to autoimmune destruction of neural tissue. Sometimes.

Strong worried sugars have been shown to show some antiviral activity against some viruses, whereas certain compounds show very different levels of antiviral activity against different viruses, That means that some show very poor antiviral activity and some show better antiviral activity when enhanced against the same virus.

There remains a need for improved pharmaceutical compositions containing oleandrin, oleanolic acid, ursolic acid, bethulinic acid, or any combination thereof that are therapeutically active against a particular viral infection.

The present invention provides pharmaceutical compositions and methods for treating and / or preventing viral infections in mammalian subjects. The invention also provides pharmaceutical compositions and methods for treating viral infections in mammalian subjects, such as viral hemorrhagic fever (VHF) infections. The present invention also provides a method of treating a viral infection in a mammal by administration of a pharmaceutical composition. We have succeeded in preparing antiviral compositions that exhibit sufficient antiviral activity to justify their use in the treatment of viral infections in humans and animals. We have developed a corresponding treatment method using a specific dosing regimen. We have developed a corresponding treatment method using a specific dosing regimen. The present invention is also a prophylactic method of treating a subject at risk of contracting a viral infection, subject to one or more doses of the antiviral composition at a repetitive frequency over a long treatment period prior to the subject becoming infected with the virus. Chronically administered to the subject, thereby comprising preventing the subject from contracting a viral infection, the antiviral composition provides a method comprising oleandrin.

In some embodiments, the antiviral composition is administered to a subject having virus-infected cells, which exhibit an enhanced ratio of alpha-3 to alpha-1 isoforms of Na, K-ATPase.
In some embodiments, the viral infection is caused by one of the following viral families: arenavirus, alter virus, bunyavirus, phyllovirus, flavivirus, orthomixovirus, paramixovirus, rabdovirus, retrovirus ( For more information, Delta Retrovirus), Coronavirus or Togavirus. In some embodiments, the virus infection is caused by (+)-ss-envRNAV or (-)-ss-envRNAV.

Some embodiments of the invention are directed to compositions and methods for treating phyllovirus infections, flavivirus infections, henipavirus infections, alphavirus infections, or togavirus infections. Viral infections that can be treated include at least Ebola virus, Marburg virus, Alpha virus, Flavivirus, Yellow fever, Deng fever, Japanese encephalitis, West Nile virus, Dicavirus, Venezuelanuma encephalomyelitis (VEE) virus, Chikungunia virus, Western horse encephalomyelitis (encephalitis) (WEE) virus, Eastern horse encephalomyelitis (encephalitis) (EEE) virus, tick-mediated encephalitis, Kasanur forest disease, Alkhurma disease, Omsk hemorrhagic fever, Hendra Examples include viruses, nipaviruses, delta retroviruses, HTLV-1 viruses, and species thereof.
Some embodiments of the present invention include arenavirus family, altervirus, bunyavirus family, phyllovirus family, flavivirus family (flavivirus genus), orthomixiovirus family, paramixovirus family, rabdovirus family, retrovirus. Compositions and methods for treating viral infections from viruses of the family (Delta Retrovirus), Coronaviridae, (+)-ss-envRNAV, (-)-ss-envRNAV or Togaviridae are targeted. ..
Some embodiments of the invention are for treating viral infections from viruses of the genus Henipavirus, Ebolavirus, Flavivirus, Marburgvirus, Deltaretrovirus, Coronavirus (CoV) or Alphavirus. The composition and method of.

In some embodiments, (+)-ss-envRNAV is a virus selected from the group consisting of Coronaviridae, Flaviviridae, Togaviridae and Alterviridae.
In some embodiments, (+)-ss-envRNAV is a coronavirus that is pathogenic to humans. In some embodiments, the coronavirus spike protein binds to the ACE2 (angiotensin converting enzyme 2) receptor in human tissue. In some embodiments, the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV229E, CoV NL63, CoV OC43, CoV HKU1 and CoV HKU20. To.
In some embodiments, (+)-ss-envRNAV is flavivirus, yellow fever virus, dengue fever virus, Japanese encephalitis virus, western Nile virus, decavirus, tick-mediated encephalitis virus, casaneur forest disease virus, alfluma ( It is a virus selected from the group consisting of Alkhurma) disease virus, Omsk hemorrhagic fever virus and Poissan virus.

In some embodiments, (+)-ss-envRNAV is an arbovirus, eastern horse encephalomyelitis virus (EEEV), western horse encephalomyelitis virus (WEEV), Venezuelanuma encephalomyelitis virus (VEEV), chikunnia. It is a Togavirus family virus selected from the group consisting of virus (CHIKV), Onyonnyon virus (ONNV), Pogosta disease virus, Sindbis virus, Ross River fever virus (RRV) and Semuliki forest virus.

In some embodiments, the (-)-(ss) -envRNAV is a group consisting of Arenavirus, Bunyavirales (Bunyavirales), Phyllovirus, Orthomyxoviridae, Paramyxoviridae, or Rabdovirus. It is a virus selected from.
In some embodiments, the arenavirus family virus is selected from the group consisting of lassavirus, aseptic meningitis, ganaritovirus, funinevirus, lujovirus, machupovirus, saviavirus and whitewater aroyovirus. Virus.
In some embodiments, the Bunyaviridae virus is selected from the group consisting of orthohantavirus and Crimean-Congo hemorrhagic fever orthonylovirus.
In some embodiments, the paramyxoviridae virus is a group consisting of mumps virus, nipavirus, hendravirus, respiratory polynuclear virus (RSV), human paramyxoviridae virus (HPIV), and Newcastle disease virus (NDV). Is selected from.
In some embodiments, the orthomixoviridae virus is influenza virus (A to C), isavirus, togotovirus, quaranja virus, H1N1 virus, H2N2 virus, H3N2 virus, H1N2 virus, Spanish cold virus, Asia. It is selected from the group consisting of cold virus, Hong Kong cold virus, and Soviet cold virus.
In some embodiments, the rhabdoviridae virus is selected from the group consisting of rabies virus, becyclovirus, lyssavirus, and cytorabdovirus.

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 myelopathy / tropical spastic vs. palsy (HAM / TSP), adult T-cell leukemia / lymphoma (ATLL), autoimmune condition, inflammation. It is selected from the group consisting of the condition, infectious dermatitis, rheumatoid arthritis, vegetative inflammation, keratoconjunctivitis dry syndrome, Sjogren's syndrome, and Strongyloids stercoralis.
The present invention also relates to a method of inhibiting infection of HTLV-1 particles released into the supernatant of a culture of treated cells, as well as by inhibiting the Env-dependent form of virological synapses. Provided are methods of reducing intracellular transmission, comprising administering an effective amount of an antiviral composition to a subject in need thereof.
In some embodiments, the invention comprises a) a particular cardiac glycoside (s), b) a plurality of triterpenes, or c) a combination of a particular cardiac glycoside (s) and a plurality of triterpenes. Provided is an antiviral composition containing (becomes essentially) an antiviral composition.

One aspect of the invention provides a method of treating a viral infection in a subject by chronic administration of the antiviral composition to the subject. Subjects are treated by chronically administering to the subject a therapeutically effective amount (therapeutic-related dose) of the composition, thereby providing relief of symptoms associated with viral infection or improvement of viral infection. Administration of the composition to the subject can be initiated immediately after infection, at any time within 1-5 days after infection, or at the earliest time after a definitive diagnosis of viral infection. The virus can be any virus described herein.
Accordingly, the invention also provides a method of treating a viral infection in a mammal, the method comprising administering to the mammal one or more therapeutically effective doses of the antiviral composition. One or more doses are given daily, weekly, or monthly. One or more doses per day may be administered. The virus can be any virus described herein.

The invention also provides a method of treating a viral infection in a subject in need of treatment.
Determining if a subject has a viral infection and
Instructing the administration of antiviral composition and
Administering the initial dose of the antiviral composition to the subject for a period of time according to the prescribed initial dosing regimen,
Periodically determining the adequacy of a subject's clinical and / or therapeutic response to treatment with an antiviral composition.
If the subject's clinical response and / or treatment response is reasonable, continue treatment with the antiviral composition as needed, or subject's clinical response and / or treatment until the desired clinical endpoint is achieved. If the response is not appropriate for the initial dose and initial dosing regimen, it comprises increasing or decreasing the dose until the desired clinical and / or therapeutic response of the subject is achieved.
Treatment of the subject with the antiviral composition is continued as needed. The dose or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint (s), such as reduction or alleviation of certain symptoms associated with the viral infection. The validity of the clinical and / or therapeutic response can be determined by a clinician familiar with viral infection.

The individual steps of the method of the invention can be performed in separate facilities or within the same facility.
The present invention provides another embodiment for all embodiments described herein, where oleandrin is used in combination with digoxin, which is replaced by digoxin. The method of the present invention may use oleandrin, digoxin, or a combination of oleandrin and digoxin. Thus, oleandrin, digoxin, oleandrin-containing compositions, digoxin-containing compositions, or oleandrin and digoxin-containing compositions can be used in the methods of the invention. Cardiac glycosides can be interpreted to mean oleandrin, digoxin or a combination thereof. Cardiac glycoside-containing compositions include oleandrin, digoxin or a combination thereof.

The present invention is also a method of treating a coronavirus infection, specifically a coronavirus infection that is pathogenic to humans, such as a SARS-CoV-2 infection, wherein the therapeutically effective dose is given to a subject having the infection. Provided are a method comprising chronic administration of a strong worried sugar (composition containing a strong worried sugar).
The present invention is also a two-way method of treating a coronavirus infection, in particular a coronavirus infection that is pathogenic to humans, eg, SARS-CoV-2 infection, in a subject having said infection. Chronic administration of a therapeutically effective dose of a coronavirus-containing composition, thereby inhibiting the replication of the coronavirus virus and infectivity of the coronavirus progeny virus. Provide methods including reduction.
The present invention also relates to a subject having the infection by repeatedly administering a therapeutically effective dose of a cardiac glycoside (a cardiac glycoside-containing composition) (the mode of administration discussed herein). A method of treating a coronavirus infection, in detail a SARS-CoV-2 infection, is provided (via either). The one or more doses may be administered daily over a day or more per week, optionally over a week or more per month, and optionally over a month or more per year.

The present invention is also a method of treating coronavirus infection in humans, in which a subject is given a dose of 1 to 10 doses of cardiac glycoside (cardiac glycoside) over a treatment period of 2 days to about 2 months. A method comprising administering (containing composition) is provided. During the treatment period, doses of 2-8, 2-6 or 4 times daily may be administered. Doses may be administered from 2 days to about 60 days, from 2 days to about 45 days, from 2 days to about 30 days, from 2 days to about 21 days, or from 2 days to about 14 days. The administration may be via any of the modes of administration discussed herein. Systemic administrations that impart therapeutically effective plasma levels of oleandrin and / or digoxin in the subject are preferred.

In some embodiments, one or more doses of oleandrin are administered daily over multiple days until the viral infection is cured. In some embodiments, one or more doses of cardiac glycosides (cardiac glycoside-containing compositions) are administered daily over multiple days and weeks until the viral infection is cured. One or more doses may be administered daily. 1, 2, 3, 4, 5, 6 or more doses are administered daily.

In some embodiments, the concentration of oleandrin and / or digoxin in the treated plasma of an infected subject, eg, showing coronavirus infection, is about 10 microg / mL or less, about 5 microg / mL or less, about 2.5 microg / mL. Hereinafter, it is about 2 microg / mL or less, or about 1 microg / mL or less. In some embodiments, the concentrations of oleandrin and / or digoxin in the plasma of treated subjects showing coronavirus infection are greater than or equal to about 0.0001 microlog / mL, greater than or equal to about 0.0005 microlog / mL, and about 0.001 microg /. mL or more, about 0.0015 microg / mL or more, about 0.01 microg / mL or more, about 0.015 microg / mL or more, about 0.1 microg / mL or more, about 0.15 microg / mL or more, about 0.05 microg / mL or more , Or about 0.075 microg / mL or higher. In some embodiments, the concentrations of oleandrin and / or digoxin in the treated plasma of the infected subject are from about 10 microg / mL to about 0.0001 microlog / mL, from about 5 microg / mL to about 0.0005 microg / mL, about. 1 microg / mL to about 0.001 microg / mL, about 0.5 microg / mL to about 0.001 microg / mL, about 0.1 microg / mL to about 0.001 microlog / mL, about 0.05 microg / mL to about 0.001 microg / ML, from about 0.01 microg / mL to about 0.001 microg / mL, from about 0.005 microlog / mL to about 0.001 microg / mL. The present invention includes all combinations and options of plasma concentration ranges specified herein.

The antiviral composition is chronic, i.e. every other day, every two days, every three days, every four days, every five days, every six days, every week, every one week, every two weeks, three weeks. It may be administered at a recurring frequency such as every, monthly, bimonthly, semi-monthly, monthly, every two months, quarterly, quarterly, seasonal, semi-annual, seasonal, semi-annual, and / or yearly. Treatment period of 1 week or more, 1 month or more, 1 quarter or more, and / or 1 year or more. An effective dose of cardiac glycoside (cardiac glycoside-containing composition) is administered at least once a day.

In some embodiments, the subject is administered 140-315 microg of cardiac glycoside per day. In some embodiments, the dose comprises a cardiac glycoside of 20 microg to 750 microg, 12 microg to 300 microg, or 12 microg to 120 microrog. The daily dose of cardiac glycoside can range from 20 microg to 750 microg, 0.01 microg to 100 mg, or 0.01 microg to 100 microg per day. The recommended daily dose of oleandrin present in the SCF extract is generally about 0.25 to about 50 microg twice daily, or about 0.9 to 5 microg twice daily or about every 12 hours. be. Dose is about 0.5 to about 100 microg / day, about 1 to about 80 microg / day, about 1.5 to about 60 microg / day, about 1.8 to about 60 microg / day, about 1.8 to about 40 microg / day. Can be. The maximum tolerated dose can be an oleander extract containing oleandrin containing about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day, or about 30 microg / day. The minimum effective dose can be about 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg / day, or about 5 microg / day. Suitable doses containing sucrose and triterpene are about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg / kg / day, about 0.05-0.22 mg / kg / day. Daily, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / day It can be kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day. In some embodiments, the dose of oleandrin is from about 1 mg to about 0.05 mg, from about 0.9 mg to about 0.07 mg, from about 0.7 mg to about 0.1 mg, from about 0.5 mg to about 0. It is 1 mg, about 0.4 mg to about 0.1 mg, about 0.3 mg to about 0.1 mg, and about 0.2 mg. The present invention includes all combinations of doses specified herein.

In some embodiments, cardiac glycosides are administered in at least two dosing phases: a loading phase and a maintenance phase. The loading phase continues until steady-state plasma levels of cardiac glycosides are nearly achieved. The maintenance phase begins at the beginning of treatment or after the loading phase is nearly complete. The dose escalation method can be performed in the loading phase and / or the maintenance phase.
All dosing regimens, dosing schedules, and doses described herein are assumed to be suitable; however, some dosing regimens, dosing schedules, and doses are more than others in some subjects. Can be suitable. The target clinical endpoint is used to induce said administration.
The composition can be administered systemically. Modes of systemic administration include parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, oral, lung, or oral. The composition can also be administered via injection or intravenously. The composition can also be administered to the same subject by two or more routes. In some embodiments, the composition is one of two or more dosage regimens selected from the group consisting of parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, oral, lung, and oral. It is administered by the combination of.

The invention also provides a sublingual formulation comprising oleandrin and a liquid carrier. The present invention is also a method of treating a viral infection, specifically, for example, a coronavirus infection as defined herein, wherein a multi-dose oleandrin-containing (digoxin-containing) composition is subjected to said viral infection. Provided are methods comprising sublingual administration to subjects with the virus. The dose of 1 or more times per day is administered for 2 days or more per week and 1 week or more per month, and optionally over 1 month or more per year.
In some embodiments, the antiviral composition comprises oleandrin (or digoxin or a combination of oleandrin and digoxin) and an oil. The oil may contain medium chain triglycerides. The antiviral composition may include one, two or more oleandrin-containing extracts, and one or more pharmaceutical excipients.

Additional cardiac glycosides, if present in the antiviral composition, may further include odroside, neritaroside, or oleandrigenin. In some embodiments, the composition is a) one or more triterpes, b) one or more steroids, c) one or more triterpene derivatives, d) one or more steroid derivatives, or e) theirs. Includes more combinations. In some embodiments, the composition is a cardiac glycoside, and a) two or three triterpenes, b) two or three triterpene derivatives, c) two or three triterpene salts, or d) them. Including combinations of. In some embodiments, the triterpenes are selected from the group consisting of oleanolic acid, ursolic acid, bethulinic acid, and salts or derivatives thereof.

Some embodiments of the invention include those in which the pharmaceutical composition comprises at least one pharmaceutical excipient and antiviral composition. In some embodiments, the antiviral composition is a) at least one cardiac glycoside and at least one triterpene, b) at least one cardiac glycoside and at least two triterpenes, c) at least one cardiac glycoside. Sugars and at least 3 triterpenes, d) excluding at least 2 triterpenes and cardiac glycosides, e) excluding at least 3 triterpenes and cardiac glycosides; or f) at least one cardiac glycoside, eg oleand Contains phosphorus and digoxin. As used herein, the general terms triterpenes and cardiac glycosides also include salts and derivatives thereof, unless otherwise stated.
Cardiac glycosides can be present in pharmaceutical compositions in pure form or as part of an extract containing one or more cardiac glycosides. The triterpenes (s) can be present in the pharmaceutical composition in pure form or as part of an extract containing the triterpenes (s). In some embodiments, cardiac glycosides are present as a major therapeutic component in pharmaceutical compositions and mean components that are primarily involved in antiviral activity. In some embodiments, triterpenes (s) are present in the pharmaceutical composition as tumor therapeutic components (s) and mean components (s) that are primarily involved in antiviral activity.

In some embodiments, the oleandrin-containing extract is obtained by extraction of plant material. Extracts may include hot water extracts, cold water extracts, supercritical fluid (SCF) extracts, subcritical liquid extracts, organic solvent extracts, or combinations thereof of plant materials. In some embodiments, the extract is prepared by subcritical liquid extraction of a Nerium plant mass (biomass) using subcritical liquid carbon dioxide, optionally containing alcohol, as the extraction fluid (biomass). there were. In some embodiments, the oleandrin-containing composition comprises two or more different types of oleandrin-containing extracts.
In the embodiment of the present invention, oleander-containing biomass (plant material) is used as Nerium sp. , Nerium oleander, Nerium oleander L (Apocynaceae), Nerium odorum, White oleander, Pink oleander, Thevetia sp. , Thevetia peruviana, Thevetia peruviana, Thevetia nerifolia, Agrobacterium tumefaciens, cell cultures (cell masses) of any of the above species, or combinations thereof. In some embodiments, the biomass comprises leaves, stems, flowers, bark, fruits, seeds, sap, and / or pods.

In some embodiments, the extract is at least one other pharmacology obtained with the cardiac glycoside during extraction, which contributes to the therapeutic efficacy of the cardiac glycoside when the extract is administered to the subject. Contains highly active agents. In some embodiments, the composition further comprises a therapeutically effective agent for one or more other non-cardiac glycosides, i.e., one or more agents that are not cardiac glycosides. In some embodiments, the composition further comprises one or more antiviral compounds (s). In some embodiments, the antiviral composition excludes pharmacologically active polysaccharides.

In some embodiments, the extract is one or more cardiac glycosides and one or more cardiac glycoside precursors (cardenolide, cardadienolide, and cardatrienolide, all of which are cardiac glycosides. Aglycon constituents of, for example, digitoxin, acetyldigitoxin, digitoxygenin, digoxin, acetyldigoxin, digoxigenin, medigoxin, strophanthin, cymarin, vabein, or strophanthidin). The extract may further contain one or more glycoside constituents of the cardiac glycoside, such as glucosides, fructosides, and / or glucuronides, as cardiac glycoside precursors. Thus, the antiviral composition is one or more cardiac glycoside precursors selected from the group consisting of one or more cardiac glycosides and one or more aglycone constituents and one or more glycoside constituents. Can include the body. The extract is also from Nerium sp. Or Thevetia sp. Can include therapeutically effective agents of one or more other non-cardiac glycosides obtained from the botanical material of.

In some embodiments, compositions comprising oleandrin (OL), oleanolic acid (OA), ursolic acid (UA), and bethulinic acid (BA) compare equivalent doses based on oleandrin content. Then it is more effective than pure oleandrin.
In some embodiments, the molar ratio of total triterpene content (OA + UA + BA) to oleandrin is from about 15: 1 to about 5: 1, or from about 12: 1 to about 8: 1, or from about 100: 1 to. About 15: 1, or about 100: 1 to about 50: 1, or about 100: 1 to about 75: 1, or about 100: 1 to about 80: 1, or about 100: 1 to about 90: 1, or The range is about 10: 1.
In some embodiments, the molar ratio of individual triterpen to oleandrin ranges from: about 2-8 (OA): about 2-8 (UA): about 0.1-1 (BA). : Approximately 0.5 to 1.5 (OL); Approximately or 3 to 6 (OA): Approximately 3 to 6 (UA): Approximately 0.3 to 8 (BA): Approximately 0.7 to 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) ): Approximately 4.4 (UA): Approximately 0.6 (BA): Approximately 1 (OL).

In some embodiments, Nerium sp. Or Thevetia sp. Other therapeutic agents, such as those obtained by extraction of plant material, are not polysaccharides obtained during the preparation of the extract, which means that it is not acidic homopolygalacturonan or arabinogaraturonan. In some embodiments, the extract excludes another therapeutic agent and / or acid homopolygalacturonan or arabinogaraturonan obtained during the preparation of the extract.
In some embodiments, Nerium sp. Or Thevetia sp. Other therapeutic agents, such as those obtained by extraction of plant material, are polysaccharides obtained during the preparation of extracts such as acidic homopolygalacturonan or arabinogaraturonan. In some embodiments, the extract comprises another therapeutic agent and / or comprises an acidic homopolygalacturonan or arabinogaraturonan obtained during the preparation of an extract from said plant material.

In some embodiments, the extracts are oleandrin, as well as strong worried sugars, glycosides, aglycones, steroids, triterpenes, polysaccharides, sugars, alkaloids, fats, proteins, neritarosides, odrosides, oleanolic acids, ursolic acids. , Bethuric acid, oleandrigenin, oleacid A, bethrin (Ursa-12-en-3β, 28-diol), 28-norursa-12-en-3β-ol, Ursa-12-en-3β-ol, 3β, 3β-hydroxy-12-oleanen-28-acid, 3β, 20α-dihydroxyursa-21-ene-28-acid, 3β, 27-dihydroxy-12-ursen-28-acid, 3β, 13β-dihydroxyursa-11- En-28-acid, 3β, 12α-dihydroxyoleanan-28,13β-oride, 3β,27-dihydroxy-12-oleanan-28-acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, coffee acid , L-quinic acid, 4-kumaloyl-CoA, 3-O-cafe oil quinic acid, 5-O-cafe oil quinic acid, cardenolide B-1, cardenolide B-2, oleagenin, nerididinoside, nerisoside, odroside-H, 3-Beta-O- (D-diginosyl) -5-beta, 14 beta-dihydroxy-cardo-20 (22) -enolidepectinic polysaccharide, composed of galacturonic acid, lambnorth, arabinose, xylose, and galactose, Polysaccharides with a MW in the range of 17,000 to 120,000D, or a MW of about 35,000D, about 3000D, about 5500D, or about 12000D, cardenolide monoglycosides, cardenolide N-1, cardenolide N-2, cardenolide N-3, cardenolide. N-4, Pregnane, 4,6-diene-3,12,20-trione, 20R-hydroxypregna-4,6-diene-3,12-dione, 16 beta, 17 beta-epoxy-12 beta-hydroxy Pregna-4,6-diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A), 20S, 21-dihydroxypregna-4, 6-diene-3,12-dione (neridienone B), neriukumalic acid, isoneriukumalic acid, oleanderoic acid, oleanderen, 8alpha-methoxylabdan-1 8-acid, 12-ursen, caneroside, neriumoside, 3β-O- (D-diginosyl) -2α-hydroxy-8,14β-epoxy-5β-carda-16: 17,20: 22-dienolide, 3β-O- (D-diginosyl) -2α, 14β-dihydroxy-5β-carda-16: 17, 20: 22-dienolide, 3β, 27-dihydroxy-Ursa-18-en-13,28-oride, 3β, 22α, 28- Trihydroxy-25-norlupu-1 (10), 20 (29) -dien-2-one, cis-calenin (3β-hydroxy-28-Z-p-kumaroyloxy-ursa-12-en-27) -Acid), trans-calenin (3-β-hydroxy-28-E-p-kumaroyloxy-ursa-12-ene-27-acid), 3beta-hydroxy-5alpha-carda-14 (15), 20 (22) -dienolide (beta-anhydroepidigitoxygenin), 3beta-O- (D-digitarosyl) -21-hydroxy-5 beta-carda-8,14,16,20 (22) -tetraenolide ( Neriumogenin-A-3 Beta-D-Digitaroside), Proceragenin, Neridienone A, 3 Beta, 27-Dihydroxy-12-Ursen-28-Acid, 3 Beta, 13 Beta-Dihydroxy Ursa-11-En-28-Acid, 3 Beta-Hydroxy Ursa-12-en-28-aldehyde, 28-Orursa-12-en-3 beta-ol, Ursa-12-en-3 beta-ol, Ursa-12-en-3 beta, 28-diol, 3 Beta, 27-Dihydroxy-12-oleanen-28-acid, (20S, 24R) -epoxydammaran-3 beta, 25-diol, 20 beta, 28-epoxy-28alpha-methoxytalaxasteran-3 beta- All, 20 beta, 28-epoxytalaxaster-21-en-3 beta-all, 28-nor-ursa-12-en-3 beta, 17 beta-diol, 3beta-hydroxyursa-12-en-28 -Aldehyde, alpha-neriursate, beta-neriursate, 3alpha-acetophenoxy-ursa-12-ene-28-acid, 3beta-acetophenoxy-ursa-12-ene-28-acid, oleanderolic acid ), Canelology, 3β-p-hydroxyphenoxy-11α-methoxy-12α-hydroxy-2 0-Ursen-28-acid, 28-hydroxy-20 (29) -lupen-3,7-dione, canerosin, 3alpha-hydroxy-ursa-18,20-diene-28-acid, D-salmentose, D- Diginose, nerididinoside, nerisoside, isolicinoleic acid, gentiobiosyl nerigoside, gentiobiosylvomontside, gentiobiosyloleandrin, holineline, 12β-hydroxy-5β-carda-8,14,16,20 ( 22) -Tetraenolide, 8β-hydroxy-digitoxygenin, Δ16-8β-hydroxy-digitoxygenin, Δ16-neriagenin, uvaol, ursolealdehyde, 27 (p-kumaroyloxy) ursoleic acid, oleanderol, 16-anhydro- Deacetyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, azigoside, aginerin, alpha-amylin, beta-citosterol, campestrol, kauchuk, capric acid, capric acid, choline, cornerin, Cortenerin, deacetyloleandrin, diacetyl-nerigoside, horiandrin, pseudochloramine, quercetin, kercetin-3-ramnoglucoside, kercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospeside, urehitoxin. ) And at least one other compound selected from the group consisting of uzarigenin. Additional components that may be 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 is also obtained from an extract of suspension culture derived from transformed callus of Agrobacterium tumefaciens. Agrobacterium hot water, organic solvents, aqueous organic solvents, or supercritical fluid extracts can be used in accordance with the present invention.
Oleander is also obtained from an extract of the Nerium oleander microculture in vitro, whereby the shoot culture is from seedlings and / or from Nerium oleander varieties such as Splendens Giganteum, Revanche or Alsace, or other varieties. You can start from the tip of the stem. Hot water, organic solvents, aqueous organic solvents, or supercritical fluid extracts of microculture Nerium oleander can be used according to the present invention.
Extracts are also obtained by extraction of cell masses of any of the plant species, such as those present in cell cultures.

The present invention also provides the use of cardiac glycosides in the manufacture of pharmaceuticals for the treatment of viral infections in a subject. In some embodiments, the manufacture of such pharmaceuticals comprises providing one or more antiviral compounds of the invention and comprising a dose of the antiviral compound (s) in pharmaceutical dosage form. Includes packaging pharmaceutical dosage forms. In some embodiments, the manufacture can be carried out as described in PCT International Application No. PCT / US06 / 29061. Manufacture also sends the packaged dosage form to the seller (retailer, wholesaler, and / or distributor), sells or otherwise provides the packaged dosage form to a subject with a virus infection, Includes one or more additional steps, such as including a label and package insert with the drug to provide instructions regarding use, dosing regimen, dosing, content, and dosage form toxicity profile. In some embodiments, treatment of a viral infection involves determining that the subject has a viral infection, instructing the subject to administer a pharmaceutical dosage form according to a dosing regimen, and one or more pharmaceuticals. One or more pharmaceutical dosage forms are administered according to a dosing regimen, including administration of the dosage form to a subject.

The pharmaceutical composition is at least one selected from the group consisting of a water-soluble (miscible) co-solvent, a water-insoluble (immiscible) co-solvent, a surfactant, an antioxidant, a chelating agent, and an absorption enhancer. Further inclusions of material combinations may be included.
The solubilizer is at least a single surfactant, which is also a) a surfactant and a water-miscible solvent; b) a surfactant and a water-miscible solvent; c) a surfactant, an antioxidant. Agents; d) surfactants, antioxidants, and water-miscible solvents; e) surfactants, antioxidants, and water-miscible solvents; f) surfactants, water-miscible solvents, and water-miscible solvents. It can be a combination of materials such as a sex solvent; or g) a combination of a surfactant, an antioxidant, a water miscible solvent, and a water immiscible solvent.
The pharmaceutical composition is a) at least one liquid carrier, b) at least one emulsifier, c) at least one solubilizer, d) at least one dispersant, e) at least one other excipient, or. f) Optional further inclusions of those combinations.

In some embodiments, the water-miscible solvent is a low molecular weight (less than 6000) PEG, glycol, or alcohol. In some embodiments, the surfactant is a pegged surfactant, meaning a surfactant containing a poly (ethylene glycol) functional group.
The present invention includes all combinations of aspects, embodiments, and subordinate embodiments of the invention disclosed herein.
The following figures form part of this specification and illustrate exemplary embodiments of the claimed invention. Those skilled in the art will be able to practice the invention without undue experimentation in the light of these figures and the description herein.

Figure 3 shows a chart summarizing the in vitro dose response antiviral activity of various compositions against Ebola virus. Figure 3 shows a chart summarizing the in vitro dose response antiviral activity of various compositions against Ebola virus. Figure 3 shows a chart summarizing the in vitro dose response antiviral activity of various compositions against Marburg virus. Figure 3 shows a chart summarizing the in vitro dose response antiviral activity of various compositions against Marburg virus. FIG. 3 shows a chart summarizing the in vitro dose response antiviral activity of oleandrin against Zika virus (SIKV strain PRVABC59) in Vero E6 cells. FIG. 3 shows a chart summarizing the in vitro dose response antiviral activity of digoxin against Zika virus (SIKV strain PRVABC59) in Vero E6 cells. FIG. 6 shows a chart summarizing the in vitro dose-responsive antiviral activity of various compositions (oleandrin, digoxin, and PBI-05204) against Ebola virus in Vero E6 cells. FIG. 6 shows a chart summarizing the in vitro dose-responsive antiviral activity of various compositions (oleandrin, digoxin, and PBI-05204) against Marburg virus in Vero E6 cells. FIG. 3 shows a chart summarizing the in vitro cell viability of Vero E6 cells in the presence of various compositions (oleandrin, digoxin, and PBI-05204). FIG. 10A shows a chart summarizing the ability of the composition (Oleandrin and PBI-05204) to inhibit Ebola virus in Vero E6 cells immediately after exposure to the virus, FIG. 10A is 2 hours after infection. FIG. 10B shows a chart summarizing the ability of the composition (Oleandrin and PBI-05204) to inhibit Ebola virus in Vero E6 cells immediately after exposure to the virus, FIG. 10B is 24 hours post-infection. FIG. 11A shows a chart summarizing the ability of the composition (Oleandrin and PBI-05204) to inhibit Marburg virus in Vero E6 cells immediately after exposure to the virus, FIG. 11A is 2 hours after infection. FIG. 11B shows a chart summarizing the ability of the composition (Oleandrin and PBI-05204) to inhibit Marburg virus in Vero E6 cells immediately after exposure to the virus, FIG. 11B is 24 hours post-infection. FIG. 12A shows a chart summarizing the ability of the composition (oleandrin and PBI-05204) to inhibit the production of infectious offspring by virus-infected Vero E6 cells exposed to oleandrin, where FIG. 12A is Ebola virus. .. FIG. 12B shows a chart summarizing the ability of the composition (oleandrin and PBI-05204) to inhibit the production of infectious offspring by virus-infected Vero E6 cells exposed to oleandrin, where FIG. 12B is Marburg virus. .. FIG. 3 shows a chart summarizing the in vitro dose-responsive antiviral activity of various compositions (oleandrin, digoxin, and PBI-05204) against Venezuelan encephalomyelitis virus (FIG. 13A) in Vero E6 cells. FIG. 3 shows a chart summarizing the in vitro dose-responsive antiviral activity of various compositions (oleandrin, digoxin, and PBI-05204) against western horse encephalomyelitis virus (FIG. 13B) in Vero E6 cells. Vehicle control, oleandrin, or N. Summarize the effects of the oleander extract on HTLV-1 replication or release of newly synthesized virus particles, as determined by the quantification of HTLV-1 p19 ^Gag (see Examples 19 and 20). The chart is shown. Untreated (UT) cells are shown for comparison. All data are representative of at least three independent experiments. The data represent the mean of the experimental ± standard deviation (error bars). Vehicle control against HTLV-1 + SLB1 lymphoma T-cell line, oleandrin, and N. The chart which summarizes the relative cytotoxicity of the oleander extract is shown. All data are representative of at least three independent experiments. The data represent the mean of the experimental ± standard deviation (error bars). FIG. 16A shows representative micrographs of the results of staining with Annexin V-FITC (green) and PI (red), showing the DIC phase difference in the composite image. Individual Annexin V-FITC and PI fluorescence channel images are also shown. Scale bar, 20 μm. FIG. 16B shows representative micrographs of the results of staining with Annexin V-FITC (green) and PI (red), showing the DIC phase difference in the composite image. Individual Annexin V-FITC and PI fluorescence channel images are also shown. Scale bar, 20 μm. FIG. 16C shows representative micrographs of the results of staining with Annexin V-FITC (green) and PI (red), showing the DIC phase difference in the composite image. Individual Annexin V-FITC and PI fluorescence channel images are also shown. Scale bar, 20 μm. (FIG. 16D) Representative micrographs of Annexin V-FITC (green) and PI (red) staining results are shown, showing DIC phase differences in synthetic images. Individual Annexin V-FITC and PI fluorescence channel images are also shown. Scale bar, 20 μm. FIG. 16E shows representative micrographs of the results of staining with Annexin V-FITC (green) and PI (red), showing the DIC phase difference in the composite image. Individual Annexin V-FITC and PI fluorescence channel images are also shown. Scale bar, 20 μm. (FIG. 16F) Representative micrographs of Annexin V-FITC (green) and PI (red) staining results are shown, showing DIC phase differences in synthetic images. Individual Annexin V-FITC and PI fluorescence channel images are also shown. Scale bar, 20 μm. Vehicle control, oleandrin, or N. oleandrin upon release of HTLV-1 replication or newly synthesized viral particles from HTLV-1 + lymphoma T-cells treated with oleandrin. The figure which summarizes the effect which the extract of oleander has is shown. Vehicle control with treated huPBMC, oleandrin, or N. et al. The chart which summarizes the relative cytotoxicity of the extract of oleander is shown. Vehicle control, oleandrin, or N. FIG. 6 shows a chart summarizing the relative inhibition of HTLV-1 transmission in a co-culture assay with huPBMC containing an extract of oleander. Representative micrographs of GFP-expressed HTLV-1 + SLB1 T-cell lines are shown: fluorescence microscopy (upper panel) and immunobrotting (lower panel). FIG. 3 shows representative micrographs of virological synapses between HTLV-1 + SLB1 / pLenti-GFP lymphoblasts (green cells) treated with huPBMC and mitomycin C. FIG. 21 shows a chart of averaging data with standard deviation (error bar) by quantification of the micrograph of FIG. 21. SARS 24 and 48 hours after "treatment" on VERO E6 cells infected with SARS-CoV-2 virus treated with oleandrin (red bar) or control vehicle (incubation medium) (black bar) A chart of log (Example 28) of -CoV-2 virus titer (PFU / mL) vs. time (h) is shown. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells were oleandrin (FIG. 23A: 1 microg / mL in 0.1% DMSO aqueous solution with RPMI 1640 medium as an aqueous component; FIG. 23C: 0.1 microg / mL in 0.01% DMSO aqueous solution with RPMI 1640. The virus titer was measured by treatment with mL) or only the control vehicle (FIG. 23B: 0.1% DMSO aqueous solution with RPMI 1640; FIG. 23D: 0.01% DMSO aqueous solution with RPMI 1640). SARS 24 and 48 hours after "treatment" on VERO E6 cells infected with SARS-CoV-2 virus treated with oleandrin (red bar) or control vehicle (incubation medium) (black bar) A chart of log (Example 28) of -CoV-2 virus titer (PFU / mL) vs. time (h) is shown. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells were oleandrin (FIG. 23A: 1 microg / mL in 0.1% DMSO aqueous solution with RPMI 1640 medium as an aqueous component; FIG. 23C: 0.1 microg / mL in 0.01% DMSO aqueous solution with RPMI 1640. The virus titer was measured by treatment with mL) or only the control vehicle (FIG. 23B: 0.1% DMSO aqueous solution with RPMI 1640; FIG. 23D: 0.01% DMSO aqueous solution with RPMI 1640). SARS 24 and 48 hours after "treatment" on VERO E6 cells infected with SARS-CoV-2 virus treated with oleandrin (red bar) or control vehicle (incubation medium) (black bar) A chart of log (Example 28) of -CoV-2 virus titer (PFU / mL) vs. time (h) is shown. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells were oleandrin (FIG. 23A: 1 microg / mL in 0.1% DMSO aqueous solution with RPMI 1640 medium as an aqueous component; FIG. 23C: 0.1 microg / mL in 0.01% DMSO aqueous solution with RPMI 1640. The virus titer was measured by treatment with mL) or only the control vehicle (FIG. 23B: 0.1% DMSO aqueous solution with RPMI 1640; FIG. 23D: 0.01% DMSO aqueous solution with RPMI 1640). SARS 24 and 48 hours after "treatment" on VERO E6 cells infected with SARS-CoV-2 virus treated with oleandrin (red bar) or control vehicle (incubation medium) (black bar) A chart of log (Example 28) of -CoV-2 virus titer (PFU / mL) vs. time (h) is shown. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells were oleandrin (FIG. 23A: 1 microg / mL in 0.1% DMSO aqueous solution with RPMI 1640 medium as an aqueous component; FIG. 23C: 0.1 microg / mL in 0.01% DMSO aqueous solution with RPMI 1640. The virus titer was measured by treatment with mL) or only the control vehicle (FIG. 23B: 0.1% DMSO aqueous solution with RPMI 1640; FIG. 23D: 0.01% DMSO aqueous solution with RPMI 1640). Percent inhibition of viral replication (Y1, left axis) and Vero-E6 cell count 24 hours after infection (Y2, right axis: potential cytotoxic expression of oleandrin to said cells) oleandrin in medium The figure of the double y-axis (Example 29) of the concentration (microg / mL) of is shown. It is for the culture of FIG. 24A, except that it was obtained 48 hours after infection. Percentage of oleandrin concentration (microg / mL) in Vero-E6 cell (cell titer) vs. medium for 24 hours after continuous exposure of cells to the indicated concentration of oleandrin (Example 30). ) Is shown. 24 hours after "treatment" with VERO CCL-81 cells infected with SARs-CoV-2 virus and then treated with oleandrin (blue circle) or control vehicle (incubation medium) (red square). Concentration of oleandrin in SARS-CoV-2 virus titer (PFU / mL) vs. medium for 48 hours (FIG. 26A) (FIG. 26A) and 48 hours (FIG. 26A); normal cells of the ceropythecus aethiops kidney; https://www.atcc.org The chart of the log (Example 31) of /products/all/CCL-81.aspx) is shown. 24 hours after "treatment" with VERO CCL-81 cells infected with SARs-CoV-2 virus and then treated with oleandrin (blue circle) or control vehicle (incubation medium) (red square). Concentration of oleandrin in SARS-CoV-2 virus titer (PFU / mL) vs. medium for 48 hours (FIG. 26A) (FIG. 26A) and 48 hours (FIG. 26A); normal cells of the ceropythecus aethiops kidney; https://www.atcc.org The chart of the log (Example 31) of /products/all/CCL-81.aspx) is shown. For the samples of FIGS. 26A and 26B, a double reduction in virus titer was determined at 24 hours (FIG. 26C) and 48 hours (FIG. 26D). For the samples of FIGS. 26A and 26B, a double reduction in virus titer was determined at 24 hours (FIG. 26C) and 48 hours (FIG. 26D). SARS-CoV- 24 and 48 hours after "treatment" in SARS-CoV-2 virus-infected VERO E6 cells treated with oleandrin (blue circle) or control vehicle (incubation medium) (red square). 2 The chart of the log (Example 28) of virus titer (PFU / mL) vs. time (h) is shown. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells in oleandrin (FIG. 27A: 0.005 microg / mL in DMSO aqueous solution (0.005%) with RPMI 1640 medium as an aqueous component; Figure 27B: in DMSO aqueous solution (0.01%) with RPMI 1640). 0.01 microg / mL; FIG. 27C: 0.05 microg / mL in DMSO aqueous solution (0.05%) with RPMI 1640; FIG. 27B: 0.1 microg in DMSO aqueous solution (0.1%) with RPMI 1640. / ML) was treated and the virus titer was measured. SARS-CoV- 24 and 48 hours after "treatment" in SARS-CoV-2 virus-infected VERO E6 cells treated with oleandrin (blue circle) or control vehicle (incubation medium) (red square). 2 The chart of the log (Example 28) of virus titer (PFU / mL) vs. time (h) is shown. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells in oleandrin (FIG. 27A: 0.005 microg / mL in DMSO aqueous solution (0.005%) with RPMI 1640 medium as an aqueous component; Figure 27B: in DMSO aqueous solution (0.01%) with RPMI 1640). 0.01 microg / mL; FIG. 27C: 0.05 microg / mL in DMSO aqueous solution (0.05%) with RPMI 1640; FIG. 27B: 0.1 microg in DMSO aqueous solution (0.1%) with RPMI 1640. / ML) was treated and the virus titer was measured. SARS-CoV- 24 and 48 hours after "treatment" in SARS-CoV-2 virus-infected VERO E6 cells treated with oleandrin (blue circle) or control vehicle (incubation medium) (red square). 2 The chart of the log (Example 28) of virus titer (PFU / mL) vs. time (h) is shown. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells in oleandrin (FIG. 27A: 0.005 microg / mL in DMSO aqueous solution (0.005%) with RPMI 1640 medium as an aqueous component; Figure 27B: in DMSO aqueous solution (0.01%) with RPMI 1640). 0.01 microg / mL; FIG. 27C: 0.05 microg / mL in DMSO aqueous solution (0.05%) with RPMI 1640; FIG. 27B: 0.1 microg in DMSO aqueous solution (0.1%) with RPMI 1640. / ML) was treated and the virus titer was measured. SARS-CoV- 24 and 48 hours after "treatment" in SARS-CoV-2 virus-infected VERO E6 cells treated with oleandrin (blue circle) or control vehicle (incubation medium) (red square). 2 The chart of the log (Example 28) of virus titer (PFU / mL) vs. time (h) is shown. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells in oleandrin (FIG. 27A: 0.005 microg / mL in DMSO aqueous solution (0.005%) with RPMI 1640 medium as an aqueous component; Figure 27B: in DMSO aqueous solution (0.01%) with RPMI 1640). 0.01 microg / mL; FIG. 27C: 0.05 microg / mL in DMSO aqueous solution (0.05%) with RPMI 1640; FIG. 27B: 0.1 microg in DMSO aqueous solution (0.1%) with RPMI 1640. / ML) was treated and the virus titer was measured. Infected with SARS-CoV-2 virus, followed by oleandrin (dark blue circles (Exp. 2) and light blue circles (Exp. 3)) or control vehicle (incubation medium) (dark red squares (Exp. 3)). SARS-CoV-2 virus titers (FIG. 28A) and 48 hours (FIG. 28B) "after treatment" in VERO 81 cells treated with .2) and pale red squares (Exp. 3)). The figure of log of the concentration of oleandrin in PFU / mL) vs. medium is shown. Exp. 2 and Exp. 3 simply repeats the assay. Infected with SARS-CoV-2 virus, followed by oleandrin (dark blue circles (Exp. 2) and light blue circles (Exp. 3)) or control vehicle (incubation medium) (dark red squares (Exp. 3)). SARS-CoV-2 virus titers (FIG. 28A) and 48 hours (FIG. 28B) "after treatment" in VERO 81 cells treated with .2) and pale red squares (Exp. 3)). The figure of log of the concentration of oleandrin in PFU / mL) vs. medium is shown. Exp. 2 and Exp. 3 simply repeats the assay. A bar graph of oleandrin concentration in virus titer vs. medium was shown, and virus titers were measured 24 hours after infection (FIG. 29A) and 48 hours (FIG. 29B). In some samples, cells were treated with oleandrin (solid blue bar) or DMSO control vehicle only (solid red bar) before and after infection (2 hours). In other samples, cells were oleandrin (broken blue bar: 12 hours after infection; white blue bar: 24 hours after infection) or DMSO control vehicle only (red dashed bar: 12 hours after infection). After; White red bar: 24 hours after infection). A bar graph of oleandrin concentration in virus titer vs. medium was shown, and virus titers were measured 24 hours after infection (FIG. 29A) and 48 hours (FIG. 29B). In some samples, cells were treated with oleandrin (solid blue bar) or DMSO control vehicle only (solid red bar) before and after infection (2 hours). In other samples, cells were oleandrin (broken blue bar: 12 hours after infection; white blue bar: 24 hours after infection) or DMSO control vehicle only (red dashed bar: 12 hours after infection). After; White red bar: 24 hours after infection). The figure which evaluates the anti-COVID-19 activity of the combination composition (PBI-A) of two extracts is shown. In FIG. 30B, it is estimated that PBI-A was supplied as a 1 mg / ml (oleandrin concentration) solution from the designation of μg / ml (oleandrin concentration). The virus titer (Log ₁₀ (PFU / mL)) vs. Log ₁₀ dilution factor (FIG. 30A) or the concentration vs. Log ₁₀ oleandrin (FIG. 30B) was determined. 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 figure which evaluates the anti-COVID-19 activity of the combination composition (PBI-A) of two extracts is shown. In FIG. 30B, it is estimated that PBI-A was supplied as a 1 mg / ml (oleandrin concentration) solution from the designation of μg / ml (oleandrin concentration). The virus titer (Log ₁₀ (PFU / mL)) vs. Log ₁₀ dilution factor (FIG. 30A) or the concentration vs. Log ₁₀ oleandrin (FIG. 30B) was determined. 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 invention is directed by chronic or acute administration of one or more effective doses of an antiviral composition (or pharmaceutical composition comprising an antiviral composition and at least one pharmaceutical excipient) to a subject virus. Provide a method of treating an infection. The composition is administered according to the optimal dosing regimen for the subject, and the dosage and dosing regimen suitability are clinically determined according to conventional clinical practices and clinical treatment endpoints of viral infection.
As used herein, the term "subject" is to be construed to mean warm-blooded animals such as mammals, such as cats, dogs, mice, guinea pigs, horses, cows, sheep, and humans.

As used herein, subjects at risk of viral infection are a) subjects living in geographic areas within the habitat of mosquitoes, especially Ixodes species (Aedes egypti, Aedes albopictus), b. ) Subjects who live with or near people with or near a person with a viral infection, c) Subjects who have a sexual relationship with a person with a viral infection, d) Mites, especially the Ixodes species (Ixodes marx, Ixodes scalaris, or). Targets living in geographical areas within the range inhabited by ticks of the Virus species), e) Targets living in geographical areas within the range inhabited by worms, f) Targets living in tropical regions, g) subjects living in Africa, h) subjects in contact with the body fluids of other subjects with viral infections, i) children, or j) subjects with a weakened immune system. In some embodiments, the subject is a woman, a woman who can become pregnant, or a pregnant woman.

Subjects treated according to the present invention will exhibit a therapeutic response. "Therapeutic response" means that a subject suffering from a viral infection has the following clinical benefits as a result of treatment with a highly worried sugar: reduction of active viral titer in the subject's blood or plasma, subject's blood. Alternatively, eradication of the active virus from the plasma, improvement of the infection, reduction of the incidence of infection-related symptoms, partial or complete remission of the infection, or increased time to progression of the infection, and / or the viral infection. It means enjoying at least one of the reductions in infectivity of the virus that causes it. The therapeutic response can be a complete or partial therapeutic response.
As used herein, "time to progression" is the time, length, or duration from the diagnosis (or treatment) of a viral infection to the onset of exacerbation of the infection. It is the period during which the level of infection is maintained without further progression of the infection, and ends when the infection begins to progress again. Disease progression is determined by "staging" subjects affected with the infection before or at the start of treatment. For example, the health of a subject is determined before or at the start of treatment. Subjects were then treated with an antiviral composition and their viral titers were regularly monitored. At some point later, the symptoms of the infection may worsen, thus marking the progression of the infection and the end of the "time to progression". The period during which the infection has not progressed, or the period during which the level or severity of the infection has not worsened, is the "time to progress".

The dosing regimen includes one or more cardiac glycosides and / or triterpenes (s) of treatment-related doses (or effective doses) administered according to the dosing schedule. Therefore, the treatment-related dose is a therapeutic dose at which the therapeutic response of the viral infection to treatment with the antiviral composition is observed and the subject can be administered the antiviral composition without an excessive amount of unwanted or adverse side effects. Is. Treatment-related doses can cause some side effects in the patient, but are not fatal to the subject. It is a dose at which the level of clinical benefit to a subject receiving the antiviral composition exceeds the level of adverse side effects experienced by the subject by administration of the antiviral composition or its constituents (s). .. Treatment-related doses will vary from subject to subject according to various established pharmacological, pharmacodynamic, and pharmacokinetic principles. However, treatment-related doses (eg, for oleandrin) are typically about 25 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms, or about 750 micrograms of anxious sugars / day. , Or it is about 25-750 micrograms of anxious sugars per dose, or about 25 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms or about 750 micrograms of anxious sugars / day. It can be in a range that cannot be exceeded. Other examples of treatment-related doses (eg, for individual or combined triterpenes) are typically about 0.1 micrograms to 100 micrograms, 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 about 500 mg / kg, about 10 to about 750 mg / kg, about It may range from 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 body weight. It is known in the art that the actual amount of antiviral composition required to provide targeted therapeutic results in a subject may vary from subject to subject according to the basic principles of the pharmacy.

Treatment with digoxin can be performed using two or more dosing phases: the loading phase and the maintenance phase. The loading phase can use the following dosing regimen until steady-state plasma levels of digoxin are achieved, and the maintenance phase can use the following dosing regimen after completion of the loading phase.

Treatment-related doses can be administered according to any dosing regimen commonly used to treat viral infections. Treatment-related doses can be administered once, twice, three times or more daily. It is every other day, every three days, every four days, every five days, every six months, every week, every other week, every three weeks, every four weeks, every month, every other month, every three months, every three months, every six months. Administered annually, annually, or according to any combination of the above to reach a suitable dosing schedule. For example, treatment-related doses can be administered at least once daily (up to 10 times daily at the highest doses) over a week or longer.

Example 15 is oleandrin (as the only activity) for the treatment of Ebolavirus (FIGS. 1 and 2) and Marburg virus (FIGS. 3 and 4) infections, both of which are filoviruses, Anvirzel ™. Provides a detailed description of the in vitro assay used to assess the efficacy of compositions comprising (a hot water extract of virus oleander) and PBI-05204 (a supercritical fluid (SCF) extract of virus oleander). do.

Hot water extracts can be administered orally, sublingually, subcutaneously, and intramuscularly. One embodiment is ANVIRZEL ™ (Nerium Biotechnology, Inc., San Antonio, TX; Salud Optical Medical Clinic, Tegucigalpa, Honduras; www. Available. For sublingual administration, a typical dosing regimen is 1.5 ml per day, or 0.5 ml at a dose of 3 times daily. For administration by infusion, a typical dosing regimen is from about 1 to about 2 ml / day, or from about 1 week to about 6 months or more, from about 0.1 to about 0.4 ml / m ² / day, or from about 1 week. About 0.4 to about 0.8 ml / m ² / day for about 6 months or longer, or about 0.8 to about 1.2 ml / m ² / day for about 1 week to about 6 months or longer. The maximum tolerated dose of ANVIRZEL ™ is so high that higher doses can be used. ANVIRZEL ™ includes polysaccharides (hot water extracts) extracted from oleandrin, oleandrigenin, and Nerium oleander. Commercially available vials are freeze-dried powders of about 150 mg of oleander extract containing about 200-about 900 microg of oleander, about 500-about 700 microg of oleander genin, and polysaccharides extracted from Nerium oleander. Included as (before reconstitution with water). The vial also contains at least one penetrant, such as mannitol, sodium chloride, at least one buffer, such as sodium ascorbate and ascorbate, and at least one preservative, such as propylparaben, methylparaben and other pharmaceutical excipients. Can include.

Experiments were prepared by adding the composition to cells at 40 microg / mL, then adding the virus and incubating for 1 hour. Upon addition of the virus to the cells, the final concentration of the composition is 20 microg / mL. Compositions containing different amounts of oleandrin can be adjusted according to the concentration of oleandrin they contain and converted to molar concentration. FIGS. 1-4 show efficacy based on the oleandrin content of the extract. The office lady itself is effective. PBI-05204, an SCF extract of Nerium oleander containing OL, OA, UA, and BA, is substantially more effective than OL itself. Anvirzel ™, a hot water extract of Nerium oleander, is more effective than OL itself. Both extracts clearly demonstrate efficacy in the nanomolar range. The proportion of oleandrin (1.74%) in the PBI-05204 extract is higher than the proportion of Anvirzel ™ (0.459%, 4.59 microg / mg). The highest dose of PBI-05204 completely inhibited EBOV and MARV infection, but Anvirzel ™ did not show complete inhibition as toxicity was observed at doses above 20 microlog / mL with Anvirzel ™. rice field. The data show the highest antiviral activity of PBI-05204 against Ebola and Marburg viruses. The combination of PBI-05204 triterpenes increased the antiviral activity of oleandrin.

Example 6 provides a detailed description of the in vitro assay used to assess the efficacy of cardiac glycosides for the treatment of Zika virus (flavivirus) infections. Vero E6 cells were infected with Zika virus (ZIKV strain PRVABC59) with a MOI of 0.2 in the presence of oleandrin (FIG. 5) or digoxin (FIG. 6). The cells were incubated with virus and cardiac glycoside for 1 hour before inoculation and non-absorbable cardiac glycoside (if any) were removed. The cells were soaked in fresh medium and incubated for 48 hours, then fixed with formalin and stained with ZIKV infection. The data show antiviral activity of both cardiac glycosides against Zika virus, however, oleandrin showed higher (almost eight-fold) antiviral activity than digoxin.
Example 14 provides a detailed description of the assay used to assess the antiviral activity of the test composition against Zika virus and dengue virus. The data show that oleandrin is effective against Zika virus and dengue virus.

FIG. 7 is a chart summarizing the in vitro dose-responsive antiviral activity of various compositions (oleandrin, digoxin, and PBI-05204) against Ebola virus (EBOV) in Vero E6 cells. FIG. 8 shows a chart summarizing the in vitro dose-responsive antiviral activity of various compositions (oleandrin, digoxin, and PBI-05204) against Marburg virus (MARV) in Vero E6 cells. FIG. 9 shows a chart summarizing the in vitro cell viability of Vero E6 cells in the presence of various compositions (oleandrin, digoxin, and PBI-05204). In FIGS. 7-8, host cells were exposed to the composition prior to viral infection. EBOV / Kik (FIG. 7, MOI = 1) or MARV / Ci67 (FIG. 8, MOI = 1) in the presence of oleandrin, digoxin, or oleandrin-containing plant extract PBI-05204 in Vero E6 cells. ) Was 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 EBOV or MARV infected cells. Infected cells were counted using Operetta.

Cell viability in the presence of the composition was tested to ensure that no false positives were observed for antiviral activity. For the data in FIG. 9, Vero E6 cells were treated with the above compounds. ATP levels were measured by CellTiter-Glo as a measure of cell viability. Oleandrin, digoxin, and PBI-05204 were determined not to reduce cell viability, which is due to the cytotoxicity of the individual compounds, the antiviral activity detailed in the other figures herein. It means that it is not due to the false positives caused.
Accordingly, the present invention provides a method of treating a viral infection in a mammal or host cell, wherein the antiviral composition is administered to the mammal or host cell prior to the morbidity of the viral infection, thereby the mammal. Upon viral infection of an animal or host cell, the antiviral composition comprises reducing viral titer and improving, reducing or eliminating 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 composition. Vero E6 cells were infected with EBOV (FIGS. 10A, 10B) or MARV (FIGS. 11A, 11B). Two hours after infection (FIGS. 10A, 11A) or 24 hours after infection (FIGS. 10B, 11B), oleandrin or PBI-05204 was added to the cells for 1 hour, then discarded and the cells returned to culture medium.

10A and 10B show a chart summarizing the ability of the composition (oleandrin and PBI-05204) to inhibit Ebola virus in Vero E6 cells immediately after exposure to the virus, FIG. 10A shows 2 hours after infection. , FIG. 10B is 24 hours after infection. When the antiviral composition is administered within 2 hours (or up to 12 hours) after virus infection, the antiviral composition provides effective treatment and reduces EBOV virus titers. Even after 24 hours, the viral composition is still effective, however, as time passes after the initial viral infection has increased, its effectiveness becomes less. The same evaluation was done on MARV. 11A and 11B show a chart summarizing the ability of the composition (oleandrin and PBI-05204) to inhibit Marburg virus in Vero E6 cells immediately after exposure to the virus, FIG. 11A shows 2 hours after infection. , FIG. 11B is 24 hours after infection. When the antiviral composition is administered within 2 hours (or up to 12 hours) after virus infection, the antiviral composition provides effective treatment and reduces MARV virus titers. Even after 24 hours, the viral composition is still effective, however, as time passes after the initial viral infection has increased, its effectiveness becomes less.

Given that the antiviral activity of the compositions herein is reduced in a single generation of virus-infected cells, eg, within 24 hours after infection, can the antiviral composition inhibit viral growth? Evaluate, which means inhibit the production of infectious offspring. Vero E6 cells were infected with EBOV or MARV in the presence of oleandrin or PBI-05204 and incubated for 48 hours. The supernatant of the infected cell culture was subcultured into fresh Vero E6 cells, incubated for 1 hour, and then discarded. Cells containing the passaged supernatant were incubated for 48 hours. Cells infected with EBOV (B) or MARV (C) were evaluated as described herein. The control infection rate was 66% for EBOV and 67% for MARV. The antiviral composition of the present invention inhibited the production of infectious offspring.
Thus, the antiviral compositions of the invention can be a) prophylactically administered prior to viral infection to inhibit viral infection after exposure to the virus, or b) viral replication and infectious progeny. Can be administered after viral infection, or a combination of c) a) and b) to inhibit or reduce the production of.

The antiviral activity of the Togavirus antiviral composition against alpha virus was assessed using VEE and WEE viruses in Vero E6 cells. 13A and 13B show in vitro compositions (oleandrin, digoxin, and PBI-05204) against Venezuelan encephalomyelitis virus (FIG. 13A) and western horse encephalomyelitis virus (FIG. 13B) in Vero E6 cells. A chart summarizing the dose-responsive antiviral activity is shown. Venezuelan equine encephalitis virus (Fig. 13A, MOI = 0.01) or Western equine encephalitis virus (Fig. 13B, MOI = 0.1) was applied to Vero E6 cells in the presence or absence of the indicated compound for 18 hours. Infected. Infected cells were detected as before and counted in Operetta. The antiviral composition of the present invention has been found to be effective.
Therefore, the present invention relates to an arenavirus family virus, a phyllovirus family virus, a flavivirus family virus (flavivirus genus), a retroviridae virus, a delta retrovirus family virus, a coronavirus family virus, and paramixo in a subject or host cell. A method of treating a viral infection caused by a virus family virus, or Toga virus family virus, in which an effective amount of an antiviral composition is administered, thereby exposing the virus to the antiviral composition, and said virus. Provide methods including treating an infection.

We, oleandrin and the extracts described herein for the treatment of HTLV-1 (human T-cell leukemia virus type 1; enveloped retrovirus; Deltaretrovirus) infection. Evaluated the use of. Purified oleandrin compounds, or N. To determine if an extractor extract can inhibit HTLV-1 provirus replication and / or the production and release of p19 ^Gag -containing viral particles, virus-generated HTLV-1-transformed SLB1 lymphoma T-cells. Strains were added to elevated concentrations of oleandrin or N. et al. It was treated with oleander extract or sterilized vehicle control (20% DMSO in ddH ₂ O treated with MilliQ) and then incubated under 10% CO ₂ at 37 ° C. for 72 hours. The cells were then pelleted by centrifugation and subjected to ELISA (Zeptometry) of anti-HTLV-1 p19 ^Gag to quantify the relative levels of extracellular p19 ^Gag -containing virus particles released into the supernatant of the culture.

FIG. 14 shows vehicle controls (1.5 μl, 7.5 μl, or 15 μl), or elevated concentrations (10 μg / ml, 50 μg / ml, and 100 μg / ml) of oleandrin compounds or N. Data are shown for quantification (Examples 19 and 20) of HTLV-1 p19 ^Gag expressed by HTLV-1 + SLB1 lymphoma T-cell lines treated with an extract of oleander for 72 hours. Viral replication and release of extracellular particles into cultures were quantified by anti-HTLV-1 p19 ^Gag ELISA (Zeptometrix). Oleandrin did not significantly inhibit the release of HTLV-1 replication or newly synthesized viral particles. We have determined that only the extract or oleandrin does not significantly inhibit viral replication or the release of p19 ^Gag -containing particles into the culture supernatant. We therefore did not anticipate further antiviral activity; however, we found that the viral particles collected from the treated cells were infectious in primary human peripheral blood mononuclear cells (huPBMC). Unexpectedly found that it showed a reduction. Unlike HIV-1, extracellular HTLV-1 particles are less infectious and virus transmission typically occurs via direct intracellular interactions via virological synapses.
The present invention is therefore a method of producing HTLV-1 virus particles with reduced infectivity, wherein the HTLV-1 virus particles are treated with the antiviral composition of the present invention to reduce infectivity. 1 Provided is a method comprising obtaining virus particles.

To ensure that the observed antiviral activity was not an artifact due to the potential cytotoxicity of the antiviral composition to HTLV-1 + SLB1 lymphoblasts, we then treated HTLV. Purified oleandrin compounds and N. et al. In a -1 + SLB1 lymphoblast culture. The cytotoxicity of various dilutions of the oleander extract was evaluated (Example 21). SLB1 T-cells are referred to as oleandrin or N. Treatment was performed with elevated concentrations of oleander extract (10, 50 and 100 μg / ml) for 72 hours. As a negative control, cells were also treated with an elevated volume (1.5, 7.5, and 15 μl) of vehicle solution used in the drug-treated culture. Cells treated with cyclophosphamide (50 μM; Sigma-Aldrich) were included as positive controls for apoptosis. Samples were then washed, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by confocal fluorescence microscopy. The relative proportions of Annexin V-FITC and / or PI-positive cells were quantified by fluorescence microscopy and the field of view was counted 3 times using a 20x objective.

Results (FIGS. 15 and 16A-16F) show the lowest concentrations (10 μg / ml) of oleandrin and N. et al. It indicates that the oleander extract did not induce significant cytotoxicity / apoptosis. However, higher concentrations (about 50 and about 100 μg / ml) of crude plant extracts, notably, induced apoptosis more than oleandrin compounds did. This is Oleandrin's N. Consistent with occupying about 1.23% of the oleander extract. The cytotoxicity caused by oleandrin was not significantly higher in treated HTLV-1 + SLB1 cells than in vehicle controls.

The inventors then described oleandrin or N. et al. It was investigated whether the oleander extract was able to inhibit virus transmission from the green fluorescent protein (GFP) -expressed HTLV-1 + lymphoma T-cell line to huPBMC in a co-culture assay (Example 20). In these studies, HTLV-1 + SLB1 lymphoma T-cells were placed in 96-well microtiter plates at elevated concentrations of oleandrin compounds or N. Treatment with oleander extract, or vehicle control, for 72 hours, then virus-containing supernatants were collected and used to directly infect primary cultured human peripheral blood mononuclear cells (huPBMC) in vitro. After 72 hours, the relative levels of extracellular p19 ^Gag -containing virus particles released into the supernatant of the culture as a result of direct infection were quantified by performing anti-HTLV-1 p19 ^Gag ELISA.

The HTLV-1 + SLB1 lymphoma T-cell line was subjected to vehicle control, or elevated concentrations (10 μg / ml, 50 μg / ml, and 100 μg / ml) of N. It was treated with an oleander extract or an organophosphorus compound for 72 hours, then the virus-containing supernatant was collected and used to directly infect the primary huPBMC. Vehicle control, N. The oleander extract, or oleandrin, was also included in the culture medium for huPBMC. After 72 hours, the supernatant of the culture was collected and anti-HTLV-1 p19 ^Gag ELISA was performed to quantify the significant amount of extracellular virus particles produced.

The data (FIG. 17) show the lowest concentrations (10 μg / ml) of oleandrin and N. et al. Even the oleander extract indicates that it inhibited the infectivity of the newly synthesized p19 ^Gag -containing virus particles released into the supernatant of the culture of treated cells as compared to the same amount of vehicle control. Both oleandrin and crude extracts inhibited virological synapse formation and transmission of HTLV-1 in vitro. Extracellular virus particles produced by HTLV-1 + lymphoma T-cells treated with oleandrin show reduced infectivity to primary huPBMC. Importantly, oleandrin exhibits antiviral activity against enveloped viruses by reducing the incorporation of enveloped glycoproteins into mature particles that represent a unique step in the retroviral infection cycle.

To ensure that the observed antiviral activity was not an artifact of the antiviral composition due to potential cytotoxicity against treated huPBMC, we also purified with treated huPBMC. Oleandrin and N. The cytotoxicity of the oleander extract was investigated in comparison with a vehicle negative control (Example 21). The primary huPBMC of the buffy coat was isolated, stimulated with phytohaemagglutinin (PHA) and cultured in the presence of recombinant human interleukin-2 (hIL-2). The cells were then subjected to elevated concentrations of oleandrin or N. Treated with oleander extract or with an elevated volume vehicle for 72 hours. Samples were subsequently stained with Annexin V-FITC and PI, and the relative proportion of apoptotic (ie Annexin V-FITC and / or PI-positive) cells per view was cofocal fluorescence microscopy and 3 counts. Quantified by.

Vehicle control, N. The cytotoxic effects of the oleander extract and the oleandrin compound were evaluated by treating the primary huPBMC for 72 hours, then the cultures were stained with Annexin V-FITC and PI. Relative proportions of apopulated (ie Annexin V-FITC and / or PI-positive) cells were quantified by fluorescence microscopy and 3 counts of the visual field using a 20x objective. The total number of cells was determined using DIC phase contrast microscopy. Cells treated with cyclophosphamide (50 μM) were included as a positive control against apoptosis. NA, which indicates the number of cells in this sample, was too low for an accurate assessment due to its high toxicity.
The data (FIG. 18) indicate oleandrin that showed moderate cytotoxicity (eg, 35-37% at the lowest concentration) in huPBMC compared to vehicle controls. In contrast, N. The oleander extract was significantly cytotoxic and induced high levels of programmed cell death even at the lowest concentrations. The huPBMC was slightly more sensitive to purified oleandrin than the HTLV-1 + SLB1 lymphoblasts; however, the huPBMC also contained other cytotoxic compounds such as triterpenes described herein. It was much more sensitive to the oleander extract.

We also have oleandrin or N. et al. It was investigated whether the oleander extract could inhibit the propagation of HTLV-1 particles to the target huPBMC in co-culture experiments (Example 22). In these studies, virus-producing HTLV-1 + SLB1 T-cell lines were used with mitomycin C, followed by increased doses of oleandrin, N. et al. Treated with oleander extract, or vehicle control, for 15 minutes or 3 hours. SLB1 cells were washed 2x in serum-free medium, the same number of huPBMCs were then added to each well, and the sample was sampled at 37 ° C. under 10% CO ₂ in a humidified incubator for 72 hours in complete medium. Co-cultured. Relative intracellular transmission of HTLV-1 was assessed by performing anti-HTLV-1 p19 ^Gag ELISA and measuring the level of extracellular virus released into the supernatant of the culture.
Primary huPBMCs were co-cultured with HTLV-1 + SLB1 lymphoma T-cells treated with mitomycin C and these were vehicle control or elevated concentrations (10 μg / ml, 50 μg / ml, and 100 μg / ml) of N. Pretreated with oleander extract or organophosphorus compounds for 15 minutes or 3 hours. Vehicle controls, extracts, and compounds were also present in co-culture groups. After 72 hours, the supernatant was collected and the amount of released extracellular virus particles was quantified by performing anti-HTLV-1 p19 ^Gag ELISA.

The results shown in FIG. 19 are oleandrin and N. et al. Both of the oleander extracts showed inhibition of HTLV-1 transmission compared to vehicle controls; there was no observed difference between 15 minutes and 3 hours of pretreatment of HTLV-1 + SLB1 cells. ..
We also investigated whether oleandrin inhibits virological synaptogenesis and transmission of HTLV-1 in co-culture assays (Example 22). The GFP-expressed HTLV-1 + SLB1 T-cell line was generated by transducing SLB1 lymphoma T-cells with the pLenti-6.2 / V5-DEST-GFP vector and blastsidine (5 μg / ml; Life Technologies). Selected for 2 weeks. GFP-positive clones were screened by fluorescence microscopy (upper panel in FIG. 20) and immunoblotting (lower panel in FIG. 20), expanded and repeatedly passaged. Images of DIC phase differences are shown for comparison.

Vehicle control or elevated doses (10 μg / ml, 50 μg / ml, and 100 μg / ml) of N. Fluorescence of the formation of viral synapses between huPBMCs pretreated with oleander extract or oleandrin compounds for 3 hours and HTLV-1 + SLB1 / pLenti-GFP lymphoblasts (green cells) treated with mitomycin C. Visualized by microscopy (Fig. 21). Virus transmission was infected (ie, HTLV-1 gp21-positive, red) The relative proportion of huPBMC (GFP-negative) was quantified in 20 fields of view (n = 20) by fluorescence microscopy using a 20x objective lens. (See the arrow in the vehicle control panel). Fluorescence microscopy data was quantified (FIG. 22). The data confirm that the antiviral composition inhibits virological synapse formation and transmission of HTLV-1 in co-culture assays.
Therefore, the present invention also inhibits (reduces) the infectivity of HTLV-1 particles released into the supernatant of the culture of treated cells, and also inhibits the Env-dependency formation of virological synapses. Provided is a method of reducing intracellular transmission of HTLV-1 by comprising treating virus-infected cells (in vitro or in vivo) with an effective amount of an antiviral composition.

The antiviral activity of the compositions herein was assessed against rhinovirus infection. Rhinoviruses belong to the family Picornavirus and the genus Enterovirus. It is an envelopeless, (+) polar ss-RNA virus. Oleandrin did not inhibit viral replication and was found to be inactive against rhinovirus at the concentrations and assays used herein.

CoV infections can be treated in vivo as detailed in Example 26 and the antiviral composition is administered to the subject as monotherapy or combination therapy. The efficacy of oleandrin for CoV was established in vivo according to Example 27. The child received 0.25 ml of reconstituted ANVIRZEL ™ as part of the orange juice. Then, every 12 hours, the child was administered 0.5 ml of reconstituted ANVIRZEL ™ over a period of about 2-3 days. The child recovered from COVID-19 infection.

Further proof of the efficacy of oleandrin (oleandrin-containing composition) against coronaviruses such as SARS-CoV-2 (COVID-19) was obtained via in vitro evaluation according to Example 28, oleanding Vero cells. It was pretreated with phosphorus and then infected with SARS-CoV-2. After infection of the cells, the extracellular virus and oleandrin were washed away and the infected cells were then oleandrin (FIG. 23A: 0.1% v / v 1 microlog / mL in DMSO aqueous solution; FIG. 23C: 0.01% v. Treatment was performed with 0.1 microg / mL in / vDMSO aqueous solution or only DMSO aqueous solution as a control vehicle (FIG. 23B: 0.1% v / vDMSO aqueous solution; FIG. 23D: 0.01% v / vDMSO aqueous solution). The results were a) oleandrin pretreatment caused a 1368-fold reduction in viral load at 24 hours and a 369-fold reduction at 48 hours; b) oleandrin was about 0.1-about. Oleandrin was even at low concentrations, eg 0.01-0.1 virus / mL, as it was more effective than the full concentration range of 1.0 microg / mL and the high dose was slightly better than the low dose. C) Oleandrin should be given repeatedly as a single dose is insufficient to completely stop viral replication; and d) Oleandrin and Using only 30 minutes of preincubation of Vero cells indicates that it is only slightly effective in reducing early viral infections and does not appear to affect the infectivity of progeny virions. The results also indicated that oleandrin at concentrations of 0.1 and 1.0 microg / mL was not overly toxic to Vero cells. The results further indicate that oleandrin inhibits progeny virus infectivity by a) about 1 log ₁₀ without continuous drug treatment; and b) about> 3 log ₁₀ with continuous drug treatment (no toxicity). Instruct.

To determine if oleandrin directly inhibits viral replication, Vero-E6 cells were infected with SARS-CoV-2 virus and treated with various concentrations of oleandrin according to Example 29. The results are shown in FIGS. 24A and 24B. At 24 hours (FIG. 24A), antiviral activity was observed in wells treated with oleandrin only during the absorption phase (pretreatment data) and the IC ₅₀ evaluated was 0.625 microlog / mL. In wells treated with oleandrin, over the duration of the assay (duration data), oleandrin significantly restricted viral entry and / or virus replication, even in the presence of large doses of inoculum. At 48 hours (FIG. 24B), minimal antiviral activity was observed by the end of the time in wells treated with oleandrin only during the absorption phase (pretreatment data). In wells treated with oleandrin over the duration of the assay (duration data), oleandrin significantly limited viral infection. Potential 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 is not due to the cytotoxicity of oleandrin to Vero-E6 cells, the cell titer was 24 hours (Figure). Judgment was made at 24A) and 48 hours (FIG. 24B). At oleandrin concentrations above 1.0 microg / mL, cytotoxicity developed and potentially interfered with the assay; however, at oleandrin concentrations below 0.625 microg / mL, cytotoxic interference was substantially reduced. And it confirmed the potent antiviral activity of oleandrin, even at very low concentrations. Additional evidence of oleandrin toxicity to Vero-E6 cells was observed in the assay of Example 30 (FIG. 25). At a oleandrin concentration of 0.625 microg / mL, about 80% of Vero cells were still viable at 24 hours, and at lower concentrations, still low toxicity was observed. It should be understood that the toxicity of oleandrin to Vero-E6 cells does not suggest that oleandrin is toxic to humans. This toxicity measurement is used only to determine the potential effects of background cell death when measuring antiviral activity.
Oleandrin therefore possesses at least two mechanisms (routes) for treating viral infections, specifically coronavirus infections, such as SARS-CoV-2 infections: a) direct inhibition of viral replication; and b) Reduction of infectivity of progeny virus.

In addition, oleandrin possesses antiviral activity even at very low doses, and oleandrin exhibits a significant protective effect. This was shown according to Example 31 and VERO CCL-81 cells were infected with SARS-CoV-2. Cells were pretreated with oleandrin prior to infection. After the first incubation 2 hours after infection, infected cells were washed to remove extracellular virus and oleandrin. Then, the recovered infected cells were treated as follows. Infected cells are treated with oleandrin (various concentrations in DMSO aqueous solution with RPMI 1640 medium as an aqueous component) or control vehicle only (DMSO aqueous solution with RPMI 164) 24 hours after "treatment" (FIG. 26A) and Virus titers were measured at 48 hours (FIG. 26B). Without oleandrin, SARS-CoV-2 reached a high (approximately 6 log ₁₀ plaque forming unit (pfu) / ml) titer by 24 hours and maintained this titer at a later time: it Consistently remained below the detection limit of the assay. Oleandrin concentrations of 1 microg / mL to 0.05 microg / mL resulted in a significant reduction in virus titers in 24 hours alone. The two higher doses reduced the virus titer to below the detection limit in principle and no cytotoxicity was observed at any of the oleandrin concentrations tested. Double unit reductions in virus titers were calculated for these samples. Double-unit reductions in virus titers ranging from about 1,000-fold to about 40,000-fold (FIGS. 26C and 26D) were observed at 48 hours, with approximately 1,000-fold to approximately 20,000-fold. , Observed at 24 hours. The 10 ng / ml dose did not show a significant effect compared to DMSO controls 24 hours after infection, but did result in a significant reduction in titers 48 hours after infection. Importantly, the reduction due to oleandrin was improved at the highest concentration compared to 24 hours when measured at 48 hours. Increased prophylactic efficacy of oleandrin is reflected over time (24 vs. 48 hours) as _EC50 values calculated at 11.98 ng / ml 24 hours after infection and 7.07 ng / ml 48 hours after infection. It had been.

The Vero81 cells described above were subjected to genomic analysis to determine if SARS-CoV-2 inhibition was at the level of total or infected particle production. RNA was extracted from the supernatant of cell cultures from prophylactic studies and genomic equivalents were quantified by qRT-PCR (Example 39). The protective effect of oleandrin first observed by the infection assay was confirmed at the level of genomic equivalents. Twenty-four hours after infection, oleandrin significantly reduced the SARS-CoV-2 genome in the supernatant at the four highest doses. The protective effect of oleandrin first observed by the infection assay was confirmed at the level of genomic equivalents. Twenty-four hours after infection, oleandrin significantly reduced the SARS-CoV-2 genome in the supernatant at the four highest doses.

Additional studies were performed to determine the dose response of COVID-19 infection to oleandrin 24 and 48 hours after infection (FIGS. 27A-27B). Observing the dose response, the increased concentration of oleandrin in the medium further reduced the viral titer; however, even the lowest concentration tested (0.05 microlog / mL) reduced the titer in 24 hours. The titer was further reduced 48 hours after infection. At the highest dose, an infectious SARS-CoV-2 titer was reduced by more than 1,000-fold, and doses of 0.5 μg / ml and 100 ng / ml caused a reduction of more than 100-fold, 50 ng / ml. The dose resulted in a 78-fold reduction.

Figures 28A and 28B show the results of repeated studies, each performed 3 times to vary the oleandrin concentration (0.005-1 microlog / mL) in the medium and to indicate the dose response to COVID-19 treatment. Judged. Significant antiviral activity was observed in Vero81 cells at concentrations above 0.01 microg / mL, even 24 and 48 hours after infection. A significant reduction in virus titers was observed even at very low concentrations of 0.05 microlog / mL.

A study according to Example 34 was performed to determine the antiviral efficacy of oleandrin after infection. Vero81 cells were not pretreated with oleandrin prior to infection. Instead, cells were infected with COVID-19 virus and then treated with oleandrin 12 and 24 hours after infection (at indicated concentrations). Virus titers were then measured 24 hours after infection (FIG. 29A) and 48 hours (FIG. 29B). The data show that oleandrin can exert antiviral activity at least 12, at least 24 hours, or at least 36 hours after infection, even with a single treatment. It is important to note that this assay is a time-saving assay compared to human viral infections. The 24-hour time point is equal to about 5-7 days after infection in humans and the 48-hour time point is equal to about 10-14 days after infection in humans.

Examples 31 and 34 using a combined composition of the two extracts (1 wt% ethanol extract dissolved in DMSO (98 wt%), PBI-A containing 1 wt% Example 36). The assay was repeated. FIG. 30A details the results for the evaluation of the combined composition of the two extracts according to the assay of Example 31, and FIG. 30B shows the evaluation of the two extracts (1 wt%) according to the assay of Example 34. The results for are detailed. The data in FIG. 30A show the relative antiviral (anti-COVID-19) efficacy of PBI-A based on the relative dilution of the original stock solution. The data in FIG. 30B is based on the relative concentration of oleandrin (μg / mL) in the assay solution. Dotted lines in each graph indicate the lowest concentration of virus that can be detected using the CFU (Viral Colony Forming Unit) assay.

Based on the results in FIGS. 30A and 30B, the combined composition of the two extracts was COVID- at a concentration containing 0.05-1.0 μg / ml, which is in the same range as that observed with pure oleandrin. It is effective as an antiviral agent against 19.
It is also important to observe that the oleandrin levels assessed in the assay are clinically relevant in terms of dose and plasma levels.
Proof of safety of the oleandrin-containing composition is further obtained by an in vitro cell assay to determine the release of lactate dehydrogenase after exposure of the cells to a solution containing different concentrations of oleandrin. It was determined that at a maximum concentration of 1 microg / mL, no further toxicity was present beyond the control vehicle.

The effectiveness of oleandrin (oleandrin-containing composition, oleandrin-containing extract) in the treatment of COVIDEO-19 virus infection is according to Example 35 under the FDA's new clinical trial drug range expansion system. It was further established by administration of the oleandrin-containing sublingual dosage form (Example 32 or 37) to the subject. Oleandrin (2) for subjects aged 18 to 78 years (y) at a 15 microg dose per day, 4 times at approximately 6 hour intervals, or at a 15 mg dose per day, 3 times at approximately 8 hour intervals. As a composition of two extracts) was administered. Prior to the start of treatment, the clinical condition and / or viral titer of the subject was observed. Some subjects were in palliative or hospice care. Clinical status and / or viral titers were routinely determined during the treatment period of 1-2 weeks, 10-14 days. The following results were observed after the start of treatment.

Additional in vivo studies were performed in Group 2 human subjects with different levels of COVID-19-related symptoms under the FDA's new clinical trial drug availability regime. Prior to the start of treatment, the subject's clinical status and / or viral titer was determined to confirm COVID-19 infection. Some subjects showed moderate to severe symptoms. Subjects in the age range received 4 doses of oleandrin (as a composition of the two extracts) at a daily dose of 15 microg at intervals of about 6 hours. Clinical status and / or viral titers were routinely determined during the treatment period of 1-2 weeks, 10-14 days. All subjects fully recovered from COVID-19 infection within 5-12 days after the start of treatment.

Oleandrin has also been shown to produce a potent anti-inflammatory response that can be beneficial in the prevention of hypersensitive inflammatory responses to SARS-CoV-2 infection.
The present invention is therefore a method of treating a COVID-19 virus infection in which multiple doses of cardiac glycosides (cardiac glycoside-containing compositions or cardiac glycoside-containing extracts) are used to treat the infection. Provided are methods including administration to a subject having a virus. Multiple doses can be divided into doses of 2 days or more per week, optionally 1 week or more per month, and optionally 1 or more doses per day for 1 month or more per year. A preferred cardiac glycoside is oleandrin.

The present invention is therefore a method of treating a coronavirus infection, in particular a coronavirus infection that is pathogenic to humans, such as SARS-CoV-2 infection, for a therapeutically effective dose to a subject having said infection. Provided are a method comprising chronic administration of a strong worried sugar (composition containing a strong worried sugar). Chronic administration can be achieved by repeated administration of one or more (plural) therapeutically effective doses of cardiac glycosides (cardiac glycoside-containing compositions). The one or more doses may be administered daily over a day or more per week, optionally over a week or more per month, and optionally over a month or more per year.

Accordingly, the present invention is a method of treating an infection with a virus, eg, CoV, in a subject in need of treatment (specifically, a human subject), wherein the subject is a) oleandrin; or b) oleandrin. , And a method comprising administering one or more doses of an antiviral composition comprising one or more other compounds extracted from the Nerium species. Oleandrin can be present as part of the Nerium species extract, which is a) supercritical fluid extract; b) hot water extract; c) organic solvent extract; d) aqueous organic solvent. Extract; e) Extract using supercritical fluid and optionally adding at least one organic solvent (extraction modifier); f) Using subcritical liquid and optionally at least one organic solvent (extraction reformer) The extract to which the agent) is added; or g) any combination of any two or more of the extracts.

PBI-05204 (US8187644B2 of Addington, issued May 29, 2012, US7402325B2, issued July 22, 2008, which is incorporated herein by reference in its entirety, March 2013, March 2013. (Described in US8394434B2 by Addington et al., Published 12th), are the major pharmacologically active constituents of the worrisome sugar (oleandrin, OL) and triterpenes (oleanolic acid (OA), ursolic acid (UA)). ), And bethulinic acid (BA)). The molar ratio of OL to total triterpenes is about 1: (10-96). The molar ratio of OA: UA: BA is about 7.8: 7.4: 1. The combination of OA, UA, and BA of PBI-05204 increases the antiviral activity of oleandrin when compared on an OL equimolar basis. PBI-04711 is a fraction of PBI-05204, but it does not contain cardiac glycosides (OLs). The molar ratio of OA: UA: BA of PBI-04711 is about 3: 2.2: 1. PBI-04711 also possesses antiviral activity. Therefore, antiviral compositions containing OL, OA, UA, and BA are more effective than compositions containing OL as the sole active ingredient, based on the equimolar content of OL. In some embodiments, the molar ratio of individual triterpen to oleandrin ranges from: about 2-8 (OA): about 2-8 (UA): about 0.1-1 (BA). : Approximately 0.5 to 1.5 (OL); or Approximately 3 to 6 (OA): Approximately 3 to 6 (UA): Approximately 0.3 to 8 (BA): Approximately 0.7 to 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) ): Approximately 4.4 (UA): Approximately 0.6 (BA): Approximately 1 (OL).

Antiviral compositions comprising oleandrin as the sole antiviral agent are within the scope of the invention. Antiviral compositions containing digoxin as a simple substance antiviral agent are within the scope of the present invention.
Antiviral compositions comprising oleandrin and a plurality of triterpenes as antiviral agents are within the scope of the present invention. In some embodiments, the antiviral composition comprises oleandrin, oleanolic acid (its free acid, salt, derivative, or prodrug), ursolic acid (its free acid, salt, derivative, or prodrug), and. Includes bethulinic acid (its free acid, salt, derivative, or prodrug). The molar ratios of the compounds are as described herein.

Antiviral compositions comprising multiple triterpenes as the major active ingredient (meaning excluding steroids, cardiac glycosides, and pharmacologically active ingredients) are also within the scope of the invention. As mentioned above, PBI-04711 is. It contains OA, UA, and BA as the major active ingredients, which exhibit antiviral activity. In some embodiments, the triterpene-based antiviral composition comprises OA, UA, and BA, each independently from its free acid form, salt form, deuterated form, and derivative form. Selected at each occurrence.

PBI-01011 is an improved triterpene-based antiviral composition comprising OA, UA, and BA with a molar ratio of OA: UA: BA of 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 to 10.5: about 0.75 to 1.25: about 0.75 to 1.25, or about 9.5 to 10.5: about 0.8 to 1.2: It is about 0.8 to 1.2, or about 9.75 to 10.5: about 0.9 to 1.1: about 0.9 to 1.1.

In some embodiments, the antiviral composition is at least oleanolic acid (its free acid, salt, derivative, or prodrug) and ursolic acid present in a molar ratio of OA to UA, as described herein. Includes acids (its free acids, salts, derivatives, or prodrugs). OA is present in large molar excesses above UA.
In some embodiments, the antiviral composition is at least oleanolic acid (its free acid, salt, derivative, or prodrug) and bethulin present in a molar ratio of OA to BA, as described herein. Includes acids (its free acids, salts, derivatives, or prodrugs). OA is present in large molar excesses above BA.
In some embodiments, the antiviral composition is at least oleanolic acid (its free acid, salt, derivative, or prodrug) present in a molar ratio of OA to UA to BA, as described herein. , Ursoleic acid (its free acid, salt, derivative, or prodrug), and bethulin acid (its free acid, salt, derivative, or prodrug). OA is present in large molar excesses above both UA and BA.
In some embodiments, the triterpene-based antiviral composition excludes cardiac glycosides.

Generally, subjects with arenavirus infection, altern virus infection, phyllovirus infection, flavivirus infection (flavivirus genus), delta retrovirus genus, coronavirus family, paramyxovirus family, orthomixovirus family, or togavirus infection. Is treated as follows. The subject is evaluated to determine if the subject is infected with the virus. Administration of antiviral composition is instructed. The initial dose of the antiviral composition is administered to the subject for a period of time (treatment period) according to the prescribed dosing regimen. The level of clinical and therapeutic response of the subject is determined on a regular basis. If the level of therapeutic response is too low at a given dose, the dose is graded according to a given dose escalation schedule until the desired level of therapeutic response in the subject is achieved. Treatment of the subject with the antiviral composition is continued as needed. Dosages or dosing regimens are adjusted as needed until the patient reaches the desired clinical endpoint (s), such as cessation of the infection itself, reduction of infection-related symptoms, and / or reduction of infection progression. obtain.

The 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's viral infection is treated with the one or more other therapeutic agents. If it is known to respond at least partially therapeutically to, the methods of the invention include a treatment-related dose of the antiviral composition and the treatment-related dose of the one or more other therapeutic agents thereof. The antiviral composition is administered according to the first dosing regimen, including administration to the subject in need, and one or more other therapeutic agents are administered according to the second dosing regimen. In some embodiments, the first and second dosing regimens are the same. In some embodiments, the first and second dosing regimens are different.
The antiviral composition of the present invention (s) can be administered as primary antiviral therapy, adjuvant antiviral therapy, or combined antiviral therapy. The methods of the invention include separate or co-administration of the antiviral composition with at least one other known antiviral composition, wherein the antiviral composition of the invention is a known antiviral composition (compound). (Multiple)) or means that it can be administered before, during, or after administration of the composition for treating symptoms associated with a viral infection. For example, agents used to treat inflammation, vomiting, nausea, headache, fever, diarrhea, nausea, urticaria, conjunctivitis, malaise, muscle pain, joint pain, seizures, or paralysis are the antiviral agents of the invention. It can be administered with or separately from the composition.

One or more other therapeutic agents may be administered according to a dose and dosing regimen that the clinician perceives to be therapeutically effective, or at a dose that the clinician perceives to be less than therapeutically effective. .. The clinical benefits and / or therapeutic effects provided by administration of the antiviral composition and the combination of one or more other therapeutic agents can be additive or synergistic, and such levels of benefits or effects are , Combined administration and comparison with administration of individual antiviral composition components (s) and administration of one or more other therapeutic agents. Food and Drug Administration, World Health Organization, European Medicines Agency (EMEA), Drug and Drug Administration (TGA, Australia), National Health Organization (PAHO), Medsafe, New Zealand ), Or one or more other therapeutic agents can be administered according to doses and dosing regimens as proposed or described by various Ministry of Health around the world.

Other exemplary therapeutic agents that may be included in the antiviral compositions of the invention for treating viral infections are antiretroviral agents, interferon α (IFN-a), zidovudine, ramibdin, cyclosporine A, arsenic trioxide. CHOP, sodium valproate, methotrexate, azathiopurine, one or more symptom-relieving drugs (s), steroid sparing drugs, corticosteroids, cyclophosphamide, immunosuppressants, anti-inflammatory agents, Janus Kinase inhibitor, tofacitinib, calcinulin inhibitor, tacrolimus, mTOR inhibitor, sirolimus, everolimus, IMDH inhibitor, azathiopurine, reflunomide, mycophenolic acid, biologics, avatacept, adalimumab, anakinla, sertrizumab, etanelcept, golymab , Natalizumab, rituximab, sekkinumab, tosirizumab, ustequinumab, vedrizumab, monoclonal antibody, baciliximab, dacrizumab, polyclonal antibody, nucleoside analog, reverse transcriptase inhibitor, emtricitabine, terbibdin, abacavir , Azithromycin, macrolide antibiotics, protease inhibitors, interferons, immune response modifiers, mRNA synthesis inhibitors, protein synthesis inhibitors, thiazolide, CYP3A4 inhibitors, heterocyclic biguanidine, CCR5 receptor inhibitors, and them. Including combinations of. The treatments studied also include plasma separation and / or irradiation. Antibodies to a particular virus can also be administered to a subject treated with the antiviral composition of the invention. Plasma obtained from the blood of survivors infected with the virus for the first time can be administered to other subjects with the same type of viral infection, who are also administered the antiviral composition of the invention. For example, plasma from a survivor of COVID-19 infection may be administered to another subject with COVID-19 infection, said other subject also receiving the antiviral composition of the invention.

Example 5 provides an exemplary procedure for the treatment of Zika virus infection in mammals. Example 12 provides an exemplary procedure for the treatment of filovirus infections (Ebola virus, Marburg virus) in mammals. Example 13 is flavivirus infection, yellow fever, dengue fever, Japanese encephalitis, western Nile virus, decavirus, tick-borne encephalitis, Cassanur forest disease, Alkhurma disease, omsk hemorrhagic fever, Powassan virus infection in mammals. Provide an exemplary procedure for the treatment of the virus. Example 25 shows an exemplary procedure for treating a Deltaretrovirus (HTLV-1) infection.

The antiviral compounds (s) present in the pharmaceutical composition (triterpenes (s), cardiac glycosides (s), etc.) are in their unmodified form, salt form, derivative form, or a combination thereof. Can exist in. As used herein, the term "derivative" is a) a chemical that is structurally related to and then theoretically derivatable from the first chemical; b) formed from a similar first compound. Compounds to be compounded, or compounds that may result from another first compound if one atom of the first compound is replaced by another atom or group of atoms; c) derived or obtained from the parent compound. , A compound containing the essential elements of the parent compound; or d) interpreted to mean a chemical compound that can be produced from a first compound of similar structure in one or more steps. For example, the derivative may include its polyhydrogenated, oxidized, dehydrated, unsaturated, polymer complexed, or glycosylated forms, or ester, amide, lactone, homologous, ether, thioether, cyano, amino. , Alkylamino, sulfhydryl, heterocyclic, heterocyclic condensation, polymerization, pegging, benzidenyl, triazolyl, piperazinyl, or dehydrogenylated forms thereof.

As used herein, the term "oleandrin" is to be construed to mean all known forms of oleandrin, unless otherwise stated. Oleandrin can be present in racemic, optically pure, or optically enriched forms. Nerium oleander botanical materials are available from commercial botanical suppliers, for example, Aldridge Nursery in Atascosa, Texas.

Supercritical fluid (SCF) extracts can be prepared as detailed in US7,402,325, US8394434, US8187464, or PCT International Publication No. WP2007 / 016176A2, the entire disclosure of which is hereby referred to. Incorporated in the specification. Extraction can be carried out using supercritical carbon dioxide in the presence or absence of a modifier (organic solvent) such as ethanol.

Other extracts containing cardiac glycosides, especially oleandrin, can be prepared by a variety of different processes. The extract describes the procedure for preparing a hot water extract from Dr. Extracts can be prepared according to the process developed by Huseyin Ziya Ozel (US Pat. No. 5,135,745). Aqueous extracts are reported to contain several polysaccharides with molecular weights varying from 2KD to 30KD, oleandrin, oleandrigenin, odroside and neritaroside. Polysaccharides have been reported to include acidic homopolygalacturonan or arabinogaraturonan. US Pat. No. 5,869,060 of Selvaraj et al. Discloses a hot water extract of Nerium species and a method for producing it, eg, Example 2. The resulting extract can then be lyophilized to produce a powder. U.S. Pat. No. 6,565,897 (pre-Registration No. 2002114852 and PCT International Publication No. WO 2000/016793 of Selvaraj et al.) Discloses a hot water extraction process for the preparation of substantially sterilized extracts. are doing. Erdemoglu et al. (J. Ethnopharmacol. (2003) Nov. 89 (1), 123-129) compared aqueous and ethanol extracts of plants containing Nerium oleander based on anti-nociceptive and anti-inflammatory effects. The results are disclosed. Organic solvent extracts of Nerium oleander were also found in Adobe et al. (Afr. Health Sci. (2003) Aug. 3 (2), 77-86; ethanolic extract), el-Shazly et al. (J. Egypt SoC. Parasitol. ), August 26 (2), 461-473; ethanolic extract), Begum et al. (Phytochemistry (1999) Feb. 50 (3), 435-438; metanolic extract), Zia et al. .49 (1), 33-39; mechanical extract), and Vlasenko et al. (Farmatsia. (1972) Sept.-Oct. 21 (5), 46-47; alcoholic extract). US Pre-Registration Patent Application Publication No. 20040247660 of Singh et al. Discloses the preparation of a protein-stabilized liposomal formulation of oleandrin for use in the treatment of cancer. US Pre-Registration Patent Application Publication No. 20050026849 by Singh et al. Discloses a water-soluble preparation of oleandrin containing cyclodextrin. US Pre-Registration Patent Application Publication No. 200485221 of Singh et al. Discloses the preparation of a protein-stabilized nanoparticle formulation of oleandrin from a hot water extract.

オレアンドリンはまた、Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓの形質転換カルス（Ｉｂｒａｈｉｍら、Ｅｎｚ． Ｍｉｃｒｏｂｉａｌ Ｔｅｃｈｎｏ．（２００７），４１（３），３３１－３３６の“Ｓｔｉｍｕｌａｔｉｏｎ ｏｆ ｏｌｅａｎｄｒｉｎ ｐｒｏｄｕｃｔｉｏｎ ｂｙ ｃｏｍｂｉｎｅｄ Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ ｍｅｄｉａｔｅｄ ｔｒａｎｓｆｏｒｍａｔｉｏｎ ａｎｄ ｆｕｎｇａｌ ｅｌｉｃｉｔａｔｉｏｎ ｉｎ Ｎｅｒｉｕｍ It is obtained from an extract of suspension cultures derived from "oleander cell cells", the entire disclosure of which is incorporated herein by reference). Agrobacterium hot water, organic solvents, aqueous organic solvents, or supercritical fluid extracts can be used in accordance with the present invention.

Oleander is also obtained in vitro from an extract of the Nerium oleander microculture, whereby shoot cultures are cultivated from seedlings and / or from the stem tips of the Nerium oleander variety Splendens Giganteum, Revanche or Alsace, or other varieties. It can be initiated (Vila et al., HeartScience (2010), 45 (1), 98-102 "Microculture of Owner (Nerium seeder L.)", the entire disclosure of which is incorporated herein by reference). Hot water, organic solvents, aqueous organic solvents, or supercritical fluid extracts of microculture Nerium oleander can be used according to the present invention.

The extracts also differ in their polysaccharide and carbohydrate content. Hot water extracts contain 407.3 glucose equivalents of carbohydrates relative to a standard curve prepared with glucose, while analysis of SCF CO ₂ extracts is viewed at very low levels below the quantification limit. Found the carbohydrate level given. However, the amount of carbohydrates in the hot water extract of Nerium oleander was at least 100 times higher than that of the SCF CO ₂ extract. The polysaccharide content of the SCF extract can be 0%, <0.5%, <0.1%, <0.05%, or <0.01% by weight. In some embodiments, the SCF extract excludes the polysaccharides obtained during the extraction of the plant mass.

Partial compositions of SCF CO ₂ and hot water extracts are JEOL AccuTOF-DART mass spectrometers (JEOL USA, Peabody, MA, USA) with DART TOF-MS (real-time time direct for flight mass spectrometry). It was determined by analysis).
SCF extracts of Nerium or Thevetia are mixtures of pharmacologically active compounds such as oleandrin and triterpenes. The extract obtained by the SCF process is a viscous semi-solid that is substantially water insoluble at ambient temperature (after removing the solvent). The SCF extract contains many different constituents with a variety of different ranges of water solubility. Extracts from supercritical fluid processes are 0.9% by weight to 2.5% by weight oleandrin or 1.7% to 2.1% by weight oleandrin or 1.7% by weight. Includes a theoretical range of 2.0 wt% oleandrin. SCF extracts containing different amounts of oleandrin have been obtained. In one embodiment, the SCF extract contains about 2% by weight oleandrin. The SCF extract contains 3-10 times higher concentrations of oleandrin than the hot water extract. This was confirmed by both HPLC and LC / MS / MS (tandem mass spectrometry) analysis.

The SCF extract contains oleandrin and triterpene oleanolic acid, bethulinic acid and ursolic acid, and optionally other constituents described herein. The contents of oleandrin and triterpenes may vary from batch to batch, however, the degree of variation is not excessive. For example, a batch of SCF extract (PBI-05204) was analyzed for these four components and found to contain an approximate amount of each of the following:

The content of the individual constituents can vary by ± 25%, ± 20%, ± 15%, ± 10%, or ± 5% with respect to the values shown. Therefore, the content of oleandrin in the SCF extract is in the range of 20 mg ± 5 mg (± 25% of 20 mg) per 1 mg of the SCF extract.

Oleandrin, oleanolic acid, ursolic acid, bethulinic acid, and derivatives thereof can also be purchased from Sigma-Aldrich (www.sigmaaldrich.com; St. Louis, MO, USA). Digoxin is described in 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, 0.25 mg / mL), West-Ward Pharmaceuticals International LTD (NDA A083391, Injection, 0.25 mg / mL), Covis Pharma BV (NDA N09330, 0.1 mg / mL, 0.25 mg). / ML), Impax Laboratories (NDA A078556, Tablets, 0.125 mg, 0.25 mg), Jerome Stevens Pharmaceuticals Inc. (NDA A076268, Tablets, 0.125 mg, 0.25 mg), Mylan Pharmaceuticals Inc. (NDA A040282, Tablets, 0.125 mg, 0.25 mg), Sun Pharmaceutical Industries Inc. (NDA A076363, Tablets, 0.125 mg, 0.25 mg), Concordia Pharmaceuticals Inc. (NDA A020405, Tablets, 0.0625, 0.125 mg, 0.1875 mg, 0.25 mg, 0.375 mg, 0.5 mg, LANOXIN), GlaxoSmithKline LLC (NDA 018118, Capsules, 0.05 mg, 0.1 mg, It is commercially available from 0.15 mg, 0.2 mg, LANOXICAPS).

As used herein, individually named triterpenes, in their natural (unmodified, free acid) form, their salt form, derivative form, prodrug form, or a combination thereof, at each development. Be selected. Compositions comprising deuterated forms of triterpenes and methods of use thereof are also within the scope of the invention.

Derivatives, prodrugs, and salts of oleanolic acid are described in Gribble et al. US2015 / 0011627A1 published January 8, 2015, and Rong et al. US2014 / 0343108A1, published November 20, 2014, November 2014. Xu et al.'S US2014 / 0343064A1 published on 20th, Anderson et al.'S US2014 / 0179928A1 published on June 26, 2014, Bender et al.'S US2014 / 0100227A1, 2014 3 published on April 10, 2014. Jiang et al. US2014 / 088188A1 published on 27th March 2014, Jiang et al. US2014 / 088163A1 published on 27th March 2014, Jiang et al. US2014 / 0066408A1, 2013 published on 6th March 2014. Anderson et al. US2013 / 0317007A1 published on November 28, Gribble et al. US2013 / 0303607A1 published on November 14, 2013, Anderson et al. US2012 / 0245374, 2012 published on September 27, 2012. Jiang et al. US2012 / 0238767A1 published on September 20, 2012, Shode et al. US2012 / 0237629A1 published on September 20, 2012, Anderson et al. US2012 / 0214814A1 published on August 23, 2012, Lee et al.'S US2012 / 0165279A1 published on June 28, 2012, Arntzen et al.'S US2011 / 0294752A1 published on December 1, 2011, and Majeed et al.'S US2011 / 0091398A1 published on April 21, 2011. , US2010 / 0189824A1 by Arntzen et al. Published on July 29, 2010, US2010 / 0048911A1 by Jiang et al. Published on February 25, 2010, and US2006 by Arntzen et al. Published on April 6, 2006. / 0073222A1 and the entire disclosure thereof is incorporated herein by reference.

Derivatives, prodrugs, and salts of ursoleic acid are described in Gribble et al. US2015 / 00122627A1, published January 8, 2015, Gribble et al. US2013 / 0303607A1, published November 14, 2013, August 2015. Yoon et al. US2015 / 0218206A1 published on 6th, Fritzche et al. US6824811 published on November 30, 2004, Ochiai et al. US7718635 published on May 8, 2010, May 20, 2014. It is disclosed in Lin et al. US8729055, published in Lin et al., And in Yoon et al. US9120839, published September 1, 2015, the entire disclosure of which is incorporated herein by reference.

Derivatives, prodrugs, and salts of bethuric acid are described in Gribble et al. US2015 / 00122627A1, published January 8, 2015, Gribble et al. US2013 / 0303607A1, published November 14, 2013, September 2012. US2012 / 0237629A1 of Shode et al. Published on 20th, US2017 / 0204133A1 of Regueiro-Ren et al. Published on July 20, 2017, US2017 / 0996446A1, 2015 of Nitz et al. Published on April 6, 2017. Prathasaradhhi Reddy et al.'S US2015 / 0337004A1 published on November 26, 2015, Reddy et al.'S US2015 / 0119373A1 published on April 30, 2015, and Yan et al.'S US2014 / 0296546A1 published on October 2, 2014. , US2014 / 0243298A1 of Swidorski et al. Published on August 28, 2014, US2014 / 0221328A1 of Reddy et al. Published on August 7, 2014, US2014 / 2014 of Leunis et al. Published on March 6, 2014. 0066416A1, Durst et al. US2013 / 0065868A1 published on March 14, 2013, Regueiro-Ren et al. US2013 / 0029954A1 published on January 31, 2013, Zhang et al. Published on November 29, 2012. US2012 / 0302530A1, Power et al. US2012 / 0214775A1 published on August 23, 2012, Honda et al. US2012 / 0110149A1 published on April 26, 2012, Bulllock published on September 15, 2011. US2011 / 0224182, Hemp et al. US2011 / 0313191A1 published on December 22, 2011, Picchet et al. US2011 / 0224159A1 published on September 15, 2011, published on September 8, 2011. Saradhi Reddy et al. US2011 / 0218204, Safe et al. US2009 / 0203661A1 published on August 13, 2009, Krasutsky et al. US2009 / 0131714A1 published on May 21, 2009, published March 19, 2009. US2009 / 0076290, 200 by Krasutsky et al. Leunis et al.'S US2009 / 0068257A1 published on March 12, 1997, Mukherjee et al.'S US2008 / 0293682 published on November 27, 2008, and Pezuto et al.'S US2007 / 0072835A1 published on March 29, 2007. , Jansen et al. US2006 / 0252733A1 published November 9, 2006, and O'Neill et al. US2006 / 025274A1 published November 9, 2006, for full reference. Is incorporated herein by.

The antiviral composition can be formulated into any suitable pharmaceutically acceptable dosage form. Parenteral, ear, eye, nose, inhalation, 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, troches, sachets, solutions, suspensions, dispersions, vials, bags, bottles, injections, i. v. (Intravenous), i. m. (Intramuscular) or i. p. Includes (intraperitoneal) administrable liquids and other such dosage forms known to those of skill in the art of pharmaceutical science.

Since viral infection can affect multiple organs simultaneously and cause failure in multiple organs, it may be advantageous to administer the composition by more than one route. For example, COVID-19 is known to affect the lungs, heart, gastrointestinal tract, and brain. Therefore, the strongly worried sugar-containing compositions are advantageously inhalable and oral compositions, sublingual and oral compositions, inhalable and sublingual compositions, inhalable compositions. And can be administered as parenteral compositions, sublingual and parenteral compositions, oral and parenteral compositions, or other such combinations.

Suitable dosage forms comprising the antiviral composition are described herein as the antiviral composition or described in Pi et al. ｄｉｓｓｏｌｕｔｉｏｎ ｒａｔｅ ａｎｄ ｂｉｏａｖａｉｌａｂｉｌｉｔｙ ｅｎｈａｎｃｅｍｅｎｔ：ｉｎｆｌｕｅｎｃｅ ｏｆ ｄｉｆｆｅｒｅｎｔ ｐａｒｔｉｃｌｅ ｓｉｚｅ”）、Ｙａｎｇら（Ｉｎｔ．Ｊ．Ｎａｎｏｍｅｄ．（２０１３），８（１），２９１７－２９２６の“Ｓｅｌｆ－ｍｉｃｒｏｅｍｕｌｓｉｆｙｉｎｇ ｄｒｕｇ ｄｅｌｉｖｅｒｙ ｓｙｓｔｅｍ ｆｏｒ ｉｍｐｒｏｖｅｄ ｏｒａｌ ｂｉｏａｖａｉｌａｂｉｌｉｔｙ ｏｆ ｏｌｅａｎｏｌｉｃ ａｃｉｄ ：ｄｅｓｉｇｎ ａｎｄ ｅｖａｌｕａｔｉｏｎ”）、Ｌｉら（Ｂｉｏｌ．Ｐｈａｒｍ．Ｂｕｌｌ．（２０１４），３７（６），９２６－９３７のＤｅｖｅｌｏｐｍｅｎｔ ａｎｄ ｅｖａｌｕａｔｉｏｎ ｏｆ ｏｐｔｉｍｉｚｅｄ ｓｕｃｒｏｓｅ ｅｓｔｅｒ ｓｔａｂｉｌｉｚｅｄ ｏｌｅａｎｏｌｉｃ ａｃｉｄ ｎａｎｏｓｕｓｐｅｎｓｉｏｎｓ ｐｒｅｐａｒｅｄ ｂｙ ｗｅｔ ｂａｌｌ ｍｉｌｌｉｎｇ ｗｉｔｈ ｄｅｓｉｇｎ ｏｆ ｅｘｐｅｒｉｍｅｎｔｓ” ), Zhang et al. (J. Pharma. Sci. (June 2014), 103 (6), 1711-1719, "Enhancement of oral bioavailability of triterpene neurotherapy, ripid nanospheres: One (Mar 2014), 9 (3): e89919 "Approaches to impact the oral bioavailability and effects of novel antiviral drug 14 derb erbene" , 21 (6), 467-479, “Preparation and characterization of betulin nanoparticles for oral hypoglycemic drug drug by antisold prescription”), Yang et al. (Fo). (May 2012), 132 (1), 319-325 “Physicochemicals and oral bioavailability of ursolic acid nanoparticles using sing suppercital. (8), 2127-2135, "Ethylene glycol-linked amino acid diester prodrugs of oral acid acid for PEPT1-mediated transport: synthesis, integral2016" ), 2020-2033, "Formulation, bioavailable and pharmacokinetic studios of solvent ester-stabilized nanosuspensions of oleanolic acid"), 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”）、Ｘｉら（ＡＡＰＳ ＰｈａｒｍＳｃｉＴｅｃｈ（２００９），１０（１），１７２－１８２のＦｏｒｍｕｌａｔｉｏｎ ｄｅｖｅｌｏｐｍｅｎｔ ａｎｄ ｂｉｏａｖａｉｌａｂｉｌｉｔｙ ｅｖａｌｕａｔｉｏｎ of a self-nanoemulsified drug delivery system of oleanolic acid "), Chen et al. (J. Pharma. Pharmacol. (Feb 2005), 57 (2), 259-264". They can be prepared by mixing with the pharmaceutically acceptable excipients described in “Oleanolic acid nanosuspensations: preparation, in-vitro characterization and enhanced hepatotropic effects”), the entire disclosure of which can be prepared by reference. Incorporated into the specification.

Suitable dosage forms are also in accordance with Addington's US8187644B2, published May 29, 2012, Addington's US7402325B2, published July 22, 2008, and Addington et al., US8394434B2, published March 12, 2013. They can be made and their entire disclosure is incorporated herein by reference. Suitable dosage forms can also be made as described in Examples 13-15.

Effective or therapeutic amounts of antiviral compounds (cardiac glycosides, triterpenes, or combinations thereof) are specifically contemplated. It is understood that the term "effective amount" contemplates a pharmaceutically effective amount. A pharmaceutically effective amount is an amount or amount of active ingredient sufficient for the required or desired therapeutic response, in other words, an amount sufficient to elicit a significant biological response when administered to the patient. Significant biological responses can occur as a result of single or multiple doses of the active substance. The dose may include one or more dosage forms. Specific dose levels for any patient include the indication to be treated, the severity of the indication, the patient's health, age, gender, weight, diet, pharmacological response, specific dosage form used, and others thereof. It will be understood that it depends on various factors including such factors.

The desired dose for oral administration is up to 5 dosage forms, but only 1 and up to 10 dosage forms may be administered as a single dose. Exemplary dosage forms are totaling 0.1-500 mg per dose (dose level of 1-10) and may comprise from about 0.01-100 mg or 0.01-100 micrologs of antiviral composition per dosage form. Doses are administered according to a dosing regimen that can be predetermined and / or adjusted to achieve a particular therapeutic response or clinical benefit in the subject.
Cardiac glycosides may be present in dosage form in sufficient amounts to provide an initial dose of oleandrin of about 20 to about 100 microg, about 12 microg to about 300 microg, or about 12 microg to about 120 microrog. Dosage forms include oleandrin, oleandrin extract or Nerium oleander extract containing oleandrin from about 20 to about 100 microg, from about 0.01 microlog to about 100 microlog or from about 0.01 microg to about 100 microg. Can include.
Antiviral agents are included in oral dosage forms and sold. Some embodiments of the dosage form are not enteric coated and release their charge of the antiviral composition within a period of 0.5 to 1 hour or less. Some embodiments of the dosage form are enteric coated and release their charge of the antiviral composition downstream of the stomach such as from the jejunum, ileum, small intestine, and / or large intestine (colon). The enteric coated dosage form releases the antiviral composition into the systemic circulation within 1-10 hours after oral administration.
The antiviral composition can be of rapid release, immediate release, controlled release, sustained release, long-acting release, long-term release, burst release, continuous release, delayed release, or pulsed release dosage form, or a type of release thereof. It may be included in a dosage form showing two or more. The release profile of the antiviral composition from the dosage form can be zero-order, pseudo-zero, primary, pseudo-primary, or S-shaped release profile. The plasma concentration profile of triterpenes in subjects receiving the antiviral composition can show one or more maximums.

Based on human clinical data, 50% to 75% of the dose of oleandrin administered is expected to be orally bioavailable, hence about 10 to about 20 micrologs per dosage form, Provide oleandrin of about 20 to about 40 microg, about 30 to about 50 microg, about 40 to about 60 microg, about 50 to about 75 microg, and about 75 to about 100 microg. Considering the average blood volume of a 5 liter adult, the expected oleandrin plasma concentration is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng / mL, about 0.005 to about 8 ng / ml. mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng / mL, or about 0.05 to about 2. It will be in the range of .5 ng / mL. The recommended daily dose of oleandrin present in the SCF extract is generally from about 0.2 microlog to about 4.5 microg / kg body weight twice daily. The dose of oleandrin is about 0.2 to about 1 microg / kg body weight / day, about 0.5 to about 1.0 microg / kg body weight / day, about 0.75 to about 1.5 microg / kg body weight / day, About 1.5 to about 2.52 microg / kg body weight / day, about 2.5 to about 3.0 microg / kg body weight / day, about 3.0 to 4.0 microg / kg body weight / day, or about 3.5 to It can be 4.5 microg oleandrin / kg body weight / day. The maximum tolerated dose of oleandrin can be from about 3.5 microg / kg body weight / day to about 4.0 microg / kg body weight / day. The minimum effective dose can be about 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg / day, or about 5 microg / day.

The antiviral composition can be administered in low to high doses depending on the combination of the triterpenes present and the molar ratio in which they are present. A therapeutically effective dose to humans is an antiviral composition of about 100-1000 mg / kg body weight or about 100-1000 micrologs. Such doses can be administered up to 10 times over a 24-hour period. Other suitable dosing ranges are specified below.

It should be noted that the compounds herein may have one or more functions in the compositions or formulations of the invention. For example, the compound can serve as both a detergent and a water-miscible solvent, or as both a detergent and a water-miscible solvent.
The liquid composition may comprise one or more pharmaceutically acceptable liquid carriers. The liquid carrier can be aqueous, non-aqueous, polar, non-polar, and / or organic carrier. Liquid carriers include, but are not limited to, water-miscible solvents, water-immiscible solvents, water, buffers, and mixtures thereof, for example.

As used herein, the terms "water-soluble solvent" or "water-soluble solvent" used interchangeably do not form a two-phase mixture with water or are sufficiently soluble in water. Refers to an organic liquid that provides an aqueous solvent mixture containing at least 5% solvent without separating the liquid phase. The solvent is suitable for administration to humans or animals. Exemplary water-soluble solvents include, without limitation, PEG (poly (ethylene glycol)), PEG400 (poly (ethylene glycol) having an approximate molecular weight of about 400), ethanol, acetone, alkanol, alcohol, ether. , Ethylene glycol, glycerin, triacetin, poly (propylene glycol), PVP (poly (vinylpyrrolidone)), dimethylsulfoxide, N, N-dimethylformamide, formamide, N, N-dimethylacetamide, pyridine, propanol, N-methylacetamide , Butanol, solvent (2-pyrrolidone), pharmacosolve (N-methyl-2-pyrrolidone).

As used herein, the term "water-insoluble solvent" or "water-immiscible solvent" is used interchangeably to form a two-phase mixture with water or a solvent in water. Refers to an organic liquid that provides phase separation when the concentration of the solvent exceeds 5%. The solvent is suitable for administration to humans or animals. Exemplary water-insoluble solvents include, by way of example, medium / long chain triglycerides, oils, castor oil, corn oil, vitamin E, vitamin E derivatives, oleic acid, fatty acids, olive oil, softistan 645 (diglyceryl). Caprilate / caplate / stearate / hydroxyistearelate adipate), migliol, captex (Captex350: glyceryl tricaprilate / caplate / laurate triglyceride; Captex355: glyceryl tricaprelate / capplate triglyceride; Captex355 EP / NF: glyceryl tricaprylate / caplate medium chain triglyceride) is included.

Suitable solvents are "International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Guidance For Industry 1 Is recommended to be considered safe in. Exemplary solvents are listed as Class 2 or Class 3 solvents. Class 3 solvents include, for example, acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formate, heptane, acetate. Includes isobutyl, isopropyl acetate, methyl acetate, methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentan, 1-pentanol, 1-propanol, 2-propanol, or propyl acetate.

Other materials that can be used as the water immiscible solvent in the present invention include Captex100: propylene glycol dicaplate; Captex200: propylene glycol dicaprylate / dicaplate; Captex200P: propylene glycol dicaprylate / dicaplate; propylene glycol di. Capriloca Plate; Captex300: Glyceryl Tricaprilate / Caplate; Captex300EP / NF: Glyceryl Tricaprilate / Caplate Medium Chain Triglyceride; Captex350: Glyceryl Tricaprilate / Caplate / Laurate; : Glyceryl tricaprylate / caplate medium chain triglyceride; Captex500: triacetin; Captex500P: triacetin (pharmaceutical grade); Captex800: propylene glycol di (2-ethytexanoate); Captex810D: glyceryl tricaprelate / caplate / linoleate; Captex1000: Glyceryl Trica Plate; Captex CA: Medium Chain Triglyceride; Captex MCT-170: Medium Chain Triglyceride; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP / NF: Glyceryl Monooleate; Capmul MCM: Medium Chain Mono and Diglyceride; Capmul MCM C8: Glyceryl Monooleate; Capmul MCM C10: Glyceryl Monoca Plate; Capmul PG-8: Propylene Glycol Monocaprelate; Capmul PG-12: Propylene Glycol Monolaurate; Caprol 10G10O: Decaglycerol Decaoleate; Caprol 3GO: Triglycerol monooleate; Caprol ET: polyglycerol ester of mixed fatty acid; Caprol MPGO: hexaglycerol dioleate; Caprol PGE 860: decaglycerol mono-, geolate.

As used herein, "surfactant" refers to a compound that comprises a polar or charged hydrophilic moiety as well as a non-polar hydrophobic (lipophilic) moiety, i.e., the surfactant is amphipathic. be. The term surfactant may refer to one or a mixture of compounds. The surfactant can be a solubilizer, emulsifier, or dispersant. Surfactants can be hydrophilic or hydrophobic.

The hydrophilic surfactant can be any hydrophilic surfactant suitable for use in pharmaceutical compositions. Such surfactants can be anionic, cationic, zwitterionic or nonionic, but nonionic hydrophilic surfactants are currently preferred. As discussed above, these nonionic hydrophilic surfactants generally have an HLB value of 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. In general, suitable hydrophobic surfactants have an HLB value of less than about 10. Mixtures of hydrophobic surfactants are also within the scope of the present invention.

Examples of additional suitable solubilizers include ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediol and its isomers, glycerol, pentaerythritol, sorbitol, mannitol, transctor, dimethylisosorbide, polyethylene. Alcohols and polyols such as glycols, polypropylene, polyvinyl alcohols, hydroxypropylmethylcellulose and other cellulose derivatives, cyclodextrin and cyclodextrin derivatives; tetrahydrofurfuryl alcohol PEG ether (commercially available from BASF under the trade name Tetraglycol, glycoflor) or Ethers of polyethylene glycol having an average molecular weight of about 200 to about 6000 such as methoxyPEG (Union Carbide); 2-pyrrolidone, 2-piperidone, caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, Amids such as N-alkylcaprolactam, dimethylacetamide, and polybinipyrrolidone; ethyl propionate, tributyl citrate, acetyltriethyl citrate, acetyltributyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin , Esters such as propylene glycol monoacetate, propylene glycol diacetate, caprolactone and its isomers, valerolactone and its isomers, butyrolactone and isomers; and dimethylacetamide, dimethylisosorbide (Arlasolve DMI (ICI)), N-methylpyrrolidone. (Pharmasolve (ISP)), monooctanoin, diethylene glycol nonoethyl ether (available from Gattefose under the trade name Transtool), and other solubilizers known in the art such as water. Mixtures of solubilizers are also within the scope of the present invention.

Except as indicated, the compounds referred to herein are readily available from standard commercial sources.
Although not required, the composition or formulation is one or more chelating agents, one or more preservatives, one or more antioxidants, one or more adsorbents, one or more acidifying agents, one. 1 or more alkalizing agents, 1 or more defoaming agents, 1 or more buffering agents, 1 or more colorants, 1 or more electrolytes, 1 or more salts, 1 or more stabilizers, 1 The above tension modifiers, one or more diluents, or combinations thereof may be further included.

The compositions of the invention also include oils such as fixed oils, peanut oils, sesame oils, cotton seed oils, corn oils, and olive oils; fatty acids such as oleic acid, stearic acid, and isostearic acid; and ethyl oleate, isopropyl myristate, fatty acids. It may contain fatty acids, and fatty acid esters such as acetylated fatty acid glycerides. The composition is an alcohol such as ethanol, isopropanol, hexadecyl alcohol, glycerol, and propylene glycol; a glycerol ketal such as 2,2-dimethyl-1,3-dioxolan-4-methanol; an ether such as poly (ethylene glycol) 450. It may contain petroleum-based hydrocarbons such as mineral oil and glycerin; water; pharmaceutically suitable surfactants, suspending agents, or emulsifiers; or mixtures thereof.

It should be understood that compounds used in the technical field of pharmaceutical formulations generally serve a variety of functions or purposes. Thus, if a compound named herein is mentioned only once or is used herein to define more than one term, its purpose or function is its named purpose ( It should not be construed as being limited to (s) or functions (s).
One or more components of a pharmaceutical product may be present in its free base, free acid, or pharmaceutically or analytically acceptable salt form. As used herein, "pharmaceutically or analytically acceptable salt" refers to a compound modified by reacting the compound with an acid as needed to form an ionic bond pair. Examples of acceptable salts include, for example, conventional non-toxic salts formed from non-toxic inorganic or organic acids. Suitable non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, sulfamic acid, phosphoric acid, nitric acid, and others known to those of skill in the art. Is done. Amino acids, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanyl Salts prepared from acids, organic acids such as 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and others known to those of skill in the art. On the other hand, when the pharmacologically active component has an acid functional group, a pharmaceutically acceptable base is added to form a pharmaceutically acceptable salt. A list of other suitable salts can be found in Remington's Pharmaceutical Sciences, ^17th . ed. , Mack Publishing Company, Easton, PA, 1985, p. Found in 1418, the relevant disclosure is incorporated herein by reference.

The phrase "pharmaceutically acceptable" is suitable for use in contact with human and animal tissues, within sound medical judgment, and is excessively toxic, irritating, allergic, or optional. Refers to a compound, material, composition, and / or dosage form that corresponds to a reasonable benefit / risk ratio without other problems or complications.

Dosage forms can be made by any conventional means known in the pharmaceutical industry. Liquid dosage forms can be prepared by providing the container with at least one liquid carrier and antiviral composition. One or more other excipients can be included in the liquid dosage form. Solid dosage forms can be prepared by providing at least one solid carrier and antiviral composition. One or more other excipients can be included in the solid dosage form.
Dosage forms can be packaged using traditional packaging equipment and materials. It can be placed in packs, bottles, vias, bags, syringes, envelopes, packets, blister packs, boxes, ampoules, or other such containers.
The composition of the present invention is included in any dosage form and sold. Certain dosage forms include solid or liquid dosage forms. Exemplary suitable dosage forms include tablets, capsules, pills, caplets, troches, sachets, and other such dosage forms known to those of skill in the art of pharmaceutical science.

In view of the above description and the following examples, one of ordinary skill in the art will be able to carry out the claimed invention without undue experimentation. The above will be better understood with reference to the following examples detailing certain procedures for the preparation of embodiments of the invention. All references made in these examples are for illustration purposes only. The following examples should not be considered exhaustive, but should be considered merely as illustrations of only a few of the many embodiments contemplated by the present invention.
Vero CCL81 cells were used in prophylactic and therapeutic assays (ATCC, Manassas, VA). The plaque assay was performed on Vero E6 cells favored by Vineet Menarchy (UTMB, Galveston, TX). Cells were maintained in an incubator with 5% CO ₂ and 37 ° C. Cells were supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Rawrenceville, GA) and 1% penicillium / streptomycin (Gibco, Grand Island, NY) Dulbecco-modified Eagle's medium (Gibco, Gnla). It was used and propagated. Maintenance medium reduced FBS to 2%, but was otherwise identical. SARS-CoV-2 strain USA_WA1 / 2020 (Genbank accession MT020880) was provided by World Reference Center for Emerging Viruses and Arboviruses. The NextGen sequence Vero4 passage strain of SARS-CoV-2 was utilized in all studies.

(Example 1)
Method for extracting supercritical fluid from powdered oleander leaves A. Uses carbon dioxide.
Oleander leaf material is harvested, washed and dried, and then Oleander leaf material is presented in US Pat. Nos. 5,236,132, 5,598,979, 6,517,015, And by passing through a crushing and dehydrating apparatus such as those described in Nos. 6,715 and 705, powdered oleander leaves were prepared. The starting material used weighed 3.94 kg.
The starting material was mixed with pure CO ₂ in an extractor at a pressure of 300 bar (30 MPa, 4351 psi) and a temperature of 50 ° C. (122 ° F). A total of 197 kg of CO ₂ was used to obtain a 50: 1 solvent-to-raw material ratio. The mixture of CO ₂ and raw material was then passed through a separator, where the pressure and temperature of the mixture was varied to separate the extract from carbon dioxide.

The extract (65 g) was obtained as a brownish, sticky, viscous material with a good scent. The color is likely caused by chlorophyll and other residual chromogenic compounds. For accurate yield determination, the tube and separator were rinsed with acetone and the acetone was evaporated to give 9 g of additional extract. The total amount of extraction was 74 g. Based on the weight of the starting material, the yield of the extract was 1.88%. The content of oleandrin in the extract was calculated to be 560.1 mg, or 0.76% yield, using high performance liquid chromatography and mass spectrometry.

Method B. Oleander leaf material is harvested, washed and dried using a mixture of carbon dioxide and ethanol, followed by Oleander leaf material in U.S. Pat. Nos. 5,236,132, 5,598,979, Powdered oleander leaves were prepared by passing through a crushing and dehydrating apparatus such as those described in Nos. 6,517,015 and 6,715,705. The starting material used weighed 3.85 kg.
The starting material was mixed with pure CO ₂ and 5% ethanol as modifiers at a pressure of 280 bar (28 MPa, 4061 psi) and a temperature of 50 ° C. (122 ° F) in an extractor. A total of 160 kg of CO ₂ and 8 kg of ethanol was used to obtain a 43.6: 1 solvent-to-raw material ratio. The mixture of CO ₂ , ethanol, and raw materials was then passed through a separator, where the pressure and temperature of the mixture was varied to separate the extract from carbon dioxide.
After removal of ethanol, the extract (207 g) was obtained as a dark green, sticky, viscous mass with apparently some chlorophyll. Based on the weight of the starting material, the yield of the extract was 5.38%. The content of oleandrin in the extract was calculated using high performance liquid chromatography and mass spectrometry in yields of 1.89 g, or 0.91%.

(Example 2)
Hot water extraction of powdered oleander leaves (comparative example)
Hot water extraction is typically used to extract oleandrin and other active ingredients from the leaves of oleander. 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. 10 volumes of boiling water (weight of oleander starting material) was added to the powdered oleander leaves and the mixture was stirred constantly for 6 hours. The mixture was then filtered and leaf residues were collected and extracted again under the same conditions. The filtrate was mixed and lyophilized. The appearance of the extract was brown. The weight of the dried extract was about 1.44 g. 34.21 mg of extract material was dissolved in water and oleandrin content analysis was performed using high performance liquid chromatography and mass spectrometry. The amount of oleandrin was determined to be 3.68 mg. Based on the amount of extract, the yield of oleandrin was calculated to be 0.26%.

賦形剤を瓶に分注し、Ｎｅｗ Ｂｒｕｎｓｗｉｃｋ Ｓｃｉｅｎｔｉｆｉｃ Ｃ２４ＫＣ冷蔵インキュベーターシェーカーで、６０℃で２４時間振盪して、均一性を確実にした。次に、試料を引き抜き、可溶化について視覚的に検査した。２４時間後、全ての製剤について、賦形剤および抗ウイルス組成物の両方が完全に溶解した。 (Example 3)
Preparation of pharmaceutical compositions.
Method A. Cremofol-based drug delivery system The following ingredients were provided in the indicated amounts.

Excipients were dispensed into bottles and shaken in a New Brunswick Scientific C24KC refrigerated incubator shaker at 60 ° C. for 24 hours to ensure uniformity. The sample was then drawn and visually inspected for solubilization. After 24 hours, both the excipient and the antiviral composition were completely dissolved for all formulations.

方法Ａの手順に従った。 Method B. GMO / Cremofol-based drug delivery system The following ingredients were provided in the indicated amounts.

The procedure of method A was followed.

方法Ａの手順に従った。 Method C. Labrazole-based drug delivery system The following ingredients were provided in the indicated amounts.

The procedure of method A was followed.

方法Ａの手順に従った。 Method D. Vitamin E-TPGS-based micelle formation system The following ingredients were provided in the indicated amounts.

The procedure of method A was followed.

方法Ａの手順に従った。 Method E. Multiconstituent Drug Delivery System The following ingredients were provided in the indicated amounts.

The procedure of method A was followed.

方法Ａの手順に従った。 Method F. Multiconstituent Drug Delivery System The following ingredients were provided in the amounts indicated to be contained in the capsule.

The procedure of method A was followed.

ゼラチン溶液を完全に混合し、１～２時間膨潤させた。膨潤後、溶液をしっかりと覆い、５５℃のオーブンに入れて液化させた。ゼラチン溶液全体が液体になると、バンディングを行った。 (Example 4)
Preparation of enteric coated capsules Step I: Preparation of liquid-filled capsules Hard gelatin capsules (50 counts, 00 size) were filled with the liquid composition of Example 3. These capsules were manually filled with 800 mg of the pharmaceutical product and then sealed by hand with 50% ethanol / 50% aqueous solution. The capsules were then hand banded with a 22% gelatin solution containing the following ingredients in the indicated amounts.

The gelatin solution was thoroughly mixed and inflated for 1-2 hours. After swelling, the solution was covered tightly and placed in an oven at 55 ° C. for liquefaction. Banding was performed when the entire gelatin solution became liquid.

The gelatin solution was applied to the capsules using a pointed round 3/0 artist brush. I used the banding kit provided by Shionogi. After banding, the capsules were held in ambient conditions for 12 hours to cure the band.

ステップＩのバンディングしたカプセルを使用した場合、分散液を、２０．０ｍｇ／ｃｍ²のコーティングレベルまでカプセルに塗布した。以下の条件を使用して、カプセルをコーティングした。

Step II: Coating of liquid-filled capsules A coating dispersion was prepared from the components listed in the table below.

When the banded capsules of Step I were used, the dispersion was applied to the capsules to a coating level of 20.0 mg / cm ² . Capsules were coated using the following conditions:

(Example 5)
Treatment method for Zika virus infection in the subject A. Antiviral composition therapy Antiviral compositions are prescribed to subjects presenting with Zika virus infection, and treatment-related doses are administered to the subjects for a period of time according to the prescribed dosing regimen. The level of treatment response of the subject is determined on a regular basis. The level of therapeutic response can be measured by determining the Zika virus titer of the subject in blood or plasma. If the level of therapeutic response is too low at a given dose, the dose is graded according to a given dose escalation schedule 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 can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Another Agent Follow Method A above, except that the subject is prescribed and administered one or more other therapeutic agents for the treatment of Zika virus infection or its symptoms. One or more other therapeutic agents can then be administered before, after, or with the antiviral composition. Dose escalation (or tapering) of one or more other therapeutic agents can also be performed.

(Example 6)
In vitro evaluation method of antiviral activity against Zika virus infection A. Presence of worried sugars in pure compound Vero E6 cells (ATCC number CRL-1586; https: //www.atcc.org/Products/All/CRL-1586.aspx, also known as Vero C1008 cells) Below, ZIKV (Zika virus strain PRVABC59; ATCC VR-1843; https: //www.atcc.org/Products/All/VR-1843.aspx) was infected with a MOI (multiplicity of infection) of 0.2. .. The cells were incubated with the virus and compound for 1 hour before discarding the inoculum and compound. The cells were fed fresh medium, incubated for 48 hours, then fixed with formalin and stained with ZIKV infection. The typical infection rates of oleandrin (FIG. 1A) and digoxin (FIG. 1B) as determined by scintigraphy are shown. Other compounds were evaluated under the same conditions and showed very different levels of antiviral activity against Zika virus.

Method B. Compounds in Extract Form The extract containing the target compound being tested is evaluated as detailed in Method A, except that the amount of extract is normalized to the amount of target compound in the extract. do. For example, an extract containing 2% by weight oleandrin contains 20 micrologs of oleandrin per 1 mg of extract. Therefore, if the intended amount of oleandrin for evaluation is 20 micrologs, 1 mg of extract is used in the assay.

サイロイド２４４ＦＰは、Ｇｒａｃｅ Ｄａｖｉｓｏｎによって製造されたコロイド状二酸化ケイ素である。コロイド状二酸化ケイ素は、吸着剤、流動促進剤、および錠剤崩壊剤などのいくつかの機能を提供するために一般的に使用される。サイロイド２４４ＦＰを、その重量の３倍の油を吸着するその能力およびその５．５ミクロンの粒子サイズのために選択した。 (Example 7)
Preparation of Tablets Containing Antiviral Composition An initial tableted mixture of 3% siloid 244FP and 97% microcrystalline cellulose (MCC) was mixed. Existing batches of compositions prepared according to Example 3 were then incorporated into the syroid / MCC mixture via wet granulation. This mixture is labeled "Initial Tableting Mixture" in the table below. Additional MCC was added in the form of additional granules to increase compressibility. This addition to the initial tableted mixture was labeled as "further granular addition". The mixture obtained from the further granular addition was the same composition as the "final tableted mixture".

Cyroid 244FP is a colloidal silicon dioxide produced by Grace Davison. Colloidal silicon dioxide is commonly used to provide several functions such as adsorbents, flow promoters, and tablet disintegrants. Cyloid 244FP was selected because of its ability to adsorb three times its weight of oil and its 5.5 micron particle size.

(Example 8)
HPLC analysis of solutions containing oleandrin Samples (oleandrin standard, SCF extract, and hot water extract) were analyzed by HPLC (Waters) using the following conditions: Symmetric C18 column (5.0 μm, 150'4.6 mm inner diameter; Waters); MeOH: mobile phase of water = 54:46 (v / v) and flow rate 1.0 ml / min. The detection wavelength was set to 217 nm. Samples were prepared by dissolving the compound or extract in a constant amount of HPLC solvent to achieve the approximate target concentration of oleandrin. The retention time of oleandrin can be determined by using an internal standard. The concentration of oleandrin can be determined / calibrated by creating a signal response curve using an internal standard.

(Example 9)
Preparation of pharmaceutical composition The pharmaceutical composition of the present invention can be prepared by any of the following methods. Mixing can be done under wet or dry conditions. The pharmaceutical composition can be compressed, dried, or both during preparation. The pharmaceutical composition can be divided into dosage forms.

Method A.
At least one pharmaceutical excipient is mixed with at least one antiviral compound disclosed herein.

Method B.
At least one pharmaceutical excipient is mixed with at least two antiviral compounds disclosed herein.
Method C.
At least one pharmaceutical excipient is mixed with at least one cardiac glycoside disclosed herein.

Method D.
At least one pharmaceutical excipient is mixed with at least two triterpenes disclosed herein.
Method E.
At least one pharmaceutical excipient is mixed with at least one cardiac glycoside disclosed herein and at least two triterpenes disclosed herein.
Method D.
At least one pharmaceutical excipient is mixed with at least three triterpenes disclosed herein.

各組成物について、３つの異なるそれぞれの溶液を作製し、よって各溶液中のトリテルペンの総濃度は、約９μＭ、１８μＭ、または３６μＭであった。

(Example 10)
Preparation of Triterpene Mixtures The following compositions were made by mixing the specified triterpenes at the indicated approximate molar ratios.

For each composition, three different solutions were made, thus the total concentration of triterpenes in each solution was about 9 μM, 18 μM, or 36 μM.

(Example 11)
Preparation of antiviral composition An antiviral composition can be prepared by mixing its individual triterpene components to form a mixture. The triterpene mixture prepared above, which provided acceptable antiviral activity, was formulated into an antiviral composition.

Antiviral Compositions Containing Oleanolic Acid and Ursolic Acid A known amount of oleanolic acid and ursolic acid were mixed according to a given molar ratio of constituents as defined herein. The constituents may be mixed in solid form or solvent (s) such as methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N- It was mixed with methylpyrrolidone (NMP), water, or a mixture thereof. The resulting mixture contained constituents in the relative molar ratios described herein.
For pharmaceutically acceptable antiviral compositions, at least one pharmaceutically acceptable excipient was mixed with a pharmacologically active agent. The antiviral composition is formulated for administration to mammals.

Antiviral Compositions Containing Oleanolic Acid and Betulinic Acid A known amount of oleanolic acid and betulinic acid were mixed according to a given molar ratio of the constituents defined herein. The constituents may be mixed in solid form or solvent (s) such as methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N- It was mixed with methylpyrrolidone (NMP), water, or a mixture thereof. The resulting mixture contained constituents in the relative molar ratios described herein.
For pharmaceutically acceptable antiviral compositions, at least one pharmaceutically acceptable excipient was mixed with a pharmacologically active agent. The antiviral composition is formulated for administration to mammals.

Antiviral Compositions Containing Oleanolic Acid, Ursolic Acid, and Betulinic Acid A known amount of oleanolic acid, ursolic acid, and betulinic acid were mixed according to a given molar ratio of the constituents defined herein. The constituents may be mixed in solid form or solvent (s) such as methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N- It was mixed with methylpyrrolidone (NMP), water, or a mixture thereof. The resulting mixture contained constituents in the relative molar ratios described herein.
For pharmaceutically acceptable antiviral compositions, at least one pharmaceutically acceptable excipient was mixed with a pharmacologically active agent. The antiviral composition is formulated for administration to mammals.

Antiviral Compositions Containing Oleadrin, Oleanolic Acid, Ursolic Acid, and Betulinic Acid A known amount of oleandrin oleanolic acid, ursolic acid, and bethulinic acid are mixed according to a given molar ratio of the constituents defined herein. bottom. The constituents may be mixed in solid form or solvent (s) such as methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N- It was mixed with methylpyrrolidone (NMP), water, or a mixture thereof. The resulting mixture contained constituents in the relative molar ratios described herein.
For pharmaceutically acceptable antiviral compositions, at least one pharmaceutically acceptable excipient was mixed with a pharmacologically active agent. The antiviral composition is 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 Antiviral compositions are prescribed to subjects present with filovirus infection, and treatment-related doses are administered to the subjects for a period of time according to the prescribed dosing regimen. The level of treatment response of the subject is determined on a regular basis. The level of therapeutic response can be measured by determining the filovirus titer of the subject in blood or plasma. If the level of therapeutic response is too low at a given dose, the dose is graded according to a given dose escalation schedule 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 can be adjusted as needed until the patient reaches the desired clinical endpoint.
Method B. Combination Therapy: Antiviral Composition with Another Agent Follow Method A above, except that the subject is prescribed and administered one or more other therapeutic agents for the treatment of a phyllovirus infection or its symptoms. One or more other therapeutic agents can then be administered before, after, or with the antiviral composition. Dose escalation (or tapering) of one or more other therapeutic agents can also be performed.

(Example 13)
Treatment of flavivirus infections in subjects Examples of flavivirus infections include yellow fever, dengue fever, Japanese encephalitis, western Nile virus, decavirus, tick-borne encephalitis, Kasanur forest disease, alfluma disease, chikungunia virus, omsk hemorrhagic fever, Includes Poissan virus infection.
Method A. Antiviral composition therapy Antiviral compositions are prescribed to subjects presenting with flavivirus infection, and treatment-related doses are administered to the subjects for a period of time according to the prescribed dosing regimen. The level of treatment response of the subject is determined on a regular basis. The level of therapeutic response can be measured by determining the flavivirus titer of the subject in blood or plasma. If the level of therapeutic response is too low at a given dose, the dose is graded according to a given dose escalation schedule 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 can be adjusted as needed until the patient reaches the desired clinical endpoint.
Method B. Combination Therapy: Antiviral Composition with Another Agent Follow Method A above, except that the subject is prescribed and administered one or more other therapeutic agents for the treatment of flavivirus infection or its symptoms. One or more other therapeutic agents can then be administered before, after, or with the antiviral composition. Dose escalation (or tapering) of one or more other therapeutic agents can also be performed.

(Example 14)
Assessment of antiviral activity against Zika and dengue viruses CPE-based antiviral assays were performed by infecting target cells at concentrations in the presence or absence of the test composition. Infection of target cells results in cytopathic effects and cell death. In this type of assay, a reduction in CPE in the presence of the test composition and a corresponding increase in cell viability are used as indicators of antiviral activity. For CPE-based assays, cell viability was determined by neutral red readings. Live cells integrate neutral red into their lysosomes. The uptake of neutral red depends on the ability of living cells to maintain a lower pH in their lysosomes than in the cytoplasm, and this active process requires ATP. Once inside the lysosome, the neutral red dye is charged and retained intracellularly. After incubating with neutral red (0.033%) for 3 hours, extracellular dye was removed, cells were washed with PBS and intracellular neutral red was solubilized with a solution of 50% ethanol + 1% acetic acid. The amount of neutral red in solution was quantified by reading the absorbance (optical density) of each well at 490 nm.

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

Compare the antiviral activity of the test composition with the level of infection or viability of cells under test conditions (for immunostaining-based assays) or viability (for CPE-based assays) with the level of infection or viability of uninfected cells. It was judged by doing. Cytotoxic effects were assessed in uninfected cells by comparing viability in the presence of inhibitors to viability of simulated treated cells. Cytotoxicity was determined by the XTT viability assay, which was performed at the same time as the corresponding infection assay readout.
The test composition was dissolved in 100% methanol. Starting at 50 μM as the highest concentration to be tested and making 8-fold dilutions, composition of 8 concentrations was produced (by replication). The highest test concentration (50 μM) of the composition resulted in a final concentration of methanol 0.25% (v / v%) in the culture medium. A series of 8-fold dilutions of the methanol vehicle are included on the plates of each assay and the concentration reflects the final concentration of methanol under the test conditions of each composition. Where possible, GraphPad Prism software was used to determine the EC50 and CC50 of the composition for each assay.

Antiviral activity was assessed by the degree of protection against virus-induced cytopathic effects (CPE). Cells were attacked with virus in the presence of controls or compositions of different concentrations. The degree of protection against CPE was determined by quantifying cell viability under different test conditions 6 days (ZIKV, Zika virus) or 7 days (DENV, dengue virus) after infection, and values were only for untreated cells and vehicles (infection medium). ) Was monitored by comparison with the values of the treated cells.

Quality control of the neutralization assay was performed on all plates, i) measured by signal (S / B) value to background, ii) inhibition by known inhibitors, and ii) coefficient of variation (CV) of all data points. The variation of the assay to be performed was determined. The overall variation of the infection assay ranges from 3.4% to 9.5%, the overall variation of the viability assay ranges from 1.4% to 3.2%, and all CVs. Calculated as the average of the values. The signal-to-background (S / B) of the infection assay ranged from 2.9 to 11.0, while the signal-to-background (S / B) of the viability assay ranged from 6.5 to 11.0. It was in the range of 29.9.

Protection of DENV2-induced cytopathic effect (CPE) by neutral red reading: In the DENV2 antiviral assay, 08-10381 Montserrat strain was used. Virus stock was generated in C6 / 36 insect cells. Vero cells (epithelial kidney cells derived from Cercopisecus aethios) were maintained with MEM (MEM5) containing 5% FBS. For both infection and viability assays, cells were seeded on 96-well clear flat bottom plates with 10,000 cells per well and maintained at 37 ° C. for 24 hours at MEM5. On the day of infection, samples were diluted 8-fold on U-bottom plates using MEM containing 1% bovine serum albumin (BSA). A diluted solution of the test substance was prepared at a final concentration of 1.25 times, and 40 μl was incubated with the target cells at 37 ° C. for 30 minutes. After preincubation of the test material, 10 μl of virus diluted solution prepared by MEM containing 1% BSA was added to each well (final volume of 50 μl per well), and the plate was placed in a humidified incubator with 5% CO2 at 37 ° C. Incubated for 3 hours. The volume of virus used in the assay was previously determined to produce a linear range of signals inhibited by compound A3, a known inhibitor of ribavirin and DENV2. After infection incubation, cells were washed with PBS followed by MEM (MEM2) containing 2% FBS to remove unbound virus. Subsequently, 50 μl of medium containing the inhibitor diluent prepared at 1-fold concentration in MEM2 was added to each well. Plates were incubated in an incubator (5% CO2) at 37 ° C. for 7 days. Virus-free controls (“simulated infection”), medium-only cultured infected cells, vehicle-only (methanol) -infected infected cells, and cell-free wells (to determine background) were placed on assay plates. included. Control wells containing 50 μM ribavirin and 0.5 μM compound A3 were also included in the assay plate. Seven days after infection, cells were stained with Neutral Red and cell viability was monitored. Test materials were evaluated by replication using a continuous 8-fold dilution in infectious medium. Controls included cells incubated without virus (“simulated infection”), infected cells incubated with medium alone, or infected cells in the presence of ribavirin (0.5 μM) or A3 (0.5 μM). A complete replication inhibition curve using only methanol vehicle was included in the same assay plate.

Protection of ZIKV-Induced Cytopathic Effect (CPE) by Neutral Red Reading: The PLCal_ZV strain was used in the ZIKV antiviral assay. Vero cells (epithelial kidney cells derived from Cercopisecus aethios) were maintained with MEM (MEM5) containing 5% FBS. For both infection and viability assays, cells were seeded on 96-well clear flat bottom plates with 10,000 cells per well and maintained at 37 ° C. for 24 hours at MEM5. On the day of infection, samples were diluted 8-fold on U-bottom plates using MEM containing 1% bovine serum albumin (BSA). A diluted solution of the test substance was prepared at a final concentration of 1.25 times, and 40 μl was incubated with the target cells at 37 ° C. for 30 minutes. After preincubation of the test material, 10 μl of virus diluted solution prepared by MEM containing 1% BSA was added to each well (final volume of 50 μl per well), and the plate was placed in a humidified incubator with 5% CO2 at 37 ° C. Incubated for 3 hours. After infection incubation, cells were washed with PBS followed by MEM (MEM2) containing 2% FBS to remove unbound virus. Subsequently, 50 μl of medium containing the inhibitor diluent prepared at 1-fold concentration in MEM2 was added to each well. Plates were incubated in an incubator (5% CO2) at 37 ° C. for 6 days. Virus-free controls (“simulated infection”), medium-only cultured infected cells, vehicle-only (methanol) -infected infected cells, and cell-free wells (to determine background) were placed on assay plates. included. Six days after infection, cells were stained with Neutral Red and cell viability was monitored. Test materials were evaluated by replication using a continuous 8-fold dilution in infectious medium. Controls included cells incubated without virus (“simulated infection”), infected cells incubated with medium alone, or infected cells in the presence of A3 (0.5 μM). A complete replication inhibition curve using only methanol vehicle was included in the same assay plate.

Analysis of CPE-based viability data: In the neutral red assay, cell viability was determined by monitoring the absorbance at 490 nm. Mean signals obtained in cell-free wells were subtracted from all samples. All data points were then calculated as the percentage of mean signals observed in 8 wells of simulated (non-infected) cells on the same assay plate. Infected cells treated with medium alone averaged 4.2% (for HRV), 26.9% (for DENV), and 5.1% (for ZIKV) of the signals observed in non-infected cells. The signal was reduced to the average of. The signal vs. background (S / B) for this assay was 2.9 (for DENV) and 7.2 (for ZIKV), a "simulation" compared to the viability of infected cells treated with vehicle alone. Determined as viability of "infected" cells.

Viability Assay (XTT) for assessing compound-induced cytotoxicity: Simulated infected cells using the same experimental settings and inhibitor concentrations as used in the corresponding infection assay, inhibitor diluent (or medium only). ) Was incubated with. The incubation temperature and length of the incubation period reflected the conditions of the corresponding infection assay. Cell viability was evaluated by the XTT method. The XTT assay measures mitochondrial activity and is based on cleavage of the yellow tetrazolium salt (XTT) that forms the orange formazan pigment. The reaction occurs only in living cells with active mitochondria. Formazan dyes are directly quantified using a scanning multi-well spectrophotometer. Background levels from cell-free wells were subtracted from all data points. Methanol vehicle-only controls (at 7 concentrations reflecting the final percent of methanol in each oleandrin test well) were included in the viability assay plate. The degree of survival was monitored by measuring the absorbance at 490 nm.

Analysis of Cytotoxicity Data: In the XTT assay, cell viability was determined by monitoring the absorbance at 490 nm. Mean signals obtained in cell-free wells were subtracted from all samples. All data points were then calculated as the percentage of mean signals observed in 8 wells of simulated (non-infected) cells on the same assay plate. The signal vs. background (S / B) for this assay is 29.9 (for IVA), 8.7 (for HRV), 6.5 (for DENV), and 6.7 (for ZIKV). And determined as the viability of "simulated infected" cells compared to the signals observed in the cell-free wells.

(Example 15)
Evaluation method of antiviral activity against filovirus (Ebola virus and Marburg virus) A.
Vero E6 cells were given EBOV / Kik (A, MOI = 1) or MARV / Ci67 (B, MOI = 1) in the presence of oleandrin, digoxin, or oleandrin-containing plant extract PBI-05204. 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 EBOV or MARV infected cells. Infected cells were counted using Operetta. C) Vero E6 cells were treated with the above compounds. ATP levels were measured by CellTiter-Glo as a measure of cell viability.
Method B.
Vero E6 cells were infected with EBOV (A, B) or MARV (C, D). Two hours after infection (A, C) or 24 hours after infection (B, D), oleandrin or PBI-05204 was added to the cells for 1 hour, then discarded and the cells returned to culture medium. Forty-eight hours after infection, infected cells were analyzed as shown in FIG.
Method C.
Vero E6 cells were infected with EBOV or MARV in the presence of oleandrin or PBI-05204 and incubated for 48 hours. The supernatant of the infected cell culture was subcultured into fresh Vero E6 cells, incubated for 1 hour, and then discarded (as shown in A). Cells containing the passaged supernatant were incubated 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)
Evaluation of antiviral activity against togaviridae virus (alpha virus: VEEV and WEEV)
Vero E6 cells are 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 compound. rice field. Infected cells were detected as described herein and counted in Operetta.

(Example 17)
Treatment of Paramyxoviridae Infections Exemplary paramyxoviridae virus infections include henipavirus infections, nipavirus infections, or Hendravirus infections.
Method A. Antiviral composition therapy Antiviral compositions are prescribed to subjects presenting with Paramyxoviridae infection, and treatment-related doses are administered to the subjects for a period of time according to the prescribed dosing regimen. The level of treatment response of the subject is determined on a regular basis. The level of therapeutic response can be measured by determining the viral titer of the subject in blood or plasma. If the level of therapeutic response is too low at a given dose, the dose is graded according to a given dose escalation schedule 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 can be adjusted as needed until the patient reaches the desired clinical endpoint.
Method B. Combination Therapy: Antiviral Composition with Another Drug Method A above, except that the subject is prescribed and administered one or more other therapeutic agents for the treatment of a paramyxoviridae infection or its symptoms. Follow. One or more other therapeutic agents can then be administered before, after, or with the antiviral composition. Dose escalation (or tapering) of one or more other therapeutic agents can also be performed.

Example 18
Primary huPBMC Cell Lines and Isolation Virus-Generating HTLV-1-Transformed (HTLV-1 +) SLB1 Lymphotropic T-Cell Lines (Arnold et al., 2008; P. Green, The Ohio State United-Comprehensive Center In a humidified incubator at 37 ° C. under 10% CO ₂ , 10% heat-inactivated fetal bovine serum (FBS; Biowest), 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, and 20 μg / ml gentamicin. The cells were cultured in a modified Dulvecco medium (IMDM; ATCC No. 30-2005) of Iskove supplemented with sulfate (Life Technologies).

Primary human peripheral blood mononuclear cells (huPBMC) are whole blood donated to the SMU Memorial Health Center under a protocol approved by the SMU Examination Review Board and in accordance with the principles of the Declaration of Helsinki. Isolated from the sample. Briefly, 2 ml of whole blood is mixed in a polypropylene conical tube (Corning) with an equal volume of sterilized phosphate buffered saline (PBS), pH 7.4, and then the sample is separated into 3 ml of lymphocytes. It was gently laminated on the medium (MP Biomedicals). Samples were centrifuged at 400 xg for 30 minutes in a swing bucket rotor at room temperature. Buffy coat huPBMC was subsequently aspirated, washed 2x in RPMI-1640 medium (ATCC No. 30-2001) and pelleted by centrifugation at 260xg for 7 minutes. Cells were 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). It was resuspended in RPMI-1640 medium, stimulated with 10 ng / ml phytohemaglutinin (PHA; Sigma-Aldrich) for 24 hours, and grown in a humidified incubator at 37 ° C. under 10% CO ₂ . The next day, cells were pelleted by centrifugation at 260 × g for 7 minutes, washed 2 × with RPMI-1640 medium to remove PHA, then resuspended, antibiotics and 50 U / ml hu-IL-2. The cells were cultured in the supplemented complete medium.

Example 19
Generation of GFP-expressing HTLV-1 + SLB1 / pLenti-GFP T-cell clones To generate GFP-expressing HTLV-1 + SLB1 T-cell clones, 2 × 10 ⁶ SLB1 cells were added to 60 mm ² tissue culture dish (Corning), 10%. Sowed in heat-inactivated FBS and IMDM supplemented with antibiotics, then transduced lentivirus particles containing pLenti-6.2 / V5-DEST-green fluorescent protein expression vector also carrying the blastsidin resistance gene. After 6 hours, transduced cells were 2x washed with serum-free IMDM pelleted by centrifugation at 260 xg for 7 minutes at room temperature and supplemented with 5 μg / ml Blast Sciences. The cells were resuspended in complete medium and divided into 96-well microtiter plates (Corning). Cultures were selected and maintained with Blasticidin for 2 weeks in a humidified incubator at 37 ° C. and 10% CO ₂ . GFP-expressing lymphoblasts were screened by fluorescence microscopy and then sown by limited dilution in 96-well microtiter plates to obtain homogeneous GFP-expressing cell clones. The resulting HTLV-1 + SLB1 / pLenti-GFP T-lymphocyte clones were swelled and repeatedly passaged; GFP expression was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and rabbit polyclonal anti-GFP (FL). ) Confirmed by immunobrotting using an antibody (Santa Cruz Biotechnology).

Example 20
Anti-HTLV-1 p19 ^Gag ELISA virus generation and quantification of particle infectivity oleandrin or N. et al. During HTLV-1 provirus replication and release of newly synthesized extracellular virus particles. To determine the effect of the extractor extract, HTLV-1 + SLB1 lymphoma T-cell lines were placed in 300 μl antibiotic-supplemented complete medium in 96-well microtiter plates with 2 × 10 ⁴ cells per well. Was sown and incubated at 37 ° C. under 10% CO ₂ . Purified oleandrin compounds and N. An extract of oleander (Phoenix Biotechnology; see Singh et al., 2013) in a vehicle solution (MilliQ distillation / deionized _H2O in 20% v / v dimethyl sulfoxide, DMSO) at a stock concentration of 2 mg / ml. It was resuspended and then sterilized using a Luerlock 0.2 μm syringe filter (Millipore). HTLV-1 + SLB1 cells with oleandrin or N. at concentrations of 10, 50, and 100 μg / ml. Treatment was performed with an oleander extract or an elevated amount (1.5, 7.5, and 15 μl) of vehicle control for 72 hours. 96-well microtiter plates are then centrifuged using an Eppendorf A-2-DWP swing plate rotor at 260 xg for 7 minutes at room temperature to pellet the cells and release into the supernatant of the culture. Levels of extracellular p19 ^Gag -containing HTLV-1 particles were quantified relative to the p19 ^Gag protein standard by performing a colorimetric anti-p19 ^Gag enzyme-linked immunosorbent assay (ELISA). Samples were analyzed with a Berthold Tristar LB 941 multimode microplate reader in absorbance mode 450 nm using three replicas.

To assess the infectivity of newly synthesized extracellular HTLV-1 particles collected from oleandrin-treated cells, 2 x 10 ⁴ HTLV-1 + SLB1 T-lymphotropic cells were used as antibiotics. Sow in 300 μl complete medium supplemented with oleandrin or N. et al. At elevated concentrations (10, 50, and 100 μg / ml). Treatment with oleander extract, or vehicle control (1.5, 7.5, and 15 μl) for 72 hours. Then, 50 μl of virus-containing supernatant was used to directly infect huPBMC, which was supplemented with antibiotics and hu-IL-2 at a density of 2 × 10 ⁴ cells per well in a 96-well microtiter plate. It was sown in the complete medium. Oleandrin compounds, N. The oleander extract, or vehicle control, was maintained in huPBMC medium to control possible reinfection events with newly generated particles. After 72 hours, the relative levels of extracellular p19 ^Gag -containing HTLV-1 virions released into the culture supernatant by infected huPBMC were quantified via anti-HTLV-1 p19 ^Gag ELISA.

Example 21
Measurement of Cell Apoptosis Oleandrin compounds in treated cell cultures, N. et al. 300 μl of antibiotic-supplemented 2 × 10 ⁴ HTLV-1 + SLB1 lymphoma T-cells or activated / cultured huPBMC to assess the relative cytotoxicity of the oleander extract, or vehicle control. Was sown in complete medium and maintained in a humidified incubator at 37 ° C. under 10% CO ₂ . Cultures were added to elevated concentrations (10, 50, and 100 μg / ml) of oleandrin or N. It was treated with oleander extract or vehicle control (1.5, 7.5, 15 ml) and incubated for 72 hours. Cells treated with cyclophosphamide (50 μM; Sigma-Aldrich) were included as positive controls for apoptosis. Cells were then aspirated and sown on Permanox 8-chamber tissue culture slides (Nalge) pretreated with a 0.01% sterilized solution of poly-L-lysine and concanavalin A (1 mg / ml; Sigma-Aldrich). Samples were subsequently stained using a microscopic apoptosis detection kit using Annexin V (Annexin V-FITC) conjugated with fluorescein isothiocyanate and propidium iodide (PI; BD-Pharmingen). Relative rate of apoptosis (ie Annexin V-FITC and / or PI-positive) cells were quantified three times by cofocal fluorescence microscopy using a 20x objective lens. The total number of cells per field of view was quantified by microscopy using a DIC retardation filter.

Example 22
HTLV-1 Transmission and Virological Synapse Formation in Co-Culture Assays HTLV-1 transmission is typically via virological synapses, via direct contact between infected and uninfected target cells. Since it occurs (Igakura et al., 2003; Pais-Correia et al., 2010; Gross et al., 2016; Omsland et al., 2018; Majorovits et al., 2008), we have described Oleandrin, N. et al. It was tested whether oleander extract, or vehicle control, could affect the formation of virological synapses and / or the transmission of infectious HTLV-1 particles via intracellular interactions in vitro. In these experiments, 2 × 10 ⁴ virus-producing HTLV-1 + SLB1 T-cells were sown on 96-well microtiter plates in 300 μl complete medium with mitomycin C (100 μg / ml) under 10% CO ₂ . Treatment at 37 ° C. for 2 hours (Bryja et al., 2006). The culture medium was then removed and the cells were washed 2x with serum-free IMDM and the cells were subjected to elevated doses (10, 50, and 100 μg / ml) of oleandrin, or N. et al. Treatment with oleander extract, or vehicle control (1.5, 7.5, and 15 μl) for 15 minutes or 3 hours. Alternatively, 2 × 10 ⁴ GFP-expressing HTLV-1 + SLB1 / pLenti-GFP T-cells are sown in 300 μl complete medium in an 8-chamber tissue culture slide, treated with mitomycin C, and 2 × with serum-free IMDM. Washed and then oleandrin, N. et al., As described in the confocal microscopy experiment. Treated with oleander extract or vehicle control. We then aspirate the medium, wash the HTLV-1 + SLB1 cells 2 × with serum-free medium, and add 2 × 10 ⁴ huPBMCs in each well, 20% FBS, antibiotics and 50 U / ml hu. -The cells were added to 300 μl RPMI-1640 medium supplemented with IL-2 and then co-cultured the cells in a humidified incubator at 37 ° C. under 10% CO ₂ for an additional 72 hours (cells co-cultured for 6 hours). Then, confocal microscopy using SLB1 / pLenti-GFP lymphoblasts visualized viral synapse formation and transmission of the virus). As a negative control, huPBMC was cultured solely without virus-producing cells. Oleandrin, N. et al. The oleander extract and vehicle were maintained in co-medium. Relative levels of extracellular p19 ^Gag -containing HTLV-1 particles released into the supernatant of the co-culture were quantified by performing anti-HTLV-1 p19 ^Gag ELISA as a result of intracellular virus transmission. Viral synapses formed between GFP-positive HTLV-1 + SLB / pLenti-GFP cells and huPBMC stain fixed samples with anti-HTLV-1 gp21 ^Env primary antibody and rhodamine red conjugate secondary antibody. This was visualized using immunofluorescence co-microscopy. Diamidino-2-phenyl-indole dihydrochloride (DAPI; Molecular Probes) nuclear staining was included for comparison and visualization of uninfected (ie, HTLV-1-negative) cells. Intracellular transmission of HTLV-1 to huPBMC in a co-culture assay is to count the relative proportions of HTLV-1 gp21 ^Env -positive (and GFP-negative) huPBMC in 20 fields of view using a 20x objective. Quantified by.

Example 23
Microscopy To quantify cell apoptosis and cytotoxicity, Annexin V-FITC / PI-stained samples were used with a Plan-Apochromat 20 × / 0.8 objective lens and Zeiss ZEN system software (Carl Zeiss Microscopy). Visualized by cofocal fluorescence microscopy on a Zeiss LSM800 instrument equipped with an Airyscan detector and a stage CO ₂ incubator. Relative proportions of virological synaptic formation and virus transmission (ie, anti-HTLV-1 gp21 ^Env -positive huPBMC) between HTLV-1 + SLB1 / pLenti-GFP lymphoblasts treated with mitomycin C and cultured huPBMC. (Determined by quantification) was visualized by immunofluorescence-confocal microscopy using a Plan-Apochromat 20x / 0.8 objective lens. Relative fluorescence intensity, anti-HTLV-1 gp21 ^Env -specificity (Rhodamine red-positive) and GFP signal of DAPI were quantified as a graph using a Zen 2.5D analysis tool (Carl Zeiss Microscopy). GFP-expressing HTLV-1 + SLB1 / pLenti-GFP T-cell clones with a Nikon Eclipse TE2000-U inverted microscope and a D-Eclipse confocal imaging system with 633 nm and 543 nm He / Ne, and a 488 nm Ar laser. Screening was performed by confocal fluorescence microscopy using a × / 0.30 objective lens and a DIC Phase Difference Filter (Nikon Instruments).

Example 24
Statistical analysis Statistical significance of experimental datasets was determined using unpaired two-sided Student's t-test (alpha = 0.05) using the Shapiro-Wilk normality test and Graphpad Prism 7.03 software. The P-value was calculated. The P-values were defined as: 0.1234 (ns), 0.0332 ( ^* ), 0.0021 ( ^** ), 0.0002 ( ^*** ), <0.0001 ( ^**** ). Unless otherwise noted, error bars represent SEMs from at least three independent experiments.

Example 25
Treatment of Deltaretrovirus Infection in Subjects Exemplary Deltaretrovirus infections include HTLV-1.
Method A. Antiviral composition therapy Antiviral compositions are prescribed to subjects presenting with HTLV-1 infection and treatment-related doses are administered to the subjects for a period of time according to the prescribed dosing regimen. The level of treatment response of the subject is determined on a regular basis. The level of therapeutic response can be measured by determining the HTLV-1 virus titer of the subject in blood or plasma. If the level of therapeutic response is too low at a given dose, the dose is graded according to a given dose escalation schedule 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 can be adjusted as needed until the patient reaches the desired clinical endpoint.
Method B. Combination Therapy: Antiviral Composition with Another Drug Follow Method A above, except that the subject is prescribed and administered one or more other therapeutic agents for the treatment of HTLV-1 infection or its symptoms. .. One or more other therapeutic agents can then be administered before, after, or with the antiviral composition. Dose escalation (or tapering) of one or more other therapeutic agents can also be performed. Other exemplary therapeutic agents are described herein.

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 HKU 20.
Method A. Antiviral composition therapy Antiviral compositions are prescribed to subjects presenting with CoV infection and treatment-related doses are administered to the subjects for a period of time according to the prescribed dosing regimen. The level of treatment response of the subject is determined on a regular basis. The level of therapeutic response can be measured by determining the CoV virus titer of the subject in blood or plasma. If the level of therapeutic response is too low at a given dose, the dose is graded according to a given dose escalation schedule 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 can be adjusted as needed until the patient reaches the desired clinical endpoint.
Method B. Combination Therapy: Antiviral Composition with Another Agent Follow Method A above, except that the subject is prescribed and administered one or more other therapeutic agents for the treatment of CoV infection or its symptoms. One or more other therapeutic agents can then be administered before, after, or with the antiviral composition. Dose escalation (or tapering) of one or more other therapeutic agents can also be performed. Other exemplary therapeutic agents are described herein.

Example 27
Treatment of COVID-19 infection in subjects using ANVIRZEL ™ Treatment of COVID-19-related symptoms by administering ANVIRZEL ™ as follows to children (pediatrics) presenting COVID-19. bottom. Prior to administration of ANVIRZEL ™, the subject's viral infection was exacerbated. Subjects were prescribed and administered ANVIRZEL ™ according to the dosing regimen below: initial dose-0.25 mL reconstituted ANVIRZEL ™ every 12 hours for 2-3 days, followed by 0.5 mL reconstituted ANVIRZEL. (trademark). The subject's COVID-19 infection was resolved and no drug-related toxicity was observed.

Example 28
In vitro assessment of oleandrin against COVIDEO-19 virus The purpose of this study was to determine the effect of oleandrin on the infectivity of progeny virions.

培養物中の未感染Ｖｅｒｏ細胞（目標初期細胞数１×１０⁶個）を、バイアル中の指示されたインキュベーション培地のそれぞれにおいて、３７℃にて３０分インキュベートした。ＳＡＲＳ－ＣｏＶ－２のウイルス接種を、次いで各バイアルに追加して、目標初期ウイルス力価（約ＰＦＵ／ｍＬ １×１０⁴個）を達成した。約０．１の目標ＭＯＩ（感染多重度）。溶液を３７℃にて２時間さらにインキュベートして、Ｖｅｒｏ細胞の感染を達成した。感染Ｖｅｒｏ細胞を、次いで、対照ビヒクルで洗浄して、細胞外ウイルスおよびオレアンドリンを除去した。各インキュベーション培地の新たなアリコートを、それぞれの感染Ｖｅｒｏ細胞の各バイアルに添加した。第２のアリコートにおいてオレアンドリンを受けたものは、「感染後＋処理」として示し、第２のアリコートにおいてオレアンドリンを受けていないものは、「感染後－処理」として示した（図２３Ａ～２３Ｄ）。各バイアルでのウイルス力価は、感染後約２４時間および約４８時間で判定した。
Ｖｅｒｏ細胞に対するオレアンドリンの潜在的毒性を判定する手段として、上記のものに基づいて、未感染Ｖｅｒｏ細胞の平行培養物を調製した。
獲得したデータには、感染および未感染細胞で生成されたウイルスの量、子孫ウイルスの感染性、およびオレアンドリンの相対的安全性（非毒性）が含まれていた。 A stock solution of oleandrin in methanol (10 mg oleandrin / mL) was prepared. Using stock solutions, DMSO (0.1% or 0.01% v / v in RPMI 1640 in aqueous medium and oleandrin (20 microg / mL, 10 microg / mL, 1.0 microg / mL, or 0.1 microg /). mL) -containing incubation medium was prepared. The incubation solution is as follows.

Uninfected Vero cells (target initial cell number 1 × 10 ⁶ cells) in culture were incubated at 37 ° C. for 30 minutes in each of the indicated incubation media in vials. SARS-CoV-2 virus inoculation was then added to each vial to achieve the target initial virus titer (approximately PFU / mL 1 × 10 ⁴ ). Target MOI (multiplicity of infection) of about 0.1. The solution was further incubated at 37 ° C. for 2 hours to achieve Vero cell infection. Infected Vero cells were then washed with a control vehicle to remove extracellular virus and oleandrin. A new aliquot of each incubation medium was added to each vial of each infected Vero cell. Those who received oleandrin in the second aliquot were shown as "post-infection + treatment", and those who did not receive oleandrin in the second aliquot were shown as "post-infection-treatment" (FIG. 23A). ~ 23D). Virus titers in each vial were determined approximately 24 hours and approximately 48 hours after infection.
Based on the above, parallel cultures of uninfected Vero cells were prepared as a means of determining the potential toxicity of oleandrin to Vero cells.
The data obtained included the amount of virus produced in infected and uninfected cells, the infectivity of progeny virus, and the relative safety (non-toxicity) of oleandrin.

Example 29
In vitro evaluation of oleandrin against COVIDEO-19 virus The purpose of this assay was to determine the direct antiviral activity of oleandrin against SARS-CoV-2.

Growth medium was removed from the dense monolayer of approximately 106 Vero CCL81 cells in a ⁶ -well plate. Oleandrin was serially diluted in culture medium and added to Vero-E6 cells seeded in 96-well plates. Growth medium in 200 μl maintenance medium containing 1.0 μg / ml, 0.5 μg / ml, 100 ng / ml, 50 ng / ml, 10 ng / ml, or 5 ng / ml oleandrin, or a matched DMSO-only control. Replaced. Plates were incubated at 37 ° C. for about 30 minutes prior to virus addition.
SARS-CoV-2 virus was added to oleandrin-treated and untreated cells with an MOI (multiplicity of infection) of 0.4 (invasion assay) or 0.02 (replication assay). Oleandrin remained in the wells during the 1 hour incubation at 37 ° C.
One hour after absorption, the inoculum was removed and washed once with PBS (standard phosphate buffered saline).
Medium only (without oleandrin) was returned to wells treated with oleandrin designated as "pretreatment" on the data slide. Medium with the indicated concentration of oleandrin was returned to the well designated as "persistent" on the data slide.
Plates were fixed 24 hours (invasion assay) or 48 hours (replication assay) post-infection and immunostained with virus-specific antibody and fluorescently labeled secondary antibody.
Cells were imaged using Operetta and the data were analyzed using a custom algorithm in Harmonia software to determine the percentage of infected cells in each well.
The results are shown in FIGS. 24A and 24B.

Example 30
In vitro assessment of oleandrin toxicity to Vero-E6 cells The purpose of this assay was to determine the relative potential toxicity of oleandrin to Vero-E6 cells.
Oleandrin was serially diluted in culture medium, added to Vero-E6 cells seeded in 96-well plates and incubated at 37 ° C. for about 24 hours. Cell counts were obtained using the cell titer Glo assay.
The results are shown in FIG.

Example 31
In vitro evaluation of oleandrin against COVID-19 virus The purpose of this study was to determine the dose response of COVID-19 virus to treatment with oleandrin.
The procedure of Example 28 was repeated, except that lower concentrations of oleandrin were used: 1 microg / mL, 0.5 microg / mL, 0.1 microg / mL, 0.05 microg / mL, 0.01 microg / mL, and 0.005 microg / mL. In addition, VERO CCL-81 cells were used in place of VERO E6 cells.
The virus titer was determined according to Example 28, and the reduction in virus titer by a factor of 2 was calculated by comparison with the control sample. Results are shown in FIGS. 26A-26D, 27A-27D, and 28A and 28B.

Example 32
Sublingual liquid dosage forms Sublingual dosage forms containing oleandrin are oleandrin or oleandrin-containing compositions (eg, oleandrin-containing extracts; 2% by weight) and medium-chain triglycerides (95% by weight) and flavor. Made by mixing the agent (3% by weight). The oleandrin content in the dosage form was about 25 microg / mL.

Example 33
Preparation of Nerium oleander subcritical fluid extract An improved method for preparing oleandrin-containing extracts has been developed by using subcritical fluid extraction rather than supercritical fluid extraction of Nerium oleander biomass.
The dried and powdered biomass was placed in an extraction chamber, which was then sealed. Carbon dioxide (about 95% by weight) and alcohol (about 5% by weight; methanol or ethanol) were injected into the chamber. The internal temperature and pressure of the chamber are such that the extraction medium is in the subcritical liquid phase rather than the supercritical fluid phase for most or substantially all of the extraction time: about 2 ° C to about 16 ° C (about 7 ° C to about). The temperature was maintained in the range of 8 ° C.) and the pressure was maintained in the range of about 115 to about 135 bar (about 124 bar). The extraction period was about 4 hours to about 12 hours (about 6 to about 10 hours). The extraction environment was then filtered and the supernatant was collected. Carbon dioxide was released from the supernatant and the resulting crude extract was diluted in ethanol (about 9 parts ethanol: about 1 part extract) and frozen at about −50 ° C. for at least 12 hours. The solution was thawed and filtered (100 micron pore size filter). The filtrate was concentrated to about 10% of its original volume and then sterilized and filtered (filter with pore size 0.2 micron). Concentration The extract was then diluted with 50% aqueous ethanol to a concentration of about 1.5 mg of extract per 1 mL of solution.
The resulting subcritical liquid (SbCL) extract contains oleandrin, and one or more other compounds that can be extracted from the Nerium oleander, wherein the one or more other compounds are described herein. Defined in.

Example 34
In vitro assessment of oleandrin against COVIDEO-19 virus The purpose of this study is to determine the effect of oleandrin on the infectivity of progeny virions without pretreatment with oleandrin (as in Example 28). Met.
The procedure of Example 28 was repeated, except that the cells were not pretreated with oleandrin prior to infection. Instead, infected cells were treated with oleandrin or a control vehicle 12 and 24 hours after infection. In addition, VERO CCL-81 cells were used instead of VERO E6 cells and lower concentrations of oleandrin were used: 1 microg / mL, 0.5 microg / mL, 0.1 microg / mL, and 0.05 microg / mL. .. The data are shown in FIGS. 29A and 29B.

Example 35
In vivo Assessment of Oleandrin Against 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 COVID-19 virus.
Subjects presenting COVID-19 infection were evaluated to represent a broad demographic distribution to determine clinical status prior to sublingual, buccal or oral administration of OCE prepared according to the dosage form of Example 32. .. The composition was safely administered to the subject by placing a droplet in the subject's oral cavity. The dosing regimen is approximately 0.5 mL per dose and 4 times per day (about once every 6 hours), which is close to about 50 micrologs of oleandrin per day. Alternatively, half of the total daily dose was administered. All subjects experienced full recovery.

Example 36
Preparation of Ethanol Extract of Nerium oleander The purpose of this was to prepare an ethanol extract by extracting the Nerium oleander biomass with an aqueous ethanol solution.
The crushed dried leaves were repeatedly treated with an aqueous ethanol solution (90% v / v ethanol; 10% v / v water). Crude ethanol extract containing about 25 mg oleandrin / mL extract by combining the combined ethanol supernatants, filtering and then concentrating by evaporating in vacuum to reduce the amount of ethanol and water in it. (Has an approximately 50% v / v ethanol content) was obtained.

Example 37
Preparation of Dosage Form Containing Combination of Nerium oleander Extracts The purpose of this is to use a portion (1 wt%) of the ethanol extract of Example 36 and a portion (1 wt%) of the SbCL extract of Example 33. The dosage form was to be prepared according to Example 32, except in combination with medium chain triglycerides (95% by weight) and flavoring agents (3% by weight).

Example 38
In vivo Evaluation of Digoxin Against COVID-19 Virus The purpose of this study was to determine the efficacy of a digoxin-containing composition (DCC) in the treatment of subjects already infected with COVID-19 virus. Purchase a commercial dosage form containing digoxin.
Subjects present with COVID-19 infection are evaluated to determine clinical status prior to oral or systemic administration of DCC. Commercially available compositions are described herein. Safe dosing regimens for each are described in their respective NDAs and package inserts. The composition is safely administered to each subject according to the intended route of administration. Clinical monitoring was performed to determine the treatment response and the dose was titrated in an appropriate manner.

Example 39
Determining the Ratio of Genome to Infected Particles in SARS-CoV-2 Infection Treated with Oleandrin The purpose of this study was whether inhibition of SARS-CoV-2 by oleandrin was at the level of total or infected particle production. Is to judge.
To quantify the genomic copy to the sample, 200 μl of sample was extracted with 5: 1 volume ratio TRIzol LS (Ambion, Carlsbad, CA) using standard manufacturer protocol and resuspended in 11 μl water. .. The extracted RNA was tested for SARS-CoV-2 by qRT-PCR according to the previously published assay (26). Briefly, the N gene was amplified using the following primers and probes: forward primer [5'-TAATCAGACAAGGAACTGATTA-3'] (SEQ ID NO. 1); reverse primer [5'-CGAGGTGTGACTTCCATG-3']. (SEQ ID NO. 2); and probe [5'-FAM-GCAAATTGTGCAATTTGCG-TAMRA-3'; (SEQ ID NO. 3)]. A 20 μl reaction mixture was prepared using the iTaq Universal probes One-Step kit (BioRad, Hercules, CA) according to the manufacturer's instructions: reaction mix (2 ×: 10 μL), iScript reverse transcriptase (0.5 μL). , Primer (10 μM: 1.0 μL), probe (10 μM: 0.5 μL), extracted RNA (4 μL) and water (3 μL). The qRT-PCR reaction was performed using a thermal cycler StepOnePlus ™ Real-Time PCR Systems (Applied Biosystems). The reaction was incubated 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. A positive control RNA sequence (COVID-2019 genome of nucleotides 26,044-29,883) was used to assess the number of RNA copies of the N gene in the sample under evaluation.

As used herein, the term "about" or "approximately" is to be construed to mean ± 10%, ± 5%, ± 2.5%, or ± 1% of the specified value. As used herein, the term "substantially" is to be construed to mean "most" or "at least most" or "more than 50%".

The above is a detailed description of a particular embodiment of the invention. Although specific embodiments of the invention have been described herein for illustrative purposes, it should be appreciated that various modifications can be made without departing from the spirit and scope of the invention. Therefore, the present invention is not limited except by the scope of the appended claims. All of the embodiments disclosed and claimed herein can be created and performed in the light of the present disclosure without undue experimentation.

１１． 前記１回以上の用量の投与後、前記対象におけるオレアンドリンの血漿中濃度が、血漿１ｍＬあたりのオレアンドリンの量として、約０．０５～約２ｎｇ／ｍｌ、約０．００５～約１０ｎｇ／ｍL、約０．００５～約８ｎｇ／ｍL、約０．０１～約７ｎｇ／ｍL、約０．０２～約７ｎｇ／ｍL、約０．０３～約６ｎｇ／ｍL、約０．０４～約５ｎｇ／ｍL、または約０．０５～約２．５ｎｇ／ｍLの範囲である、上記１～１０のいずれか一項に記載の方法。
１２． 治療を必要とする対象においてウイルス感染を治療する方法であって、オレアンドリンを含む１回以上の用量の抗ウイルス組成物を前記対象に投与することを含み、前記ウイルス感染が、プラスセンス一本鎖エンベロープＲＮＡウイルス（（＋）－ｓｓ－ｅｎｖＲＮＡ－Ｖ）によって引き起こされ、
用量が、前記対象の体重１Ｋｇあたり約１００～１０００ｍｇまたは約１００～１０００ｍｉｃｒｏｇの抗ウイルス組成物を含み；または、１日あたり投与されるオレアンドリンの量が、１４０ｍｉｃｒｏｇ～３１５ｍｉｃｒｏｇ、２０ｍｉｃｒｏｇ～７５０ｍｉｃｒｏｇ、１２ｍｉｃｒｏｇ～３００ｍｉｃｒｏｇ、１２ｍｉｃｒｏｇ～１２０ｍｉｃｒｏｇ、０．０１ｍｉｃｒｏｇ～１００ｍｇ、０．０１ｍｉｃｒｏｇ～１００ｍｉｃｒｏｇ、約０．５～約１００ｍｉｃｒｏｇ、約１～約８０ｍｉｃｒｏｇ、約１．５～約６０ｍｉｃｒｏｇ、約１．８～約６０ｍｉｃｒｏｇ、もしくは約１．８～約４０ｍｉｃｒｏｇからなる群から選択され；または、前記抗ウイルス組成物の用量が、１日あたり対象の体重１ｋｇあたりの抗ウイルス組成物の単位量に基づいて、約０．０５～０．５ｍｇ／ｋｇ／日、約０．０５～０．３５ｍｇ／ｋｇ／日、約０．０５～０．２２ｍｇ／ｋｇ／日、約０．０５～０．４ｍｇ／ｋｇ／日、約０．０５～０．３ｍｇ／ｋｇ／日、約０．０５～０．５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．３５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．２２ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．４ｍｉｃｒｏｇ／ｋｇ／日、もしくは約０．０５～０．３ｍｉｃｒｏｇ／ｋｇ／日を含み；
前記１回以上の用量の投与後、前記対象におけるオレアンドリンの血漿中濃度が、血漿１ｍＬあたりのオレアンドリンの量として、約０．０５～約２ｎｇ／ｍｌ、約０．００５～約１０ｎｇ／ｍＬ、約０．００５～約８ｎｇ／ｍＬ、約０．０１～約７ｎｇ／ｍＬ、約０．０２～約７ｎｇ／ｍＬ、約０．０３～約６ｎｇ／ｍＬ、約０．０４～約５ｎｇ／ｍＬ、または約０．０５～約２．５ｎｇ／ｍＬの範囲である、方法。
１３． 前記（＋）－ｓｓ－ｅｎｖＲＮＡＶが、コロナウイルス科、フラビウイルス科、トガウイルス科、およびアルテルウイルス科からなる群から選択されるウイルスである、上記１～１２のいずれか一項に記載の方法。
１４． 前記（＋）－ｓｓ－ｅｎｖＲＮＡＶが、ヒトに対して病原性であるコロナウイルスである、上記１～１３のいずれか一項に記載の方法。
１５． 前記コロナウイルスが、ＳＡＲＳ－ＣｏＶ、ＭＥＲＳ－ＣｏＶ、ＣＯＶＩＤ－１９（ＳＡＲＳ－ＣｏＶ－２）、ＣｏＶ ２２９Ｅ、ＣｏＶ ＮＬ６３、ＣｏＶ ＯＣ４３、ＣｏＶ ＨＫＵ１、およびＣｏＶ ＨＫＵ２０からなる群から選択される、上記１４に記載の方法。
１６． 前記（＋）－ｓｓ－ｅｎｖＲＮＡＶが、フラビウイルス、黄熱ウイルス、デング熱ウイルス、日本脳炎ウイルス、西ナイルウイルス、ジカウイルス、ダニ媒介性脳炎ウイルス、キャサヌール森林病ウイルス、アルフルマ（Ａｌｋｈｕｒｍａ）病ウイルス、オムスク出血熱ウイルス、およびポワッサンウイルスからなる群から選択されるウイルスである、上記１～１３のいずれか一項に記載の方法。
１７． 前記（＋）－ｓｓ－ｅｎｖＲＮＡＶが、アルボウイルス、東部ウマ脳脊髄炎ウイルス（ＥＥＥＶ）、西部ウマ脳脊髄炎ウイルス（ＷＥＥＶ）、ベネズエラウマ脳脊髄炎ウイルス（ＶＥＥＶ）、チクングニアウイルス（ＣＨＩＫＶ）、オニョンニョンウイルス（ＯＮＮＶ）、ポゴスタ病ウイルス、シンドビスウイルス、ロスリバー熱ウイルス（ＲＲＶ）およびセムリキ森林ウイルスからなる群から選択されるトガウイルス科ウイルスである、上記１～１３のいずれか一項に記載の方法。
１８． 治療を必要とする対象においてウイルス感染を治療する方法であって、オレアンドリン、ジゴキシンまたはそれらの組み合わせを含む１回以上の用量の抗ウイルス組成物を前記対象に投与することを含み、前記ウイルス感染が、マイナスセンス一本鎖エンベロープＲＮＡウイルス（（－）－ｓｓ－ｅｎｖＲＮＡ－Ｖ）によって引き起こされる、方法。
１９． ウイルス感染に罹るリスクのある対象を治療する予防方法であって、前記対象が前記ウイルス感染に罹る前に長期治療期間にわたって繰り返しの頻度で１回以上の用量の抗ウイルス組成物を前記対象に慢性的に投与し、それによって前記対象が前記ウイルス感染に罹ることを予防することを含み、前記抗ウイルス組成物が、オレアンドリン、ジゴキシンまたはそれらの組み合わせを含み、前記ウイルス感染が、マイナスセンス一本鎖エンベロープＲＮＡウイルス（（－）－ｓｓ－ｅｎｖＲＮＡ－Ｖ）によって引き起こされる、方法。
２０． 前記対象が前記ウイルス感染を有するかどうかを判定することと、
前記抗ウイルス組成物の投与を指示することと、
処方された初期投与計画に従って一定期間、前記抗ウイルス組成物の初期用量を前記対象に投与することと、
前記抗ウイルス組成物による治療に対する対象の臨床応答および／または治療応答の妥当性を定期的に判定することと、
前記対象の臨床応答および／または治療応答が妥当である場合、所望の臨床エンドポイントが達成されるまで、必要に応じて前記抗ウイルス組成物による治療を継続すること、または
前記対象の臨床応答および／または治療応答が前記初期用量および初期投与計画で妥当でない場合、前記対象の所望の臨床応答および／または治療応答が達成されるまで、前記用量を漸増または漸減させることと、を含む、上記１８に記載の方法。
２１． 用量が、前記対象の体重１ｋｇあたり約１００～１０００ｍｇまたは約１００～１０００ｍｉｃｒｏｇの抗ウイルス組成物を含む、上記１８～２０のいずれか一項に記載の方法。
２２． １日あたり投与される前記組成物の一部としてのオレアンドリンの量が、１４０ｍｉｃｒｏｇ～３１５ｍｉｃｒｏｇ、２０ｍｉｃｒｏｇ～７５０ｍｉｃｒｏｇ、１２ｍｉｃｒｏｇ～３００ｍｉｃｒｏｇ、１２ｍｉｃｒｏｇ～１２０ｍｉｃｒｏｇ、０．０１ｍｉｃｒｏｇ～１００ｍｇ、０．０１ｍｉｃｒｏｇ～１００ｍｉｃｒｏｇ、約０．５～約１００ｍｉｃｒｏｇ、約１～約８０ｍｉｃｒｏｇ、約１．５～約６０ｍｉｃｒｏｇ、約１．８～約６０ｍｉｃｒｏｇ、または約１．８～約４０ｍｉｃｒｏｇからなる群から選択される、上記１８～２１のいずれか一項に記載の方法。
２３． 前記抗ウイルス組成物の用量が、１日２回または約１２時間毎投与され、前記用量におけるオレアンドリンの量が、約０．２５～約５０ｍｉｃｒｏｇまたは約０．９～５ｍｉｃｒｏｇである、上記１８～２２のいずれか一項に記載の方法。
２４． 前記抗ウイルス組成物の用量が、１日あたり対象の体重１ｋｇあたりの抗ウイルス組成物の単位量に基づいて、約０．０５～０．５ｍｇ／ｋｇ／日、約０．０５～０．３５ｍｇ／ｋｇ／日、約０．０５～０．２２ｍｇ／ｋｇ／日、約０．０５～０．４ｍｇ／ｋｇ／日、約０．０５～０．３ｍｇ／ｋｇ／日、約０．０５～０．５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．３５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．２２ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．４ｍｉｃｒｏｇ／ｋｇ／日、または約０．０５～０．３ｍｉｃｒｏｇ／ｋｇ／日を含む、上記１８～２３のいずれか一項に記載の方法。
２５． ａ）前記抗ウイルス組成物におけるオレアンドリンの１日用量が、最高で約１００ｍｉｃｒｏｇ／日、約８０ｍｉｃｒｏｇ／日、約６０ｍｉｃｒｏｇ／日、約４０ｍｉｃｒｏｇ／日、約３８．４ｍｉｃｒｏｇ／日もしくは約３０ｍｉｃｒｏｇ／日であり、かつ／またはｂ）前記抗ウイルス組成物におけるオレアンドリンの１日用量が、最小で約０．５ｍｉｃｒｏｇ／日、約１ｍｉｃｒｏｇ／日、約１．５ｍｉｃｒｏｇ／日、約１．８ｍｉｃｒｏｇ／日、約２ｍｉｃｒｏｇ／日、もしくは約５ｍｉｃｒｏｇ／日である、上記１８～２４のいずれか一項に記載の方法。
２６． 前記１回以上の用量の投与後、前記対象におけるオレアンドリンの血漿中濃度が、血漿１ｍＬあたりのオレアンドリンの量として、約０．０５～約２ｎｇ／ｍｌ、約０．００５～約１０ｎｇ／ｍＬ、約０．００５～約８ｎｇ／ｍＬ、約０．０１～約７ｎｇ／ｍＬ、約０．０２～約７ｎｇ／ｍＬ、約０．０３～約６ｎｇ／ｍＬ、約０．０４～約５ｎｇ／ｍＬ、または約０．０５～約２．５ｎｇ／ｍＬの範囲である、上記１８～２５のいずれか一項に記載の方法。
２７． 治療を必要とする対象においてウイルス感染を治療する方法であって、
オレアンドリンを含む１回以上の用量の抗ウイルス組成物を前記対象に投与することを含み、前記ウイルス感染が、マイナスセンス一本鎖エンベロープＲＮＡウイルス（（－）－ｓｓ－ｅｎｖＲＮＡ－Ｖ）によって引き起こされ、
用量が、前記対象の体重１Ｋｇあたり約１００～１０００ｍｇもしくは約１００～１０００ｍｉｃｒｏｇの抗ウイルス組成物を含み；または、１日あたり投与されるオレアンドリンの量が、１４０ｍｉｃｒｏｇ～３１５ｍｉｃｒｏｇ、２０ｍｉｃｒｏｇ～７５０ｍｉｃｒｏｇ、１２ｍｉｃｒｏｇ～３００ｍｉｃｒｏｇ、１２ｍｉｃｒｏｇ～１２０ｍｉｃｒｏｇ、０．０１ｍｉｃｒｏｇ～１００ｍｇ、０．０１ｍｉｃｒｏｇ～１００ｍｉｃｒｏｇ、約０．５～約１００ｍｉｃｒｏｇ、約１～約８０ｍｉｃｒｏｇ、約１．５～約６０ｍｉｃｒｏｇ、約１．８～約６０ｍｉｃｒｏｇ、もしくは約１．８～約４０ｍｉｃｒｏｇからなる群から選択され；または、前記抗ウイルス組成物の用量が、１日あたり対象の体重１ｋｇあたりの抗ウイルス組成物の単位量に基づいて、約０．０５～０．５ｍｇ／ｋｇ／日、約０．０５～０．３５ｍｇ／ｋｇ／日、約０．０５～０．２２ｍｇ／ｋｇ／日、約０．０５～０．４ｍｇ／ｋｇ／日、約０．０５～０．３ｍｇ／ｋｇ／日、約０．０５～０．５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．３５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．２２ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．４ｍｉｃｒｏｇ／ｋｇ／日、もしくは約０．０５～０．３ｍｉｃｒｏｇ／ｋｇ／日を含み；
前記１回以上の用量の投与後、前記対象におけるオレアンドリンの血漿中濃度が、血漿１ｍＬあたりのオレアンドリンの量として、約０．０５～約２ｎｇ／ｍｌ、約０．００５～約１０ｎｇ／ｍＬ、約０．００５～約８ｎｇ／ｍＬ、約０．０１～約７ｎｇ／ｍＬ、約０．０２～約７ｎｇ／ｍＬ、約０．０３～約６ｎｇ／ｍＬ、約０．０４～約５ｎｇ／ｍＬ、または約０．０５～約２．５ｎｇ／ｍＬの範囲である、方法。
２８． 前記（－）－ｓｓ－ｅｎｖＲＮＡＶが、アレナウイルス科ウイルス、ブニヤウイルス科（ブニヤウイルス目）ウイルス、フィロウイルス科ウイルス、オルトミクソウイルス科ウイルス、パラミクソウイルス科ウイルス、およびラブドウイルス科ウイルスからなる群から選択されるウイルスである、上記１８～２７のいずれか一項に記載の方法。
２９． 前記（－）－ｓｓ－ｅｎｖＲＮＡＶが、ラッサウイルス、無菌性髄膜炎、ガナリトウイルス、フニンウイルス、ルジョウイルス、マチュポウイルス、サビアウイルスおよびホワイトウォーターアロヨウイルスからなる群から選択されるアレナウイルス科ウイルスである、上記１８～２８のいずれか一項に記載の方法。
３０． 前記（－）－ｓｓ－ｅｎｖＲＮＡＶが、ハンタウイルス、クリミア－コンゴ出血熱オルトナイロウイルスからなる群から選択されるブニヤウイルス科ウイルスである、上記１８～２８のいずれか一項に記載の方法。
３１． 前記（－）－ｓｓ－ｅｎｖＲＮＡＶが、ムンプスウイルス、ニパウイルス、ヘンドラウイルス、呼吸器多核体ウイルス（ＲＳＶ）、ヒトパラインフルエンザウイルス（ＨＰＩＶ）、およびＮＤＶからなる群から選択されるパラミクソウイルス科ウイルスである、上記１８～２８のいずれか一項に記載の方法。
３２． 前記（－）－ｓｓ－ｅｎｖＲＮＡＶが、インフルエンザウイルス（ＡからＣ）、イサウイルス、トゴトウイルス、クアランジャウイルス、Ｈ１Ｎ１、Ｈ２Ｎ２、Ｈ３Ｎ２、Ｈ１Ｎ２、スペイン風邪、アジア風邪、ホンコン風邪、ソ連風邪からなる群から選択されるオルトミクソウイルス科ウイルスである、上記１８～２８のいずれか一項に記載の方法。
３３． 前記（－）－ｓｓ－ｅｎｖＲＮＡＶが、狂犬病ウイルス、ベシクロウイルス、リッサウイルス、サイトラブドウイルスからなる群から選択されるラブドウイルス科ウイルスである、上記１８～２８のいずれか一項に記載の方法。
３４． ウイルス感染を有する対象に、オレアンドリン、ジゴキシンまたはそれらの組み合わせを含む１回以上の用量の強心配糖体を舌下投与することを含む、本明細書における上記１～３３のいずれか一項に記載のウイルス感染を治療する方法。
３５． コロナウイルス感染を治療する方法であって、前記感染を有する対象に、複数回の治療有効用量のオレアンドリン、ジゴキシンまたはそれらの組み合わせを投与することを含む、方法。
３６． 前記複数回の用量が、１週間あたり２日以上にわたって１日あたり投与される１回以上の用量である、上記３４または３５に記載の方法。
３７． 投与が１カ月あたり１週間以上継続する、上記３６に記載の方法。
３８． 投与が１年あたり１カ月以上継続する、上記３７に記載の方法。
３９． 前記コロナウイルスが、ヒトに対して病原性である、上記３４～３８のいずれか一項に記載の方法。
４０． 前記コロナウイルスが、ＳＡＲＳ－ＣｏＶ、ＭＥＲＳ－ＣｏＶ、ＣＯＶＩＤ－１９（ＳＡＲＳ－ＣｏＶ－２）、ＣｏＶ ２２９Ｅ、ＣｏＶ ＮＬ６３、ＣｏＶ ＯＣ４３、ＣｏＶ ＨＫＵ１、およびＣｏＶ ＨＫＵ２０からなる群から選択される、上記３４～３９のいずれか一項に記載の方法。
４１． 前記対象の血漿におけるオレアンドリンの濃度が、オレアンドリンの投与後、血漿１ｍＬあたりのオレアンドリンの量として、ａ）約１０ｍｉｃｒｏｇ／ｍＬ以下、約５ｍｉｃｒｏｇ／ｍＬ以下、約２．５ｍｉｃｒｏｇ／ｍＬ以下、約２ｍｉｃｒｏｇ／ｍＬ以下、もしくは約１ｍｉｃｒｏｇ／ｍＬ以下；ｂ）約０．０００１ｍｉｃｒｏｇ／ｍＬ以上、約０．０００５ｍｉｃｒｏｇ／ｍＬ以上、約０．００１ｍｉｃｒｏｇ／ｍＬ以上、約０．００１５ｍｉｃｒｏｇ／ｍＬ以上、約０．０１ｍｉｃｒｏｇ／ｍＬ以上、約０．０１５ｍｉｃｒｏｇ／ｍＬ以上、約０．１ｍｉｃｒｏｇ／ｍＬ以上、約０．１５ｍｉｃｒｏｇ／ｍＬ以上、約０．０５ｍｉｃｒｏｇ／ｍＬ以上、もしくは約０．０７５ｍｉｃｒｏｇ／ｍＬ以上；ｃ）項目ａ）の任意の上限と、項目ｃ）の任意の下限との組み合わせ；または、ｄ）約０．０５～約２ｎｇ／ｍｌ、約０．００５～約１０ｎｇ／ｍＬ、約０．００５～約８ｎｇ／ｍＬ、約０．０１～約７ｎｇ／ｍＬ、約０．０２～約７ｎｇ／ｍＬ、約０．０３～約６ｎｇ／ｍＬ、約０．０４～約５ｎｇ／ｍＬ、もしくは約０．０５～約２．５ｎｇ／ｍＬの範囲である、上記３４～４０のいずれか一項に記載の方法。
４２． 用量が、前記対象の体重１Ｋｇあたり約１００～１０００ｍｇまたは約１００～１０００ｍｉｃｒｏｇの抗ウイルス組成物を含む、上記３４～４０のいずれか一項に記載の方法。
４３． １日あたり投与される前記組成物の一部としてのオレアンドリンの量が、１４０ｍｉｃｒｏｇ～３１５ｍｉｃｒｏｇ、２０ｍｉｃｒｏｇ～７５０ｍｉｃｒｏｇ、１２ｍｉｃｒｏｇ～３００ｍｉｃｒｏｇ、１２ｍｉｃｒｏｇ～１２０ｍｉｃｒｏｇ、０．０１ｍｉｃｒｏｇ～１００ｍｇ、０．０１ｍｉｃｒｏｇ～１００ｍｉｃｒｏｇ、約０．５～約１００ｍｉｃｒｏｇ、約１～約８０ｍｉｃｒｏｇ、約１．５～約６０ｍｉｃｒｏｇ、約１．８～約６０ｍｉｃｒｏｇ、または約１．８～約４０ｍｉｃｒｏｇからなる群から選択される、上記３４～４２のいずれか一項に記載の方法。
４４． 前記抗ウイルス組成物の用量が、１日２回または約１２時間毎投与され、前記用量におけるオレアンドリンの量が、約０．２５～約５０ｍｉｃｒｏｇまたは約０．９～５ｍｉｃｒｏｇである、上記３４～４３のいずれか一項に記載の方法。
４５． 前記抗ウイルス組成物の用量が、１日あたり対象の体重１ｋｇあたりの抗ウイルス組成物の単位量に基づいて、約０．０５～０．５ｍｇ／ｋｇ／日、約０．０５～０．３５ｍｇ／ｋｇ／日、約０．０５～０．２２ｍｇ／ｋｇ／日、約０．０５～０．４ｍｇ／ｋｇ／日、約０．０５～０．３ｍｇ／ｋｇ／日、約０．０５～０．５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．３５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．２２ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．４ｍｉｃｒｏｇ／ｋｇ／日、または約０．０５～０．３ｍｉｃｒｏｇ／ｋｇ／日を含む、上記３４～４４のいずれか一項に記載の方法。
４６． ａ）前記抗ウイルス組成物におけるオレアンドリンの１日用量が、最高で約１００ｍｉｃｒｏｇ／日、約８０ｍｉｃｒｏｇ／日、約６０ｍｉｃｒｏｇ／日、約４０ｍｉｃｒｏｇ／日、約３８．４ｍｉｃｒｏｇ／日もしくは約３０ｍｉｃｒｏｇ／日であり、かつ／またはｂ）前記抗ウイルス組成物におけるオレアンドリンの１日用量が、最小で約０．５ｍｉｃｒｏｇ／日、約１ｍｉｃｒｏｇ／日、約１．５ｍｉｃｒｏｇ／日、約１．８ｍｉｃｒｏｇ／日、約２ｍｉｃｒｏｇ／日、もしくは約５ｍｉｃｒｏｇ／日である、上記３４～４５のいずれか一項に記載の方法。
４７． 強心配糖体が、少なくとも２つの投与相：負荷相および維持相、で投与される、上記１～４６のいずれか一項に記載の方法。
４８． 前記強心配糖体が、ジゴキシンであり、前記負荷相および維持相の前記投与計画が、以下の通りである、上記４７に記載の方法。

４９． 前記抗ウイルス組成物が、ａ）オレアンドリン；ｂ）オレアンドリン、オレアノール酸（遊離酸、塩、またはプロドラッグ）およびウルソール酸（遊離酸、塩、またはプロドラッグ）の組み合わせ；ｃ）オレアンドリン、オレアノール酸（遊離酸、塩、またはプロドラッグ）およびベツリン酸（遊離酸、塩、またはプロドラッグ）の組み合わせ；ｄ）オレアンドリン、オレアノール酸（遊離酸、塩、またはプロドラッグ）、ウルソール酸（遊離酸、塩、またはプロドラッグ）およびベツリン酸（遊離酸、塩、またはプロドラッグ）の組み合わせ；ｅ）オレアンドリン、オレアノール酸（遊離酸またはその塩）、ウルソール酸（遊離酸または塩）、およびベツリン酸（遊離酸または）の組み合わせ；またはｆ）オレアンドリン、ならびにオレアノール酸（遊離酸、塩、またはプロドラッグ）、ウルソール酸（遊離酸、塩、またはプロドラッグ）、ベツリン酸（遊離酸、塩、またはプロドラッグ）からなる群から選択される少なくとも２つのトリテルペンの組み合わせを含む、上記１～４８のいずれか一項に記載の方法。
５０． 抗ウイルス組成物の用量において、オレアンドリン対前記トリテルペンのモル比が、以下のいずれかから選択される、上記４９に記載の方法。

５１． 総トリテルペン含有量（オレアノール酸＋ウルソール酸＋ベツリン酸）対オレアンドリンのモル比が、約１５：１～約５：１、または約１２：１～約８：１、または約１００：１～約１５：１、または約１００：１～約５０：１、または約１００：１～約７５：１、または約１００：１～約８０：１、または約１００：１～約９０：１、または約１０：１の範囲である、上記４９に記載の方法。
５２． 個々のトリテルペン（オレアノール酸（ＯＡ）：ウルソール酸（ＵＡ）：ベツリン酸（ＢＡ））対オレアンドリン（ＯＬ）のモル比が、以下の範囲である：２～８（ＯＡ）：２～８（ＵＡ）：０．１～１（ＢＡ）：０．５～１．５（ＯＬ）；または３～６（ＯＡ）：３～６（ＵＡ）：０．３～８（ＢＡ）：０．７～１．２（ＯＬ）；または４～５（ＯＡ）：４～５（ＵＡ）：０．４～０．７（ＢＡ）：０．９～１．１（ＯＬ）；４．６（ＯＡ）：４．４（ＵＡ）：０．６（ＢＡ）：１（ＯＬ）；約９～１２（ＯＡ）：最大約２（ＵＡ）：最大約２、または約１０（ＯＡ）：約１（ＵＡ）：約１、または約９～１２（ＯＡ）：約０．１～２（ＵＡ）：約０．１～２（ＢＡ）、または約９～１１（ＯＡ）：約０．５～１．５（ＵＡ）：約０．５～１．５（ＢＡ）、または約９．５～１０．５（ＯＡ）：約０．７５～１．２５（ＵＡ）：約０．７５～１．２５（ＢＡ）、または約９．５～１０．５（ＯＡ）：約０．８～１．２（ＵＡ）：約０．８～１．２（ＢＡ）、または約９．７５～１０．５（ＯＡ）：約０．９～１．１（ＵＡ）：約０．９～１．１（ＢＡ）、上記４９に記載の方法。
５３． 前記抗ウイルス組成物が、バイオマスの抽出物を含む、上記１～５２のいずれか一項に記載の方法。
５４． 前記抽出物が、熱水抽出、冷水抽出、有機溶媒抽出、超臨界流体抽出、亜臨界液体抽出、またはそれらの組み合わせにより調製される、上記５３に記載の方法。
５５． 前記バイオマスが、Ｎｅｒｉｕｍ種、またはＡｇｒｏｂａｃｔｅｒｉｕｍ種からの植物材料のバイオマスである、上記５３または５４に記載の方法。
５６． 前記抽出物が、オレアンドリン、および前記バイオマスから抽出された１つ以上の化合物の組み合わせを含む、上記５３～５５のいずれか一項に記載の方法。
５７． 前記抽出物が、１つ以上の強心配糖体前駆体、強心配糖体の１つ以上のグリコン構成物質、またはそれらの組み合わせをさらに含む、上記５３～５６のいずれか一項に記載の方法。
５８． 前記抽出物が、オレアンドリン、ならびに強心配糖体、グリコン、アグリコン、ステロイド、トリテルペン、多糖類、糖類、アルカロイド、脂肪、タンパク質、ネリタロシド、オドロシド、オレアノール酸、ウルソール酸、ベツリン酸、オレアンドリゲニン、オレアシドＡ、ベツリン（ウルサ－１２－エン－３β，２８－ジオール）、２８－ノルウルサ－１２－エン－３β－オール、ウルサ－１２－エン－３β－オール、３β，３β－ヒドロキシ－１２－オレアネン－２８－酸、３β，２０α－ジヒドロキシウルサ－２１－エン－２８－酸、３β，２７－ジヒドロキシ－１２－ウルセン－２８－酸、３β，１３β－ジヒドロキシウルサ－１１－エン－２８－酸、３β，１２α－ジヒドロキシオレアナン－２８，１３β－オリド、３β，２７－ジヒドロキシ－１２－オレアナン－２８－酸、ホモポリガラクツロナン、アラビノガラツロナン、クロロゲン酸、コーヒー酸、Ｌ－キナ酸、４－クマロイル－ＣｏＡ、３－Ｏ－カフェオイルキナ酸、５－Ｏ－カフェオイルキナ酸、カルデノリドＢ－１、カルデノリドＢ－２、オレアゲニン、ネリジジノシド、ネリゾシド、オドロシド－Ｈ、ガラクツロン酸、ラムノース、アラビノース、キシロース、およびガラクトースで構成される３－ベータ－Ｏ－（Ｄ－ジギノシル）－５－ベータ，１４ベータ－ジヒドロキシ－カルド－２０（２２）－エノリドペクチン性多糖類、１７０００～１２００００Ｄの範囲のＭＷ、または約３５０００Ｄ、約３０００Ｄ、約５５００Ｄ、もしくは約１２０００ＤのＭＷを有する多糖類、カルデノリドモノグリコシド、カルデノリドＮ－１、カルデノリドＮ－２、カルデノリドＮ－３、カルデノリドＮ－４、プレグナン、４，６－ジエン－３，１２，２０－トリオン、２０Ｒ－ヒドロキシプレグナ－４，６－ジエン－３，１２－ジオン、１６ベータ，１７ベータ－エポキシ－１２ベータ－ヒドロキシプレグナ－４，６－ジエン－３，２０－ジオン、１２ベータ－ヒドロキシプレグナ－４，６，１６－トリエン－３，２０－ジオン（ネリジエノンＡ）、２０Ｓ，２１－ジヒドロキシプレグナ－４，６－ジエン－３，１２－ジオン（ネリジエノンＢ）、ネリウクマル酸、イソネリウクマル酸、オレアンデロン酸、オレアンデレン、８アルファ－メトキシラブダン－１８－酸、１２－ウルセン、カネロシド、ネリウモシド、３β－Ｏ－（Ｄ－ジギノシル）－２α－ヒドロキシ－８，１４β－エポキシ－５β－カルダ－１６：１７，２０：２２－ジエノリド、３β－Ｏ－（Ｄ－ジギノシル）－２α，１４β－ジヒドロキシ－５β－カルダ－１６：１７，２０：２２－ジエノリド、３β，２７－ジヒドロキシ－ウルサ－１８－エン－１３，２８－オリド、３β，２２α，２８－トリヒドロキシ－２５－ノル－ルプ－１（１０），２０（２９）－ジエン－２－オン、ｃｉｓ－カレニン（３β－ヒドロキシ－２８－Ｚ－ｐ－クマロイルオキシ－ウルサ－１２－エン－２７－酸）、ｔｒａｎｓ－カレニン（３－β－ヒドロキシ－２８－Ｅ－ｐ－クマロイルオキシ－ウルサ－１２－エン－２７－酸）、３ベータ－ヒドロキシ－５アルファ－カルダ－１４（１５），２０（２２）－ジエノリド（ベータ－アンヒドロエピジギトキシゲニン）、３ベータ－Ｏ－（Ｄ－ジギタロシル）－２１－ヒドロキシ－５ベータ－カルダ－８，１４，１６，２０（２２）－テトラエノリド（ネリウモゲニン－Ａ－３ベータ－Ｄ－ジギタロシド）、プロセラゲニン、ネリジエノンＡ、３ベータ，２７－ジヒドロキシ－１２－ウルセン－２８－酸、３ベータ，１３ベータ－ジヒドロキシウルサ－１１－エン－２８－酸、３ベータ－ヒドロキシウルサ－１２－エン－２８－アルデヒド、２８－オルウルサ－１２－エン－３ベータ－オール、ウルサ－１２－エン－３ベータ－オール、ウルサ－１２－エン－３ベータ，２８－ジオール、３ベータ，２７－ジヒドロキシ－１２－オレアネン－２８－酸、（２０Ｓ，２４Ｒ）－エポキシダンマラン－３ベータ，２５－ジオール、２０ベータ，２８－エポキシ－２８アルファ－メトキシタラキサステラン－３ベータ－オール、２０ベータ，２８－エポキシタラキサステル－２１－エン－３ベータ－オール、２８－ノル－ウルサ－１２－エン－３ベータ，１７ベータ－ジオール、３ベータ－ヒドロキシウルサ－１２－エン－２８－アルデヒド、アルファ－ネリウルセート、ベータ－ネリウルセート、３アルファ－アセトフェノキシ－ウルサ－１２－エン－２８－酸、３ベータ－アセトフェノキシ－ウルサ－１２－エン－２８－酸、オレアンデロール酸、カネロジオン、３β－ｐ－ヒドロキシフェノキシ－１１α－メトキシ－１２α－ヒドロキシ－２０－ウルセン－２８－酸、２８－ヒドロキシ－２０（２９）－ルペン－３，７－ジオン、カネロシン、３アルファ－ヒドロキシ－ウルサ－１８，２０－ジエン－２８－酸、Ｄ－サルメントース、Ｄ－ジギノース、ネリジジノシド、ネリゾシド、イソリシノール酸、ゲンチオビオシルネリゴシド、ゲンチオビオシルボーモントシド、ゲンチオビオシルオレアンドリン、ホリネリン、１２β－ヒドロキシ－５β－カルダ－８，１４，１６，２０（２２）－テトラエノリド、８β－ヒドロキシ－ジギトキシゲニン、Δ１６－８β－ヒドロキシ－ジギトキシゲニン、Δ１６－ネリアゲニン、ウバオール、ウルソールアルデヒド、２７（ｐ－クマロイルオキシ）ウルソール酸、オレアンデロール、１６－アンヒドロ－デアセチル－ネリゴシド、９－Ｄ－ヒドロキシ－ｃｉｓ－１２－オクタデカン酸、アジゴシド、アジネリン、アルファ－アミリン、ベータ－シトステロール、カンペステロール、カウチューク、カプリン酸、カプリル酸、コリン、コルネリン、コルテネリン、デアセチルオレアンドリン、ジアセチル－ネリゴシド、ホリアンドリン、プソイドクラミン、ケルセチン、ケルセチン－３－ラムノグルコシド、ケルシトリン、ロサギニン、ルチン、ステアリン酸、スチグマステロール、ストロスペシド、ウレヒトキシン、およびウザリゲニンからなる群から選択される１つ以上の化合物を含む、上記５３～５７のいずれか一項に記載の方法。
５９． 前記抽出物の１日用量が、最高で約１００ｍｉｃｒｏｇ／日、約８０ｍｉｃｒｏｇ／日、約６０ｍｉｃｒｏｇ／日、約４０ｍｉｃｒｏｇ／日、約３８．４ｍｉｃｒｏｇ／日または約３０ｍｉｃｒｏｇ／日である、上記５３～５８のいずれか一項に記載の方法。
６０． 前記抽出物の１日用量が、最小で０．５ｍｉｃｒｏｇ／日、約１ｍｉｃｒｏｇ／日、約１．５ｍｉｃｒｏｇ／日、約１．８ｍｉｃｒｏｇ／日、約２ｍｉｃｒｏｇ／日、もしくは約５ｍｉｃｒｏｇ／日である、上記５２～５８のいずれか一項に記載の方法。
６１． 前記対象の血液または血漿中のウイルス力価が、前記治療の結果として低減されるかまたは増加しない、上記１～６０のいずれか一項に記載の方法。
６２． 前記投与が、全身、非経口、頬側、経腸、筋肉内、皮下、舌下、経口、口腔、またはそれらの組み合わせである、上記１～６１のいずれか一項に記載の方法。
６３． １回以上の用量が、毎日、毎週、または毎月の頻度で投与される、上記１～６２のいずれか一項に記載の方法。
６４． 前記抗ウイルス組成物が、感染直後、または感染後１日から５日以内の任意の時間、またはウイルス感染の確定診断後の最も早い時間に投与される、上記１～６３のいずれか一項に記載の方法。
６５． 前記抗ウイルス組成物は、一次抗ウイルス療法、補助抗ウイルス療法、または併用抗ウイルス療法として投与される、上記１～６４のいずれか一項に記載の方法。
６６． 前記投与が、前記抗ウイルス組成物の少なくとも１つの他の抗ウイルス組成物、または前記ウイルス感染に関連する症状を治療するための少なくとも１つの他の組成物との別々の投与もしくは同時投与を含む、上記１～６５のいずれか一項に記載の方法。
６７． 前記症状が、炎症、嘔吐、悪心、頭痛、発熱、下痢、悪心、蕁麻疹、結膜炎、倦怠感、筋肉痛、関節痛、発作、および麻痺からなる群から選択される、上記６６に記載の方法。
６８． オレアンドリン、ジゴキシンまたはそれらの組み合わせを含む、舌下剤形。
６９． 少なくとも１つの液体担体をさらに含む、上記６８に記載の剤形。
７０． オレアンドリンを含み、Ｎｅｒｉｕｍ ｓｐ．またはＴｈｅｖｅｔｉａ ｓｐ．のバイオマスの亜臨界液体抽出により調製されている、抽出物
７１． 前記亜臨界液体が、二酸化炭素を含み、アルコールをさらに含んでもよい、上記７０に記載の抽出物。
７２． オレアンドリンを含む抽出物を調製する方法であって、オレアンドリン含有バイオマスに、アルコールをさらに含んでもよい亜臨界液体二酸化炭素を用いて、オレアンドリンを前記バイオマスから抽出するのに十分な期間抽出を行うことを含む、方法。
７３． オレアンドリンを含む抽出物を調製する方法であって、
オレアンドリン含有バイオマスを、亜臨界液体二酸化炭素を含み、アルコールをさらに含んでもよい抽出液で、オレアンドリンを前記バイオマスから抽出するのに十分な期間抽出することと、
前記バイオマスを前記抽出液から分離する一方、前記抽出液を保持することと、次いで
抽出液を除去し、それにより前記抽出物を形成することと、を含む、方法。
７４． オレアンドリン含有抽出物の組み合わせを生成する方法であって、
第１のオレアンドリン含有バイオマスに有機溶媒抽出を行って、オレアンドリン含有有機溶媒抽出物を得ることと、
第２のオレアンドリン含有バイオマスに亜臨界液体（ＳｂＣＬ）抽出（ＳｂＣＬＥ）を行って、オレアンドリン含有ＳｂＣＬ抽出物を得ることと、
前記オレアンドリン含有有機溶媒抽出物および前記オレアンドリン含有ＳｂＣＬ抽出物を組み合わせて、前記組み合わせたオレアンドリン含有抽出物を得ることと、を含む、方法。
７５． 本明細書に定義されるオレアンドリン含有有機溶媒抽出物の一部、および本明細書に定義されるオレアンドリン含有ＳｂＣＬ抽出物の一部を含む、改善されたオレアンドリン含有組成物。
７６． コロナウイルス感染を治療する方法であって、それを必要とする対象に、少なくとも約５日～約１カ月の治療期間にわたって１日あたり複数回用量のオレアンドリンを投与することを含み、オレアンドリンが、オレアンドリン含有バイオマスから得られる少なくとも１つの抽出物を含む薬学的組成物に存在する、方法。
７７． １日あたりのオレアンドリンの総用量が、約１ｍｉｃｒｏｇ～約１８０ｍｉｃｒｏｇ、約１ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約５ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ～約８０ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ～約７５ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約８０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約３０ｍｉｃｒｏ～約９０ｍｉｃｒｏｇ、約４０ｍｉｃｒｏｇ～約７０ｍｉｃｒｏｇ、約１ｍｉｃｒｏｇ、約５ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ、約２０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ、約４０ｍｉｃｒｏｇ、約５０ｍｉｃｒｏｇ、約６０ｍｉｃｒｏｇ、約７０ｍｉｃｒｏｇ、約８０ｍｉｃｒｏｇ、約９０ｍｉｃｒｏｇ、約１００ｍｉｃｒｏｇ、約１１０ｍｉｃｒｏｇ、約１２０ｍｉｃｒｏｇ、約１３０ｍｉｃｒｏｇ、約１４０ｍｉｃｒｏｇ、約１５０ｍｉｃｒｏｇ、約１６０ｍｉｃｒｏｇ、約１７０ｍｉｃｒｏｇ、または約１８０ｍｉｃｒｏｇから、各発生時に独立して選択される、上記７６に記載の方法。
７８． 前記１日あたり複数回用量のオレアンドリンが、１日あたり２～１０回、１日あたり２～８回、１日あたり２～６回、１日あたり２～４回、１日あたり２回、１日あたり３回、１日あたり４回、１日あたり５回、１日あたり６回、１日あたり７回、１日あたり８回、１日あたり９回、または１日あたり１０回である、上記７６または７７に記載の方法。
７９． 前記薬学的組成物が、Ｎｅｒｉｕｍ種から得られる少なくとも２つの抽出物を含む、上記７６～７８のいずれか一項に記載の方法。
８０． 前記抽出物が、熱水抽出物、溶媒抽出物、超臨界流体抽出物、および亜臨界液体抽出物からなる群から、各発生時に独立して選択される、上記７９に記載の方法。
８１． 本明細書に記載されるウイルス感染を治療する方法。
８２． 本明細書に記載される組成物。
８３． 本明細書に記載される抗ウイルス組成物。
８４． 本明細書に記載されるオレアンドリン含有組成物。
８５． コロナウイルス感染を治療する方法であって、それを必要とする対象に、１日あたり１回以上の用量のオレアンドリンを、少なくとも約５日間～約１カ月以上の治療期間投与することを含み、前記コロナウイルス感染が、ヒトに対して病原性である、方法。
８６． 前記コロナウイルスが、ＳＡＲＳ－ＣｏＶ、ＭＥＲＳ－ＣｏＶ、ＣＯＶＩＤ－１９（ＳＡＲＳ－ＣｏＶ－２）、ＣｏＶ ２２９Ｅ、ＣｏＶ ＮＬ６３、ＣｏＶ ＯＣ４３、ＣｏＶ ＨＫＵ１、およびＣｏＶ ＨＫＵ２０からなる群から選択される、上記８５に記載の方法。
８７． １日あたりのオレアンドリンの総用量が、約１ｍｉｃｒｏｇ～約１８０ｍｉｃｒｏｇ、約１ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約５ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ～約８０ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ～約７５ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約８０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約３０ｍｉｃｒｏ～約９０ｍｉｃｒｏｇ、約４０ｍｉｃｒｏｇ～約７０ｍｉｃｒｏｇ、約１ｍｉｃｒｏｇ、約５ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ、約２０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ、約４０ｍｉｃｒｏｇ、約５０ｍｉｃｒｏｇ、約６０ｍｉｃｒｏｇ、約７０ｍｉｃｒｏｇ、約８０ｍｉｃｒｏｇ、約９０ｍｉｃｒｏｇ、約１００ｍｉｃｒｏｇ、約１１０ｍｉｃｒｏｇ、約１２０ｍｉｃｒｏｇ、約１３０ｍｉｃｒｏｇ、約１４０ｍｉｃｒｏｇ、約１５０ｍｉｃｒｏｇ、約１６０ｍｉｃｒｏｇ、約１７０ｍｉｃｒｏｇ、約１８０ｍｉｃｒｏｇ、約１４０ｍｉｃｒｏｇ～約３１５ｍｉｃｒｏｇ、約２０ｍｉｃｒｏｇ～約７５０ｍｉｃｒｏｇ、約１２ｍｉｃｒｏｇ～約３００ｍｉｃｒｏｇ、約１２ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約０．０１ｍｉｃｒｏｇ～約１００ｍｇ、約０．０１ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約０．５～約１００ｍｉｃｒｏｇ、約１～約８０ｍｉｃｒｏｇ、約１．５～約６０ｍｉｃｒｏｇ、約１．８～約６０ｍｉｃｒｏｇ、および約１．８～約４０ｍｉｃｒｏｇから、各発生時に独立して選択される、上記８５に記載の方法。
８８． 用量が、１日あたり対象の体重１ｋｇあたりのオレアンドリン単位量に基づいて、約０．０５～０．５ｍｇ／ｋｇ／日、約０．０５～０．３５ｍｇ／ｋｇ／日、約０．０５～０．２２ｍｇ／ｋｇ／日、約０．０５～０．４ｍｇ／ｋｇ／日、約０．０５～０．３ｍｇ／ｋｇ／日、約０．０５～０．５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．３５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．２２ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．４ｍｉｃｒｏｇ／ｋｇ／日、または約０．０５～０．３ｍｉｃｒｏｇ／ｋｇ／日を含む、上記８５に記載の方法。
８９． ａ）オレアンドリンの１日用量が、最高で約１００ｍｉｃｒｏｇ／日、約８０ｍｉｃｒｏｇ／日、約６０ｍｉｃｒｏｇ／日、約４０ｍｉｃｒｏｇ／日、約３８．４ｍｉｃｒｏｇ／日もしくは約３０ｍｉｃｒｏｇ／日であり、かつ／またはｂ）オレアンドリンの１日用量が、最小で０．５ｍｉｃｒｏｇ／日、約１ｍｉｃｒｏｇ／日、約１．５ｍｉｃｒｏｇ／日、約１．８ｍｉｃｒｏｇ／日、約２ｍｉｃｒｏｇ／日、もしくは約５ｍｉｃｒｏｇ／日である、上記８５に記載の方法。
９０． 用量が、１日２回または約１２時間毎投与され、前記用量におけるオレアンドリンの量が、約０．２５～約５０ｍｉｃｒｏｇまたは約０．９～１５ｍｉｃｒｏｇである、上記８５に記載の方法。
９１． 前記１日あたり１回以上の用量のオレアンドリンが、１日あたり２～１０回、１日あたり２～８回、１日あたり２～６回、１日あたり２～４回、１日あたり２回、１日あたり３回、１日あたり４回、１日あたり５回、１日あたり６回、１日あたり７回、１日あたり８回、１日あたり９回、または１日あたり１０回である、上記８５に記載の方法。
９２． オレアンドリンが、オレアンドリン含有バイオマスから得られる少なくとも１つの抽出物を含む組成物に存在する、上記８５に記載の方法。
９３． 前記抽出物が、熱水抽出物、溶媒抽出物、および超臨界流体抽出物からなる群から、各発生時に独立して選択される、上記９２に記載の方法。
９４． 前記１回以上の用量の投与後、前記対象におけるオレアンドリンの血漿中濃度が、血漿１ｍＬあたりのオレアンドリンの量として、約０．０５～約２ｎｇ／ｍｌ、約０．００５～約１０ｎｇ／ｍＬ、約０．００５～約８ｎｇ／ｍＬ、約０．０１～約７ｎｇ／ｍＬ、約０．０２～約７ｎｇ／ｍＬ、約０．０３～約６ｎｇ／ｍＬ、約０．０４～約５ｎｇ／ｍＬ、または約０．０５～約２．５ｎｇ／ｍＬの範囲である、上記８５に記載の方法。
９５． コロナウイルス感染を治療する方法であって、前記感染を有する対象に、複数回の治療有効用量のオレアンドリンまたはオレアンドリン含有組成物を投与することを含む、方法。
９６． 前記複数回の治療有効用量が、１週間あたり２日以上にわたって１日あたり１回以上投与される、上記９５に記載の方法。
９７． 投与が１カ月あたり１週間以上継続する、上記９６に記載の方法。
９８． 投与が１年あたり１カ月以上継続する、上記９７に記載の方法。
９９． 前記コロナウイルスが、ヒトに対して病原性である、上記９５に記載の方法。
１００． 前記コロナウイルスが、ＳＡＲＳ－ＣｏＶ、ＭＥＲＳ－ＣｏＶ、ＣＯＶＩＤ－１９（ＳＡＲＳ－ＣｏＶ－２）、ＣｏＶ ２２９Ｅ、ＣｏＶ ＮＬ６３、ＣｏＶ ＯＣ４３、ＣｏＶ ＨＫＵ１、およびＣｏＶ ＨＫＵ２０からなる群から選択される、上記９９に記載の方法。
１０１． オレアンドリンが、オレアンドリン含有バイオマスから得られる少なくとも１つの抽出物を含む組成物に存在する、上記９５に記載の方法。
１０２． 前記抽出物が、熱水抽出物、溶媒抽出物、および超臨界流体抽出物からなる群から、各発生時に独立して選択される、上記１０１に記載の方法。
１０３． 前記抽出物が、オレアンドリンおよび前記バイオマスから抽出された１つ以上の化合物の組み合わせを含む、上記１０１に記載の方法。
１０４． 前記１つ以上の化合物が、１つ以上の強心配糖体前駆体、強心配糖体の１つ以上のグリコン構成物質、またはそれらの組み合わせを含む、上記１０３に記載の方法。
１０５． 前記投与が、全身、非経口、頬側、経腸、筋肉内、皮下、舌下、経口、口腔、またはそれらの組み合わせである、上記９５に記載の方法。
１０６． 前記抗ウイルス組成物が、感染直後、または感染後１日から５日以内の任意の時間、またはウイルス感染の確定診断後の最も早い時間に投与される、上記９５に記載の方法。
１０７． 前記抗ウイルス組成物が、一次抗ウイルス療法、補助抗ウイルス療法、もしくは併用抗ウイルス療法として投与され、または前記投与が、前記組成物の少なくとも１つの他の抗ウイルス組成物、もしくは前記ウイルス感染に関連する症状を治療するための少なくとも１つの他の組成物との別々の投与もしくは同時投与を含む、上記９５に記載の方法。
１０８． １日あたりのオレアンドリンの総用量が、約１ｍｉｃｒｏｇ～約１８０ｍｉｃｒｏｇ、約１ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約５ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ～約８０ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ～約７５ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約１５ｍｉｃｒｏｇ～約８０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約３０ｍｉｃｒｏ～約９０ｍｉｃｒｏｇ、約４０ｍｉｃｒｏｇ～約７０ｍｉｃｒｏｇ、約１ｍｉｃｒｏｇ、約５ｍｉｃｒｏｇ、約１０ｍｉｃｒｏｇ、約２０ｍｉｃｒｏｇ、約３０ｍｉｃｒｏｇ、約４０ｍｉｃｒｏｇ、約５０ｍｉｃｒｏｇ、約６０ｍｉｃｒｏｇ、約７０ｍｉｃｒｏｇ、約８０ｍｉｃｒｏｇ、約９０ｍｉｃｒｏｇ、約１００ｍｉｃｒｏｇ、約１１０ｍｉｃｒｏｇ、約１２０ｍｉｃｒｏｇ、約１３０ｍｉｃｒｏｇ、約１４０ｍｉｃｒｏｇ、約１５０ｍｉｃｒｏｇ、約１６０ｍｉｃｒｏｇ、約１７０ｍｉｃｒｏｇ、約１８０ｍｉｃｒｏｇ、約１４０ｍｉｃｒｏｇ～約３１５ｍｉｃｒｏｇ、約２０ｍｉｃｒｏｇ～約７５０ｍｉｃｒｏｇ、約１２ｍｉｃｒｏｇ～約３００ｍｉｃｒｏｇ、約１２ｍｉｃｒｏｇ～約１２０ｍｉｃｒｏｇ、約０．０１ｍｉｃｒｏｇ～約１００ｍｇ、約０．０１ｍｉｃｒｏｇ～約１００ｍｉｃｒｏｇ、約０．５～約１００ｍｉｃｒｏｇ、約１～約８０ｍｉｃｒｏｇ、約１．５～約６０ｍｉｃｒｏｇ、約１．８から約６０ｍｉｃｒｏｇ、および約１．８～約４０ｍｉｃｒｏｇから、各発生時に独立して選択される、上記９５に記載の方法。
１０９． 用量が、１日あたり対象の体重１ｋｇあたりのオレアンドリン単位量に基づいて、約０．０５～０．５ｍｇ／ｋｇ／日、約０．０５～０．３５ｍｇ／ｋｇ／日、約０．０５～０．２２ｍｇ／ｋｇ／日、約０．０５～０．４ｍｇ／ｋｇ／日、約０．０５～０．３ｍｇ／ｋｇ／日、約０．０５～０．５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．３５ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．２２ｍｉｃｒｏｇ／ｋｇ／日、約０．０５～０．４ｍｉｃｒｏｇ／ｋｇ／日、または約０．０５～０．３ｍｉｃｒｏｇ／ｋｇ／日を含む、上記９５に記載の方法。
１１０． ａ）オレアンドリンの１日用量が、最高で約１００ｍｉｃｒｏｇ／日、約８０ｍｉｃｒｏｇ／日、約６０ｍｉｃｒｏｇ／日、約４０ｍｉｃｒｏｇ／日、約３８．４ｍｉｃｒｏｇ／日もしくは約３０ｍｉｃｒｏｇ／日であり、かつ／またはｂ）オレアンドリンの１日用量が、最小で０．５ｍｉｃｒｏｇ／日、約１ｍｉｃｒｏｇ／日、約１．５ｍｉｃｒｏｇ／日、約１．８ｍｉｃｒｏｇ／日、約２ｍｉｃｒｏｇ／日、もしくは約５ｍｉｃｒｏｇ／日である、上記９５に記載の方法。
１１１． 用量が、１日２回または約１２時間毎投与され、前記用量におけるオレアンドリンの量が、約０．２５～約５０ｍｉｃｒｏｇまたは約０．９～１５ｍｉｃｒｏｇである、上記９５に記載の方法。
１１２． 前記１日あたり１回以上の用量のオレアンドリンが、１日あたり２～１０回、１日あたり２～８回、１日あたり２～６回、１日あたり２～４回、１日あたり２回、１日あたり３回、１日あたり４回、１日あたり５回、１日あたり６回、１日あたり７回、１日あたり８回、１日あたり９回、または１日あたり１０回である、上記９５に記載の方法。
１１３． 前記１回以上の用量の投与後、前記対象におけるオレアンドリンの血漿中濃度が、血漿１ｍＬあたりのオレアンドリンの量として、約０．０５～約２ｎｇ／ｍｌ、約０．００５～約１０ｎｇ／ｍＬ、約０．００５～約８ｎｇ／ｍＬ、約０．０１～約７ｎｇ／ｍＬ、約０．０２～約７ｎｇ／ｍＬ、約０．０３～約６ｎｇ／ｍＬ、約０．０４～約５ｎｇ／ｍＬ、または約０．０５～約２．５ｎｇ／ｍＬの範囲である、上記９５に記載の方法。
１１４． 前記抽出物が、オレアンドリン、ならびに強心配糖体、グリコン、アグリコン、ステロイド、トリテルペン、多糖類、糖類、アルカロイド、脂肪、タンパク質、ネリタロシド、オドロシド、オレアノール酸、ウルソール酸、ベツリン酸、オレアンドリゲニン、オレアシドＡ、ベツリン（ウルサ－１２－エン－３β，２８－ジオール）、２８－ノルウルサ－１２－エン－３β－オール、ウルサ－１２－エン－３β－オール、３β，３β－ヒドロキシ－１２－オレアネン－２８－酸、３β，２０α－ジヒドロキシウルサ－２１－エン－２８－酸、３β，２７－ジヒドロキシ－１２－ウルセン－２８－酸、３β，１３β－ジヒドロキシウルサ－１１－エン－２８－酸、３β，１２α－ジヒドロキシオレアナン－２８，１３β－オリド、３β，２７－ジヒドロキシ－１２－オレアナン－２８－酸、ホモポリガラクツロナン、アラビノガラツロナン、クロロゲン酸、コーヒー酸、Ｌ－キナ酸、４－クマロイル－ＣｏＡ、３－Ｏ－カフェオイルキナ酸、５－Ｏ－カフェオイルキナ酸、カルデノリドＢ－１、カルデノリドＢ－２、オレアゲニン、ネリジジノシド、ネリゾシド、オドロシド－Ｈ、ガラクツロン酸、ラムノース、アラビノース、キシロース、およびガラクトースで構成される３－ベータ－Ｏ－（Ｄ－ジギノシル）－５－ベータ，１４ベータ－ジヒドロキシ－カルド－２０（２２）－エノリドペクチン性多糖類、１７０００～１２００００Ｄの範囲のＭＷ、または約３５０００Ｄ、約３０００Ｄ、約５５００Ｄ、もしくは約１２０００ＤのＭＷを有する多糖類、カルデノリドモノグリコシド、カルデノリドＮ－１、カルデノリドＮ－２、カルデノリドＮ－３、カルデノリドＮ－４、プレグナン、４，６－ジエン－３，１２，２０－トリオン、２０Ｒ－ヒドロキシプレグナ－４，６－ジエン－３，１２－ジオン、１６ベータ，１７ベータ－エポキシ－１２ベータ－ヒドロキシプレグナ－４，６－ジエン－３，２０－ジオン、１２ベータ－ヒドロキシプレグナ－４，６，１６－トリエン－３，２０－ジオン（ネリジエノンＡ）、２０Ｓ，２１－ジヒドロキシプレグナ－４，６－ジエン－３，１２－ジオン（ネリジエノンＢ）、ネリウクマル酸、イソネリウクマル酸、オレアンデロン酸、オレアンデレン、８アルファ－メトキシラブダン－１８－酸、１２－ウルセン、カネロシド、ネリウモシド、３β－Ｏ－（Ｄ－ジギノシル）－２α－ヒドロキシ－８，１４β－エポキシ－５β－カルダ－１６：１７，２０：２２－ジエノリド、３β－Ｏ－（Ｄ－ジギノシル）－２α，１４β－ジヒドロキシ－５β－カルダ－１６：１７，２０：２２－ジエノリド、３β，２７－ジヒドロキシ－ウルサ－１８－エン－１３，２８－オリド、３β，２２α，２８－トリヒドロキシ－２５－ノル－ルプ－１（１０），２０（２９）－ジエン－２－オン、ｃｉｓ－カレニン（３β－ヒドロキシ－２８－Ｚ－ｐ－クマロイルオキシ－ウルサ－１２－エン－２７－酸）、ｔｒａｎｓ－カレニン（３－β－ヒドロキシ－２８－Ｅ－ｐ－クマロイルオキシ－ウルサ－１２－エン－２７－酸）、３ベータ－ヒドロキシ－５アルファ－カルダ－１４（１５），２０（２２）－ジエノリド（ベータ－アンヒドロエピジギトキシゲニン）、３ベータ－Ｏ－（Ｄ－ジギタロシル）－２１－ヒドロキシ－５ベータ－カルダ－８，１４，１６，２０（２２）－テトラエノリド（ネリウモゲニン－Ａ－３ベータ－Ｄ－ジギタロシド）、プロセラゲニン、ネリジエノンＡ、３ベータ，２７－ジヒドロキシ－１２－ウルセン－２８－酸、３ベータ，１３ベータ－ジヒドロキシウルサ－１１－エン－２８－酸、３ベータ－ヒドロキシウルサ－１２－エン－２８－アルデヒド、２８－オルウルサ－１２－エン－３ベータ－オール、ウルサ－１２－エン－３ベータ－オール、ウルサ－１２－エン－３ベータ，２８－ジオール、３ベータ，２７－ジヒドロキシ－１２－オレアネン－２８－酸、（２０Ｓ，２４Ｒ）－エポキシダンマラン－３ベータ，２５－ジオール、２０ベータ，２８－エポキシ－２８アルファ－メトキシタラキサステラン－３ベータ－オール、２０ベータ，２８－エポキシタラキサステル－２１－エン－３ベータ－オール、２８－ノル－ウルサ－１２－エン－３ベータ，１７ベータ－ジオール、３ベータ－ヒドロキシウルサ－１２－エン－２８－アルデヒド、アルファ－ネリウルセート、ベータ－ネリウルセート、３アルファ－アセトフェノキシ－ウルサ－１２－エン－２８－酸、３ベータ－アセトフェノキシ－ウルサ－１２－エン－２８－酸、オレアンデロール酸、カネロジオン、３β－ｐ－ヒドロキシフェノキシ－１１α－メトキシ－１２α－ヒドロキシ－２０－ウルセン－２８－酸、２８－ヒドロキシ－２０（２９）－ルペン－３，７－ジオン、カネロシン、３アルファ－ヒドロキシ－ウルサ－１８，２０－ジエン－２８－酸、Ｄ－サルメントース、Ｄ－ジギノース、ネリジジノシド、ネリゾシド、イソリシノール酸、ゲンチオビオシルネリゴシド、ゲンチオビオシルボーモントシド、ゲンチオビオシルオレアンドリン、ホリネリン、１２β－ヒドロキシ－５β－カルダ－８，１４，１６，２０（２２）－テトラエノリド、８β－ヒドロキシ－ジギトキシゲニン、Δ１６－８β－ヒドロキシ－ジギトキシゲニン、Δ１６－ネリアゲニン、ウバオール、ウルソールアルデヒド、２７（ｐ－クマロイルオキシ）ウルソール酸、オレアンデロール、１６－アンヒドロ－デアセチル－ネリゴシド、９－Ｄ－ヒドロキシ－ｃｉｓ－１２－オクタデカン酸、アジゴシド、アジネリン、アルファ－アミリン、ベータ－シトステロール、カンペステロール、カウチューク、カプリン酸、カプリル酸、コリン、コルネリン、コルテネリン、デアセチルオレアンドリン、ジアセチル－ネリゴシド、ホリアンドリン、プソイドクラミン、ケルセチン、ケルセチン－３－ラムノグルコシド、ケルシトリン、ロサギニン、ルチン、ステアリン酸、スチグマステロール、ストロスペシド、ウレヒトキシン、およびウザリゲニンからなる群から選択される１つ以上の化合物を含む、上記１１３に記載の方法。 The above is a detailed description of a particular embodiment of the invention. Although specific embodiments of the invention have been described herein for illustrative purposes, it should be appreciated that various modifications can be made without departing from the spirit and scope of the invention. Therefore, the present invention is not limited except by the scope of the appended claims. All of the embodiments disclosed and claimed herein can be created and performed in the light of the present disclosure without undue experimentation.
Next, another preferred embodiment of the present invention will be shown.
1. 1. A method of treating a viral infection in a subject in need of treatment, comprising administering to the subject one or more doses of an antiviral composition comprising oleandrin, digoxin, or a combination thereof. A method in which viral infection is caused by Plussense single-stranded enveloped RNA virus ((+)-ss-envRNA-V).
2. 2. A prophylactic method of treating a subject at risk of contracting a virus, wherein the subject is chronically given one or more doses of an antiviral composition at a repetitive frequency over a long-term treatment period prior to the subject becoming infected with the virus. The antiviral composition comprises oleandrin, digoxin, or a combination thereof, wherein the viral infection comprises a positive sense. A method caused by a single-stranded envelope RNA virus ((+)-ss-envRNA-V).
3. 3. Determining if the subject has the virus infection and
Instructing the administration of the antiviral composition and
To administer the initial dose of the antiviral composition to the subject for a period of time according to the prescribed initial dosing regimen.
Periodically determining the adequacy of the subject's clinical and / or therapeutic response to treatment with the antiviral composition.
If the clinical and / or therapeutic response of the subject is reasonable, continue treatment with the antiviral composition as needed, or continue treatment with the antiviral composition until the desired clinical endpoint is achieved.
If the subject's clinical and / or therapeutic response is not valid at the initial dose and initial dosing regimen, the dose may be increased or decreased until the desired clinical and / or therapeutic response of the subject is achieved. , The method according to 1 above.
4. The method according to any one of 1 to 3 above, wherein the dose comprises an antiviral composition of about 100 to 1000 mg or about 100 to 1000 microg per 1 kg of body weight of the subject.
5. The amount of oleandrin as part of the composition administered daily is 140 microg to 315 microg, 20 microg to 750 microg, 12 microg to 300 microlog, 12 microg to 120 microlog, 0.01 microg to 100 mg, 0.01 microg to 100 microg, about. 1. The method described in any one of the items.
6. The dose of the antiviral composition is administered twice daily or about every 12 hours, and the amount of oleandrin in the dose is about 0.25 to about 50 micrologs or about 0.9 to 5 micrologs. The method according to any one of 5 to 5.
7. The dose of the antiviral composition is about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg / day based on the unit amount of the antiviral composition per kg body weight of the subject per day. kg / day, about 0.05 to 0.22 mg / kg / day, about 0.05 to 0.4 mg / kg / day, about 0.05 to 0.3 mg / kg / day, about 0.05 to 0. 5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0.05 The method according to any one of 1 to 6 above, which comprises ~ 0.3 microg / kg / day.
8. a) The daily dose of oleandrin in the antiviral composition is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day or about 30 microg / day. , And / or b) The daily dose of oleandrin in the antiviral composition is a minimum of 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg. The method according to any one of 1 to 7 above, which is / day or about 5 microg / day.
9. The method according to any one of 1 to 8 above, wherein the cardiac glycoside is administered in at least two administration phases: a loading phase and a maintenance phase.
10. 9. The method according to 9 above, wherein the cardiac glycoside is digoxin and the administration regimen of the loaded phase and the maintenance phase is as follows.

11. After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng The method according to any one of 1 to 10 above, which is in the range of / mL or about 0.05 to about 2.5 ng / mL.
12. A method of treating a viral infection in a subject in need of treatment, comprising administering to the subject one or more doses of an antiviral composition comprising oleandrin, wherein the viral infection is a plus sense. Caused by the double-stranded envelope RNA virus ((+)-ss-envRNA-V)
The dose comprises an antiviral composition of about 100-1000 mg or about 100-1000 microg per kg of body weight of the subject; or the amount of oleandrin administered per day is 140 microg-315 microlog, 20 microg-750 microrog, 12 microg. ~ 300 microg, 12 microg ~ 120 microg, 0.01 microg ~ 100 mg, 0.01 microg ~ 100 microg, about 0.5 ~ about 100 microg, about 1 ~ about 80 microg, about 1.5 ~ about 60 microg, about 1.8 ~ about 60 microg, or Selected from the group consisting of about 1.8 to about 40 micrologs; or the dose of the antiviral composition is from about 0.05 to about 0.05 to about 0.05 per day based on the unit amount of the antiviral composition per kg body weight of the subject. 0.5 mg / kg / day, about 0.05 to 0.35 mg / kg / day, about 0.05 to 0.22 mg / kg / day, about 0.05 to 0.4 mg / kg / day, about 0. 05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about Includes 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day;
After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng The method, which is in the range of / mL, or about 0.05 to about 2.5 ng / mL.
13. The method according to any one of 1 to 12 above, wherein the (+)-ss-envRNAV is a virus selected from the group consisting of Coronaviridae, Flaviviridae, Togaviridae, and Alterviridae. ..
14. The method according to any one of 1 to 13 above, wherein the (+)-ss-envRNAV is a coronavirus pathogenic to humans.
15. 14 above, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU 20. The method described.
16. The (+)-ss-envRNAV is flavivirus, yellow fever virus, dengue fever virus, Japanese encephalitis virus, western Nile virus, decavirus, tick-mediated encephalitis virus, Casanur forest disease virus, Alkhurma disease virus, omsk. The method according to any one of 1 to 13 above, which is a virus selected from the group consisting of hemorrhagic fever virus and poissan virus.
17. The (+)-ss-envRNAV is arbovirus, eastern horse encephalomyelitis virus (EEEV), western horse encephalomyelitis virus (WEEV), Venezuelan elephant encephalomyelitis virus (VEEV), chicungnia virus (CHIKV), o. Described in any one of 1 to 13 above, which is a Togavirus family virus selected from the group consisting of Nyonnyon virus (ONNV), Pogosta disease virus, Sindbis virus, Ross River fever virus (RRV) and Semuliki forest virus. the method of.
18. A method of treating a viral infection in a subject in need of treatment, comprising administering to the subject one or more doses of an antiviral composition comprising oleandrin, digoxin or a combination thereof, said virus. A method in which infection is caused by a negative sense single-stranded enveloped RNA virus ((-)-ss-envRNA-V).
19. A prophylactic method of treating a subject at risk of contracting a virus, wherein the subject is chronically given one or more doses of an antiviral composition at a repetitive frequency over a long-term treatment period prior to the subject becoming infected with the virus. The antiviral composition comprises oleandrin, digoxin or a combination thereof, comprising the prevention of the subject from contracting the viral infection, wherein the viral infection is negative sense. A method caused by a double-stranded enveloped RNA virus ((-)-ss-envRNA-V).
20. Determining if the subject has the virus infection and
Instructing the administration of the antiviral composition and
To administer the initial dose of the antiviral composition to the subject for a period of time according to the prescribed initial dosing regimen.
Periodically determining the adequacy of the subject's clinical and / or therapeutic response to treatment with the antiviral composition.
If the clinical and / or therapeutic response of the subject is reasonable, continue treatment with the antiviral composition as needed, or continue treatment with the antiviral composition until the desired clinical endpoint is achieved.
If the subject's clinical and / or therapeutic response is not valid at the initial dose and initial dosing regimen, the dose may be increased or decreased until the desired clinical and / or therapeutic response of the subject is achieved. , The method according to 18 above.
21. 18. The method of any one of 18-20 above, wherein the dose comprises an antiviral composition of about 100-1000 mg or about 100-1000 micrologs per kg body weight of the subject.
22. The amount of oleandrin as part of the composition administered daily is 140 microg to 315 microg, 20 microg to 750 microg, 12 microg to 300 microlog, 12 microg to 120 microlog, 0.01 microg to 100 mg, 0.01 microg to 100 microg, about. 18-21 above, selected from the group consisting of 0.5-about 100 microg, about 1-about 80 microg, about 1.5-about 60 microg, about 1.8-about 60 microg, or about 1.8-about 40 microg. The method described in any one of the items.
23. 18 above, wherein the dose of the antiviral composition is administered twice daily or about every 12 hours, and the amount of oleandrin in the dose is about 0.25 to about 50 microlog or about 0.9 to 5 microg. The method according to any one of 22 to 22.
24. The dose of the antiviral composition is about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg, based on the unit amount of the antiviral composition per kg body weight of the subject per day. / Kg / day, about 0.05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0 .5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0. The method according to any one of 18 to 23 above, which comprises 05 to 0.3 microg / kg / day.
25. a) The daily dose of oleandrin in the antiviral composition is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day or about 30 microg / day. And / or b) The daily dose of oleandrin in the antiviral composition is at least about 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day. , The method according to any one of 18 to 24 above, which is about 2 microg / day or about 5 microg / day.
26. After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng The method according to any one of 18 to 25 above, which is in the range of / mL or about 0.05 to about 2.5 ng / mL.
27. A method of treating a viral infection in a subject in need of treatment.
Containing the administration of one or more doses of the antiviral composition comprising oleandrin to the subject, the viral infection is caused by a negative sense single-stranded envelope RNA virus ((-)-ss-envRNA-V). Caused,
The dose comprises an antiviral composition of about 100-1000 mg or about 100-1000 microg per kg of body weight of the subject; or the amount of oleandrin administered per day is 140 microg-315 microlog, 20 microg-750 microrog, 12 microg. ~ 300 microg, 12 microg ~ 120 microg, 0.01 microg ~ 100 mg, 0.01 microg ~ 100 microg, about 0.5 ~ about 100 microg, about 1 ~ about 80 microg, about 1.5 ~ about 60 microg, about 1.8 ~ about 60 microg, or Selected from the group consisting of about 1.8 to about 40 micrologs; or the dose of the antiviral composition is from about 0.05 to about 0.05 to about 0.05 per day based on the unit amount of the antiviral composition per kg body weight of the subject. 0.5 mg / kg / day, about 0.05 to 0.35 mg / kg / day, about 0.05 to 0.22 mg / kg / day, about 0.05 to 0.4 mg / kg / day, about 0. 05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about Includes 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day;
After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng The method, which is in the range of / mL, or about 0.05 to about 2.5 ng / mL.
28. The (-)-ss-envRNAV is selected from the group consisting of arenavirus family virus, bunyavirus family (Bunyavirus order) virus, phyllovirus family virus, orthomyxovirus family virus, paramyxovirus family virus, and rabdovirus family virus. The method according to any one of 18 to 27 above, which is a virus to be used.
29. The (-)-ss-envRNAV is an arenavirus selected from the group consisting of lassavirus, aseptic meningitis, ganarit virus, funine virus, lujovirus, machupovirus, savia virus and whitewater aroyovirus. The method according to any one of 18 to 28 above, which is a family virus.
30. The method according to any one of 18 to 28 above, wherein the (-)-ss-envRNAV is a Bunyaviridae virus selected from the group consisting of hantavirus and Crimean-Congo hemorrhagic fever ortho-nail virus.
31. Paramyxoviridae virus selected from the group consisting of mumps virus, nipavirus, Hendra virus, respiratory polynuclear virus (RSV), human parainfluenza virus (HPIV), and NDV. The method according to any one of 18 to 28 above.
32. The (-)-ss-envRNAV consists of influenza virus (A to C), Isavirus, Togotovirus, Quaranger virus, H1N1, H2N2, H3N2, H1N2, Spanish cold, Asian cold, Hong Kong cold, Soviet cold. The method according to any one of 18 to 28 above, which is an Orthomyxoviridae virus selected from the group.
33. The method according to any one of 18 to 28 above, wherein the (-)-ss-envRNAV is a rhabdoviridae virus selected from the group consisting of rabies virus, becyclovirus, lyssavirus, and cytorabdovirus.
34. Any one of 1-33 above herein comprising sublingually administering to a subject having a viral infection one or more doses of cardiac glycosides comprising oleandrin, digoxin or a combination thereof. How to treat a viral infection as described in.
35. A method of treating a coronavirus infection comprising administering to a subject having the infection multiple therapeutically effective doses of oleandrin, digoxin or a combination thereof.
36. 34 or 35 above, wherein the plurality of doses is one or more doses administered per day over two or more days per week.
37. 36. The method according to 36 above, wherein the administration is continued for one week or more per month.
38. 37. The method according to 37 above, wherein the administration is continued for 1 month or more per year.
39. The method according to any one of 34 to 38 above, wherein the coronavirus is pathogenic to humans.
40. 34- The method according to any one of 39.
41. The concentration of oleandrin in the plasma of the subject is, after administration of oleandrin, as the amount of oleandrin per 1 mL of plasma, a) about 10 microg / mL or less, about 5 microg / mL or less, about 2.5 microg / mL. Hereinafter, about 2 microg / mL or less, or about 1 microg / mL or less; b) about 0.0001 microg / mL or more, about 0.0005 microg / mL or more, about 0.001 microg / mL or more, about 0.0015 microg / mL or more, about 0.0015 microg / mL or more. 0.01 microg / mL or more, about 0.015 microg / mL or more, about 0.1 microg / mL or more, about 0.15 microg / mL or more, about 0.05 microg / mL or more, or about 0.075 microg / mL or more; c) A combination of any upper limit of item a) and any lower limit of item c); or d) about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng / mL, about 0.005 to about. 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng / mL, or about 0.05 to The method according to any one of 34 to 40 above, which is in the range of about 2.5 ng / mL.
42. 35. The method of any one of 34-40 above, wherein the dose comprises an antiviral composition of about 100-1000 mg or about 100-1000 microg per kg of body weight of the subject.
43. The amount of oleandrin as part of the composition administered daily is 140 microg to 315 microg, 20 microg to 750 microg, 12 microg to 300 microlog, 12 microg to 120 microlog, 0.01 microg to 100 mg, 0.01 microg to 100 microg, about. 34-42 above, selected from the group consisting of 0.5-about 100 microg, about 1-about 80 microg, about 1.5-about 60 microg, about 1.8-about 60 microg, or about 1.8-about 40 microg. The method described in any one of the items.
44. 34. The method according to any one of 43.
45. The dose of the antiviral composition is about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg, based on the unit amount of the antiviral composition per kg body weight of the subject per day. / Kg / day, about 0.05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0 .5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0. The method according to any one of 34 to 44 above, comprising 05 to 0.3 microg / kg / day.
46. a) The daily dose of oleandrin in the antiviral composition is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day or about 30 microg / day. And / or b) The daily dose of oleandrin in the antiviral composition is at least about 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day. , The method according to any one of 34 to 45 above, which is about 2 microg / day or about 5 microg / day.
47. The method according to any one of 1 to 46 above, wherein the cardiac glycoside is administered in at least two administration phases: a loading phase and a maintenance phase.
48. 47. The method of 47 above, wherein the cardiac glycoside is digoxin and the dosing regimen for the loading and maintenance phases is as follows.

49. The antiviral composition is a) oleandrin; b) a combination of oleandrin, oleanolic acid (free acid, salt, or prodrug) and ursoleic acid (free acid, salt, or prodrug); c) ole. A combination of andrin, oleanolic acid (free acid, salt, or prodrug) and bethuric acid (free acid, salt, or prodrug); d) oleandrin, oleanolic acid (free acid, salt, or prodrug), Combination of ursoleic acid (free acid, salt, or prodrug) and bethuric acid (free acid, salt, or prodrug); e) oleandrin, oleanolic acid (free acid or salt thereof), ursoleic acid (free acid or salt thereof) A combination of salt) and bethuric acid (free acid or); or f) oleandrin, and oleanolic acid (free acid, salt, or prodrug), ursoric acid (free acid, salt, or prodrug), bethuric acid. The method according to any one of 1 to 48 above, comprising a combination of at least two triterpenes selected from the group consisting of (free acid, salt, or prodrug).
50. 49. The method of 49 above, wherein the molar ratio of oleandrin to the triterpene at a dose of antiviral composition is selected from any of the following:

51. The molar ratio of total triterpene content (oleanolic acid + ursolic acid + bethulinic acid) to oleandrin is 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 49. The method of 49 above, which is in the range of about 10: 1.
52. The molar ratio of individual triterpenes (oleanolic acid (OA): ursolic acid (UA): bethuric acid (BA)) to oleandrin (OL) is in the following range: 2-8 (OA): 2-8. (UA): 0.1 to 1 (BA): 0.5 to 1.5 (OL); or 3 to 6 (OA): 3 to 6 (UA): 0.3 to 8 (BA): 0. 7 to 1.2 (OL); or 4 to 5 (OA): 4 to 5 (UA): 0.4 to 0.7 (BA): 0.9 to 1.1 (OL); 4.6 ( OA): 4.4 (UA): 0.6 (BA): 1 (OL); about 9-12 (OA): up to about 2 (UA): up to about 2, or about 10 (OA): about 1 (UA): about 1, or about 9-12 (OA): about 0.1-2 (UA): about 0.1-2 (BA), or about 9-11 (OA): about 0.5- 1.5 (UA): about 0.5 to 1.5 (BA), or about 9.5 to 10.5 (OA): about 0.75 to 1.25 (UA): about 0.75 to 1. .25 (BA), or about 9.5 to 10.5 (OA): about 0.8 to 1.2 (UA): about 0.8 to 1.2 (BA), or about 9.75 to 10. .5 (OA): Approximately 0.9 to 1.1 (UA): Approximately 0.9 to 1.1 (BA), the method according to 49 above.
53. The method according to any one of 1 to 52 above, wherein the antiviral composition comprises an extract of biomass.
54. 53. The method of 53 above, wherein the extract is prepared by hot water extraction, cold water extraction, organic solvent extraction, supercritical fluid extraction, subcritical liquid extraction, or a combination thereof.
55. 53 or 54 above, wherein the biomass is a biomass of plant material from a Nerium species, or Agrobacterium species.
56. The method according to any one of 53 to 55 above, wherein the extract comprises a combination of oleandrin and one or more compounds extracted from the biomass.
57. 5. The method of any one of 53-56 above, wherein the extract further comprises one or more cardiac glycoside precursors, one or more cardiac glycoside constituents of cardiac glycosides, or combinations thereof. ..
58. The extracts are oleandrin, as well as oleandrins, glycosides, aglycones, steroids, triterpenes, polysaccharides, sugars, alkaloids, fats, proteins, neritarosides, odrosides, oleanolic acids, ursoleic acids, bethuric acids, oleandrigenins. , Oleacid A, Betulin (Ursa-12-en-3β, 28-diol), 28-Noruursa-12-en-3β-ol, Ursa-12-en-3β-ol, 3β, 3β-hydroxy-12-oleanen -28-acid, 3β, 20α-dihydroxyursa-21-en-28-acid, 3β, 27-dihydroxy-12-ursen-28-acid, 3β, 13β-dihydroxyursa-11-en-28-acid, 3β , 12α-dihydroxyoleanan-28,13β-oride, 3β,27-dihydroxy-12-oleanan-28-acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, coffee acid, L-quinic acid, 4 -Kumaloyl-CoA, 3-O-cafe oil quinic acid, 5-O-cafe oil quinic acid, cardenolide B-1, cardenolide B-2, oleagenin, nerididinenoside, nerisoside, odroside-H, galacturonic acid, ramnorth, arabinose, 3-Beta-O- (D-diginosyl) -5-beta, 14 beta-dihydroxy-cardo-20 (22) -enolidepectin glycoside composed of xylose and galactose, MW in the range of 17,000 to 120,000 D , Or a polysaccharide having a MW of about 35,000D, about 3000D, about 5500D, or about 12000D, cardenolide monoglycoside, cardenolide N-1, cardenolide N-2, cardenolide N-3, cardenolide N-4, pregnane, 4, , 6-Diene-3,12,20-Trione, 20R-Hydroxypregna-4,6-Diene-3,12-Zion, 16 Beta, 17 Beta-Epoxy-12 Beta-Hydroxypregna-4,6- Diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A), 20S, 21-dihydroxypregna-4,6-diene-3,12 -Dione (neridienone B), neriukumalic acid, isoneriukumalic acid, oleanderonic acid, oleandelene, 8alpha-methoxylabdan-18-acid, 12-ursen, caneroside, neriumoside, 3β-O- (D-diginosyl) -2α -Hydroxy-8,14β-Epoxy-5β-Carda-16: 17, 20:22-Dienolide, 3β-O- (D-diginosyl) -2α, 14β-Dihydroxy-5β-Carda-16: 17, 20:22 -Dienolide, 3β, 27-dihydroxy-Ursa-18-en-13,28-oride, 3β, 22α, 28-trihydroxy-25-norlurp-1 (10), 20 (29) -dien-2- On, cis-calenin (3β-hydroxy-28-Z-z-p-kumaloyloxy-ursa-12-ene-27-acid), trans-calenin (3-β-hydroxy-28-E-p-kumaroyloxy) -Ursa-12-en-27-acid), 3beta-hydroxy-5alpha-carda-14 (15), 20 (22) -dienolide (beta-anhydroepidigitoxygenin), 3beta-O- ( D-Digitarocil) -21-Hydroxy-5 Beta-Carda-8,14,16,20 (22) -Tetraenolide (neriumogenin-A-3 beta-D-digitaroside), proceragenin, neridienone A, 3 beta, 27-dihydroxy -12-Ursen-28-acid, 3beta, 13 beta-dihydroxyursa-11-en-28-acid, 3beta-hydroxyursa-12-en-28-aldehyde, 28-olursa-12-en-3 beta -Oll, Ursa-12-En-3 Beta-Ol, Ursa-12-En-3 Beta, 28-diol, 3beta, 27-Dihydroxy-12-oleanen-28-acid, (20S, 24R) -Epoxydan Malang-3 Beta, 25-diol, 20 Beta, 28-Epoxy-28 Alpha-methoxytalaxasteran-3 Beta-Ol, 20 Beta, 28-Epoxy Taraxaster-21-En-3 Beta-Ol, 28- Nor-Ursa-12-En-3 Beta, 17 Beta-Dolyl, 3 Beta-Hydroxy Ursa-12-En-28-aldehyde, Alpha-Neriursate, Beta-Neriursate, 3Alpha-Acetphenoxy-Ursa-12-En- 28-acid, 3beta-acetophenoxy-ursa-12-ene-28-acid, oleanderolic acid, canelology, 3β-p-hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursen-28-acid, 28-Hydroxy-20 (29) -Lupen-3,7-Dione, Canerosin, 3Alpha-Hydroxy-Urser- 18,20-dien-28-acid, D-salmentose, D-diginose, nerididinoside, nerisoside, isolicinoleic acid, gentiobiosylnerigoside, gentiobiosylvomontside, gentiobiosyloleandrin, holineline, 12β-hydroxy-5β-carda-8,14,16,20 (22) -tetraenolide, 8β-hydroxy-digitoxygenin, Δ16-8β-hydroxy-digitoxygenin, Δ16-neriagenin, uvaol, ursolealdehyde, 27 (p-kumaroyl). Oxy) Ursolic acid, oleanderol, 16-anhydro-deacetyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, azigoside, aginerin, alpha-amyrin, beta-citosterol, campesterol, kauchuk, capric acid, Caprylic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl-nerigoside, horiandrin, pseudoclamin, kercetin, kercetin-3-ramnoglucoside, cercitrin, rosaginine, rutin, stearic acid, stigmasterol, strospeside, urechtxin, The method according to any one of 53 to 57 above, comprising one or more compounds selected from the group consisting of and usaligenin.
59. The above 53-58, wherein the daily dose of the extract is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day or about 30 microg / day. The method described in any one of the items.
60. The daily dose of the extract is at least 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg / day, or about 5 microg / day. The method according to any one of 52 to 58.
61. The method according to any one of 1 to 60 above, wherein the viral titer in the blood or plasma of the subject is reduced or not increased as a result of the treatment.
62. The method according to any one of 1 to 61 above, wherein the administration is systemic, parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, oral, oral, or a combination thereof.
63. The method according to any one of 1 to 62 above, wherein one or more doses are administered at a daily, weekly, or monthly frequency.
64. The item 1 to 63 above, wherein the antiviral composition is administered immediately after infection, at any time within 1 to 5 days after infection, or at the earliest time after a definitive diagnosis of viral infection. The method described.
65. The method according to any one of 1 to 64 above, wherein the antiviral composition is administered as a primary antiviral therapy, an adjuvant antiviral therapy, or a combined antiviral therapy.
66. The administration comprises separate or co-administration with at least one other antiviral composition of the antiviral composition, or at least one other composition for treating symptoms associated with the viral infection. , The method according to any one of 1 to 65 above.
67. 66. The method of 66 above, wherein the symptom is selected from the group consisting of inflammation, vomiting, nausea, headache, fever, diarrhea, nausea, urticaria, conjunctivitis, malaise, muscle pain, arthralgia, seizures, and paralysis. ..
68. A sublingual form containing oleandrin, digoxin or a combination thereof.
69. 68. The dosage form according to 68 above, further comprising at least one liquid carrier.
70. Containing oleandrin, Nerium sp. Or Thevetia sp. Extracts prepared by subcritical liquid extraction of biomass in
71. The extract according to 70 above, wherein the subcritical liquid contains carbon dioxide and may further contain alcohol.
72. A method for preparing an extract containing oleandrin, which is sufficient to extract oleandrin from the biomass using subcritical liquid carbon dioxide which may further contain alcohol in the oleandrin-containing biomass. A method that involves performing a period extraction.
73. A method of preparing an extract containing oleandrin,
Extracting the oleandrin-containing biomass with an extract containing subcritical liquid carbon dioxide, which may further contain alcohol, for a period sufficient to extract oleandrin from the biomass.
Retaining the extract while separating the biomass from the extract, and then
A method comprising removing an extract and thereby forming the extract.
74. A method for producing a combination of oleandrin-containing extracts.
The first oleandrin-containing biomass was subjected to organic solvent extraction to obtain an oleandrin-containing organic solvent extract.
Subcritical liquid (SbCL) extraction (SbCLE) was performed on the second oleandrin-containing biomass to obtain an oleandrin-containing SbCL extract.
A method comprising combining the oleandrin-containing organic solvent extract and the oleandrin-containing SbCL extract to obtain the combined oleandrin-containing extract.
75. An improved oleandrin-containing composition comprising a portion of the oleandrin-containing organic solvent extract as defined herein and a portion of the oleandrin-containing SbCL extract as defined herein.
76. A method of treating a coronavirus infection, which comprises administering to subjects in need thereof multiple doses of oleandrin per day over a treatment period of at least about 5 days to about 1 month. A method in which phosphorus is present in a pharmaceutical composition comprising at least one extract obtained from oleandrin-containing biomass.
77. The total daily dose of oleandrin is about 1 microg to about 180 microg, about 1 microg to about 120 microg, about 5 microg to about 100 microg, about 10 microlog to about 80 microlog, about 10 microlog to about 75 microg, about 15 microg to about 120 microg. ~ About 100 microg, about 15 microg ~ about 80 microg, about 30 microg ~ about 120 microg, about 30 microlog ~ about 100 microlog, about 30 microlog ~ about 90 microlog, about 40 microlog ~ about 70 microlog, about 1 microlog, about 5 microg, about 10 microg, about 10 microg 40 micrologs, about 50 micrologs, about 60 micrologs, about 70 micrologs, about 80 micrologs, about 90 micrologs, about 100 micrologs, about 110 micrologs, about 120 micrologs, about 130 micrologs, about 140 micrologs, about 150 micrologs, about 160 micrologs 76. The method according to 76 above.
78. The multiple doses of oleandrin per day are 2 to 10 times per day, 2 to 8 times per day, 2 to 6 times per day, 2 to 4 times per day, and 2 times per day. 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or 10 times a day The method according to 76 or 77 above.
79. The method according to any one of 76 to 78 above, wherein the pharmaceutical composition comprises at least two extracts obtained from the Nerium species.
80. 79. The method of 79, wherein the extract is independently selected from the group consisting of hot water extract, solvent extract, supercritical fluid extract, and subcritical liquid extract at each outbreak.
81. A method of treating a viral infection described herein.
82. The compositions described herein.
83. The antiviral composition described herein.
84. The oleandrin-containing compositions described herein.
85. A method of treating a coronavirus infection, which comprises administering to a subject in need thereof a dose of oleandrin at least once a day for a treatment period of at least about 5 days to about 1 month or more. , The method by which the coronavirus infection is pathogenic to humans.
86. 85 above, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU 20. The method described.
87. The total daily dose of oleandrin is about 1 microg to about 180 microg, about 1 microg to about 120 microg, about 5 microg to about 100 microg, about 10 microlog to about 80 microlog, about 10 microlog to about 75 microg, about 15 microg to about 120 microg. ~ About 100 microg, about 15 microg ~ about 80 microg, about 30 microg ~ about 120 microg, about 30 microlog ~ about 100 microlog, about 30 microlog ~ about 90 microlog, about 40 microlog ~ about 70 microlog, about 1 microlog, about 5 microg, about 10 microg, about 10 microg 40 microg, about 50 microg, about 60 microg, about 70 microg, about 80 microg, about 90 microg, about 100 microlog, about 110 microlog, about 120 microlog, about 130 microlog, about 140 microlog, about 150 microg, about 160 microg, about 170 microg, about 170 microg About 20 micrologs to about 750 micrologs, about 12 micrologs to about 300 micrologs, about 12 micrologs to about 120 micrologs, about 0.01 micrologs to about 100 mg, about 0.01 micrologs to about 100 micrologs, about 0.5 to about 100 micrologs, about 1 to about 80 micrologs, about 1 The method according to 85 above, which is independently selected at each outbreak from .5 to about 60 microg, from about 1.8 to about 60 microg, and from about 1.8 to about 40 microg.
88. Dosages were about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg / kg / day, about 0. 05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day The method according to 85 above, including the day.
89. a) The daily dose of oleandrin is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microlog / day or about 30 microg / day and / or. b) The daily dose of oleandrin is a minimum of 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg / day, or about 5 microg / day. , The method according to 85 above.
90. 85. The method of 85 above, wherein the dose is administered twice daily or about every 12 hours and the amount of oleandrin in said dose is about 0.25 to about 50 micrologs or about 0.9 to 15 microrogs.
91. The above-mentioned dose of oleandrin at least once a day is 2 to 10 times a day, 2 to 8 times a day, 2 to 6 times a day, 2 to 4 times a day, and a day. 2 times, 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or 10 times a day The method according to 85 above, which is a time.
92. 85. The method of 85 above, wherein oleandrin is present in a composition comprising at least one extract obtained from oleandrin-containing biomass.
93. 92. The method of 92 above, wherein the extract is independently selected from the group consisting of hot water extract, solvent extract, and supercritical fluid extract at each outbreak.
94. After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng 85, wherein the method is in the range of / mL, or about 0.05 to about 2.5 ng / mL.
95. A method of treating a coronavirus infection, comprising administering to the subject having the infection a plurality of therapeutically effective doses of oleandrin or an oleandrin-containing composition.
96. 95. The method of 95, wherein the plurality of therapeutically effective doses are administered at least once per day over a period of 2 days or more per week.
97. The method according to 96 above, wherein the administration is continued for one week or more per month.
98. The method according to 97 above, wherein the administration is continued for 1 month or more per year.
99. 95. The method of 95, wherein the coronavirus is pathogenic to humans.
100. The above 99, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU 20. The method described.
101. 95. The method of 95 above, wherein oleandrin is present in a composition comprising at least one extract obtained from oleandrin-containing biomass.
102. 10. The method of 101 above, wherein the extract is independently selected from the group consisting of hot water extract, solvent extract, and supercritical fluid extract at each occurrence.
103. 101. The method of 101, wherein the extract comprises a combination of oleandrin and one or more compounds extracted from the biomass.
104. 10. The method of 103 above, wherein the one or more compounds comprises one or more cardiac glycoside precursors, one or more cardiac glycoside constituents of cardiac glycosides, or combinations thereof.
105. 95. The method of 95 above, wherein the administration is systemic, parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, oral, oral, or a combination thereof.
106. 95. The method of 95, wherein the antiviral composition is administered immediately after infection, at any time within 1-5 days after infection, or at the earliest time after a definitive diagnosis of viral infection.
107. The antiviral composition is administered as primary antiviral therapy, adjuvant antiviral therapy, or combined antiviral therapy, or said administration to at least one other antiviral composition of said composition, or said virus infection. 95. The method of 95 above, comprising separate or co-administration with at least one other composition for treating the associated symptom.
108. The total daily dose of oleandrin is about 1 microg to about 180 microg, about 1 microg to about 120 microg, about 5 microg to about 100 microg, about 10 microlog to about 80 microlog, about 10 microlog to about 75 microg, about 15 microg to about 120 microg. ~ About 100 microg, about 15 microg ~ about 80 microg, about 30 microg ~ about 120 microg, about 30 microlog ~ about 100 microlog, about 30 microlog ~ about 90 microlog, about 40 microlog ~ about 70 microlog, about 1 microlog, about 5 microg, about 10 microg, about 10 microg 40 microg, about 50 microg, about 60 microg, about 70 microg, about 80 microg, about 90 microg, about 100 microlog, about 110 microlog, about 120 microlog, about 130 microlog, about 140 microlog, about 150 microg, about 160 microg, about 170 microg, about 170 microg About 20 micrologs to about 750 micrologs, about 12 micrologs to about 300 micrologs, about 12 micrologs to about 120 micrologs, about 0.01 micrologs to about 100 mg, about 0.01 micrologs to about 100 micrologs, about 0.5 to about 100 micrologs, about 1 to about 80 micrologs, about 1 The method according to 95 above, which is independently selected at each outbreak from .5 to about 60 microg, from about 1.8 to about 60 microg, and from about 1.8 to about 40 microg.
109. Dosages were about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg / kg / day, about 0. 05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day The method according to 95 above, including the day.
110. a) The daily dose of oleandrin is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microlog / day or about 30 microg / day and / or. b) The daily dose of oleandrin is a minimum of 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg / day, or about 5 microg / day. , The method according to 95 above.
111. 95. The method of 95 above, wherein the dose is administered twice daily or about every 12 hours and the amount of oleandrin in said dose is about 0.25 to about 50 micrologs or about 0.9 to 15 microrogs.
112. The above-mentioned dose of oleandrin at least once a day is 2 to 10 times a day, 2 to 8 times a day, 2 to 6 times a day, 2 to 4 times a day, and a day. 2 times, 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or 10 times a day The method according to 95 above, which is a time.
113. After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng 95. The method of 95 above, which is in the range of / mL, or about 0.05 to about 2.5 ng / mL.
114. The extracts are oleandrin, as well as oleandrin, glycosides, glycosides, aglycones, steroids, triterpenes, polysaccharides, sugars, alkaloids, fats, proteins, neritarosides, odrosides, oleanolic acids, ursoleic acids, bethuric acids, oleandrigenins. , Oleacid A, Betulin (Ursa-12-en-3β, 28-diol), 28-Noruursa-12-en-3β-ol, Ursa-12-en-3β-ol, 3β, 3β-hydroxy-12-oleanen -28-acid, 3β, 20α-dihydroxyursa-21-en-28-acid, 3β, 27-dihydroxy-12-ursen-28-acid, 3β, 13β-dihydroxyursa-11-en-28-acid, 3β , 12α-dihydroxyoleanan-28,13β-oride, 3β,27-dihydroxy-12-oleanan-28-acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, coffee acid, L-quinic acid, 4 -Kumaloyl-CoA, 3-O-cafe oil quinic acid, 5-O-cafe oil quinic acid, cardenolide B-1, cardenolide B-2, oleagenin, nerididinenoside, nerisoside, odroside-H, galacturonic acid, ramnorth, arabinose, 3-Beta-O- (D-diginosyl) -5-beta, 14 beta-dihydroxy-cardo-20 (22) -enolidepectin glycoside composed of xylose and galactose, MW in the range of 17,000 to 120,000 D , Or a polysaccharide having a MW of about 35,000D, about 3000D, about 5500D, or about 12000D, cardenolide monoglycoside, cardenolide N-1, cardenolide N-2, cardenolide N-3, cardenolide N-4, pregnane, 4, , 6-Diene-3,12,20-Trione, 20R-Hydroxypregna-4,6-Diene-3,12-Zion, 16 Beta, 17 Beta-Epoxy-12 Beta-Hydroxypregna-4,6- Diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A), 20S, 21-dihydroxypregna-4,6-diene-3,12 -Dione (neridienone B), neriukumalic acid, isoneriukumalic acid, oleanderonic acid, oleandelene, 8alpha-methoxylabdan-18-acid, 12-ursen, caneroside, neriumoside, 3β-O- (D-diginosyl) -2α -Hydroxy-8,14β-Epoxy-5β-Carda-16: 17, 20:22-Dienolide, 3β-O- (D-diginosyl) -2α, 14β-Dihydroxy-5β-Carda-16: 17, 20:22 -Dienolide, 3β, 27-dihydroxy-Ursa-18-en-13,28-oride, 3β, 22α, 28-trihydroxy-25-norlurp-1 (10), 20 (29) -dien-2- On, cis-calenin (3β-hydroxy-28-Z-z-p-kumaloyloxy-ursa-12-ene-27-acid), trans-calenin (3-β-hydroxy-28-E-p-kumaroyloxy) -Ursa-12-en-27-acid), 3beta-hydroxy-5alpha-carda-14 (15), 20 (22) -dienolide (beta-anhydroepidigitoxygenin), 3beta-O- ( D-Digitarocil) -21-Hydroxy-5 Beta-Carda-8,14,16,20 (22) -Tetraenolide (neriumogenin-A-3 beta-D-digitaroside), proceragenin, neridienone A, 3 beta, 27-dihydroxy -12-Ursen-28-acid, 3beta, 13 beta-dihydroxyursa-11-en-28-acid, 3beta-hydroxyursa-12-en-28-aldehyde, 28-olursa-12-en-3 beta -Oll, Ursa-12-En-3 Beta-Ol, Ursa-12-En-3 Beta, 28-diol, 3beta, 27-Dihydroxy-12-oleanen-28-acid, (20S, 24R) -Epoxydan Malang-3 Beta, 25-diol, 20 Beta, 28-Epoxy-28 Alpha-methoxytalaxasteran-3 Beta-Ol, 20 Beta, 28-Epoxy Taraxaster-21-En-3 Beta-Ol, 28- Nor-Ursa-12-En-3 Beta, 17 Beta-Dolyl, 3 Beta-Hydroxy Ursa-12-En-28-aldehyde, Alpha-Neriursate, Beta-Neriursate, 3Alpha-Acetphenoxy-Ursa-12-En- 28-acid, 3beta-acetophenoxy-ursa-12-ene-28-acid, oleanderolic acid, canelology, 3β-p-hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursen-28-acid, 28-Hydroxy-20 (29) -Lupen-3,7-Dione, Canerosin, 3Alpha-Hydroxy-Urser- 18,20-dien-28-acid, D-salmentose, D-diginose, nerididinoside, nerisoside, isolicinoleic acid, gentiobiosylnerigoside, gentiobiosylvomontside, gentiobiosyloleandrin, holineline, 12β-hydroxy-5β-carda-8,14,16,20 (22) -tetraenolide, 8β-hydroxy-digitoxygenin, Δ16-8β-hydroxy-digitoxygenin, Δ16-neriagenin, uvaol, ursolealdehyde, 27 (p-kumaroyl). Oxy) Ursolic acid, oleanderol, 16-anhydro-deacetyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, azigoside, aginerin, alpha-amyrin, beta-citosterol, campesterol, kauchuk, capric acid, Capricic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl-nerigoside, hollyandrin, pseudoclamin, kercetin, kercetin-3-ramnoglucoside, cercitrin, rosaginine, rutin, stearic acid, stigmasterol, strospeside, urechtxin, And the method according to 113 above, comprising one or more compounds selected from the group consisting of usaligenin.

Claims (114)

A method of treating a viral infection in a subject in need of treatment, comprising administering to the subject one or more doses of an antiviral composition comprising oleandrin, digoxin, or a combination thereof. A method in which viral infection is caused by Plussense single-stranded enveloped RNA virus ((+)-ss-envRNA-V). A prophylactic method of treating a subject at risk of contracting a virus, wherein the subject is chronically given one or more doses of an antiviral composition at a repetitive frequency over a long-term treatment period prior to the subject becoming infected with the virus. The antiviral composition comprises oleandrin, digoxin, or a combination thereof, wherein the viral infection comprises a positive sense. A method caused by a single-stranded envelope RNA virus ((+)-ss-envRNA-V). Determining if the subject has the virus infection and
Instructing the administration of the antiviral composition and
To administer the initial dose of the antiviral composition to the subject for a period of time according to the prescribed initial dosing regimen.
Periodically determining the adequacy of the subject's clinical and / or therapeutic response to treatment with the antiviral composition.
If the subject's clinical and / or therapeutic response is reasonable, continue treatment with the antiviral composition as needed, or continue with the subject's clinical response and / or subject until the desired clinical endpoint is achieved. / Or, if the therapeutic response is not appropriate for the initial dose and initial dosing regimen, the dosage comprises escalating or tapering until the desired clinical and / or therapeutic response of the subject is achieved. The method according to 1. The method of any one of claims 1-3, wherein the dose comprises an antiviral composition of about 100-1000 mg or about 100-1000 microg per kg of body weight of the subject. The amount of oleandrin as part of the composition administered daily is 140 microg to 315 microg, 20 microg to 750 microg, 12 microg to 300 microg, 12 microg to 120 microlog, 0.01 microg to 100 mg, 0.01 microg to 100 microg, about. Claims 1-4, selected from the group consisting of 0.5 to about 100 microg, about 1 to about 80 microg, about 1.5 to about 60 microg, about 1.8 to about 60 microg, or about 1.8 to about 40 microg. The method according to any one of the above. Claim that the dose of the antiviral composition is administered twice daily or about every 12 hours and the amount of oleandrin in the dose is about 0.25 to about 50 micrologs or about 0.9 to 5 micrologs. The method according to any one of 1 to 5. The dose of the antiviral composition is about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg / day based on the unit amount of the antiviral composition per kg body weight of the subject per day. kg / day, about 0.05 to 0.22 mg / kg / day, about 0.05 to 0.4 mg / kg / day, about 0.05 to 0.3 mg / kg / day, about 0.05 to 0. 5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0.05 The method according to any one of claims 1 to 6, which comprises ~ 0.3 microg / kg / day. a) The daily dose of oleandrin in the antiviral composition is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day or about 30 microg / day. , And / or b) The daily dose of oleandrin in the antiviral composition is a minimum of 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg. The method according to any one of claims 1 to 7, which is / day or about 5 microg / day. The method according to any one of claims 1 to 8, wherein the cardiac glycoside is administered in at least two administration phases: a loading phase and a maintenance phase. The method of claim 9, wherein the cardiac glycoside is digoxin and the administration regimens of the loaded and maintenance phases are as follows.

After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng The method according to any one of claims 1 to 10, which is in the range of / mL or about 0.05 to about 2.5 ng / mL. A method of treating a viral infection in a subject in need of treatment, comprising administering to the subject one or more doses of an antiviral composition comprising oleandrin, wherein the viral infection is a plus sense. Caused by the double-stranded envelope RNA virus ((+)-ss-envRNA-V)
Doses include an antiviral composition of about 100-1000 mg or about 100-1000 microg per kg of body weight of the subject; or the amount of oleandrin administered per day is 140 microg-315 microlog, 20 microg-750 microrog, 12 microg. ~ 300 microg, 12 microg ~ 120 microg, 0.01 microg ~ 100 mg, 0.01 microg ~ 100 microg, about 0.5 ~ about 100 microg, about 1 ~ about 80 microg, about 1.5 ~ about 60 microg, about 1.8 ~ about 60 microg, or Selected from the group consisting of about 1.8 to about 40 micrologs; or the dose of the antiviral composition is from about 0.05 to about 0.05 per day based on the unit amount of the antiviral composition per kg body weight of the subject. 0.5 mg / kg / day, about 0.05 to 0.35 mg / kg / day, about 0.05 to 0.22 mg / kg / day, about 0.05 to 0.4 mg / kg / day, about 0. 05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about Includes 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day;
After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng The method, which is in the range of / mL, or about 0.05 to about 2.5 ng / mL. The invention according to any one of claims 1 to 12, wherein the (+)-ss-envRNAV is a virus selected from the group consisting of Coronaviridae, Flaviviridae, Togaviridae, and Alterviridae. Method. The method according to any one of claims 1 to 13, wherein the (+)-ss-envRNAV is a coronavirus pathogenic to humans. 14. 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 HKU 20. The method described in. The (+)-ss-envRNAV is flavivirus, yellow fever virus, dengue fever virus, Japanese encephalitis virus, western Nile virus, decavirus, tick-mediated encephalitis virus, Casanur forest disease virus, Alkhurma disease virus, omsk. The method according to any one of claims 1 to 13, which is a virus selected from the group consisting of hemorrhagic fever virus and poissan virus. The (+)-ss-envRNAV is arbovirus, eastern horse encephalomyelitis virus (EEEV), western horse encephalomyelitis virus (WEEV), Venezuelan elephant encephalomyelitis virus (VEEV), chicungnia virus (CHIKV), o. One of claims 1 to 13, which is a Togavirus family virus selected from the group consisting of Nyonnyon virus (ONNV), Pogosta disease virus, Sindbis virus, Ross River fever virus (RRV) and Semuliki forest virus. The method described. A method of treating a viral infection in a subject in need of treatment, comprising administering to the subject one or more doses of an antiviral composition comprising oleandrin, digoxin or a combination thereof, said virus. A method in which infection is caused by a negative sense single-stranded enveloped RNA virus ((-)-ss-envRNA-V). A prophylactic method of treating a subject at risk of contracting a virus, wherein the subject is chronically given one or more doses of an antiviral composition at a repetitive frequency over a long-term treatment period prior to the subject becoming infected with the virus. The antiviral composition comprises oleandrin, digoxin or a combination thereof, comprising the prevention of the subject from contracting the viral infection, wherein the viral infection is negative sense. A method caused by a double-stranded enveloped RNA virus ((-)-ss-envRNA-V). Determining if the subject has the virus infection and
Instructing the administration of the antiviral composition and
To administer the initial dose of the antiviral composition to the subject for a period of time according to the prescribed initial dosing regimen.
Periodically determining the adequacy of the subject's clinical and / or therapeutic response to treatment with the antiviral composition.
If the subject's clinical and / or therapeutic response is reasonable, continue treatment with the antiviral composition as needed, or continue with the subject's clinical response and / or subject until the desired clinical endpoint is achieved. / Or, if the therapeutic response is not appropriate for the initial dose and initial dosing regimen, the dosage comprises escalating or tapering until the desired clinical and / or therapeutic response of the subject is achieved. 18. The method according to 18. The method of any one of claims 18-20, wherein the dose comprises an antiviral composition of about 100-1000 mg or about 100-1000 micrologs per kg body weight of the subject. The amount of oleandrin as part of the composition administered daily is 140 microg to 315 microg, 20 microg to 750 microg, 12 microg to 300 microg, 12 microg to 120 microlog, 0.01 microg to 100 mg, 0.01 microg to 100 microg, about. Claims 18-21, selected from the group consisting of 0.5 to about 100 microg, about 1 to about 80 microg, about 1.5 to about 60 microg, about 1.8 to about 60 microg, or about 1.8 to about 40 microg. The method according to any one of the above. Claim that the dose of the antiviral composition is administered twice daily or about every 12 hours and the amount of oleandrin in the dose is about 0.25 to about 50 micrologs or about 0.9 to 5 micrologs. The method according to any one of 18 to 22. The dose of the antiviral composition is about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg, based on the unit amount of the antiviral composition per kg body weight of the subject per day. / Kg / day, about 0.05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0 .5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0. The method of any one of claims 18-23, comprising 05-0.3 microg / kg / day. a) The daily dose of oleandrin in the antiviral composition is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day or about 30 microg / day. And / or b) The daily dose of oleandrin in the antiviral composition is at least about 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day. The method according to any one of claims 18 to 24, which is about 2 microg / day or about 5 microg / day. After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng The method according to any one of claims 18 to 25, which is in the range of / mL or about 0.05 to about 2.5 ng / mL. A method of treating a viral infection in a subject in need of treatment.
Containing the administration of one or more doses of the antiviral composition comprising oleandrin to the subject, the viral infection is caused by a negative sense single-stranded envelope RNA virus ((-)-ss-envRNA-V). Caused,
The dose comprises an antiviral composition of about 100-1000 mg or about 100-1000 microg per kg of body weight of the subject; or the amount of oleandrin administered per day is 140 microg-315 microlog, 20 microg-750 microrog, 12 microg. ~ 300 microg, 12 microg ~ 120 microg, 0.01 microg ~ 100 mg, 0.01 microg ~ 100 microg, about 0.5 ~ about 100 microg, about 1 ~ about 80 microg, about 1.5 ~ about 60 microg, about 1.8 ~ about 60 microg, or Selected from the group consisting of about 1.8 to about 40 micrologs; or the dose of the antiviral composition is from about 0.05 to about 0.05 per day based on the unit amount of the antiviral composition per kg body weight of the subject. 0.5 mg / kg / day, about 0.05 to 0.35 mg / kg / day, about 0.05 to 0.22 mg / kg / day, about 0.05 to 0.4 mg / kg / day, about 0. 05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about Includes 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day;
After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng The method, which is in the range of / mL, or about 0.05 to about 2.5 ng / mL. The (-)-ss-envRNAV is selected from the group consisting of arenavirus family virus, bunyavirus family (Bunyavirus order) virus, phyllovirus family virus, orthomyxovirus family virus, paramyxovirus family virus, and rabdovirus family virus. The method according to any one of claims 18 to 27, which is a virus to be used. The (-)-ss-envRNAV is an arenavirus selected from the group consisting of lassavirus, aseptic meningitis, ganarit virus, funine virus, lujovirus, machupovirus, savia virus and whitewater aroyovirus. The method according to any one of claims 18 to 28, which is a family virus. The method according to any one of claims 18 to 28, wherein the (-)-ss-envRNAV is a Bunyaviridae virus selected from the group consisting of hantavirus and Crimean-Congo hemorrhagic fever ortho-nail virus. The (-)-ss-envRNAV is a paramyxoviridae virus selected from the group consisting of mumps virus, nipavirus, Hendra virus, respiratory polynuclear virus (RSV), human parainfluenza virus (HPIV), and NDV. The method according to any one of claims 18 to 28. The (-)-ss-envRNAV consists of influenza virus (A to C), Isavirus, Togotovirus, Quaranger virus, H1N1, H2N2, H3N2, H1N2, Spanish cold, Asian cold, Hong Kong cold, Soviet cold. The method according to any one of claims 18 to 28, which is an Orthomyxoviridae virus selected from the group. The method according to any one of claims 18 to 28, wherein the (-)-ss-envRNAV is a rhabdoviridae virus selected from the group consisting of rabies virus, becyclovirus, lyssavirus, and cytorabdovirus. .. Any one of claims 1-33 herein comprising sublingual administration of one or more doses of cardiac glycosides comprising oleandrin, digoxin or a combination thereof to a subject having a viral infection. Methods for treating viral infections as described in Section. A method of treating a coronavirus infection comprising administering to a subject having the infection multiple therapeutically effective doses of oleandrin, digoxin or a combination thereof. 34. The method of claim 34 or 35, wherein the plurality of doses is one or more doses administered per day over two or more days per week. 36. The method of claim 36, wherein the administration is continued for one week or longer per month. 37. The method of claim 37, wherein the administration is continued for one month or longer per year. The method according to any one of claims 34 to 38, wherein the coronavirus is pathogenic to humans. 34. 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 HKU 20. The method according to any one of 39. The concentration of oleandrin in the plasma of the subject is, after administration of oleandrin, as the amount of oleandrin per 1 mL of plasma, a) about 10 microg / mL or less, about 5 microg / mL or less, about 2.5 microg / mL. Below, about 2 microg / mL or less, or about 1 microg / mL or less; b) about 0.0001 microg / mL or more, about 0.0005 microg / mL or more, about 0.001 microg / mL or more, about 0.0015 microg / mL or more, about 0.01 microg / mL or more, about 0.015 microg / mL or more, about 0.1 microg / mL or more, about 0.15 microg / mL or more, about 0.05 microg / mL or more, or about 0.075 microg / mL or more; c) A combination of any upper limit of item a) and any lower limit of item c); or d) about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng / mL, about 0.005 to about. 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng / mL, or about 0.05 to The method according to any one of claims 34 to 40, which is in the range of about 2.5 ng / mL. The method of any one of claims 34-40, wherein the dose comprises an antiviral composition of about 100-1000 mg or about 100-1000 microg per kg of body weight of the subject. The amount of oleandrin as part of the composition administered daily is 140 microg to 315 microg, 20 microg to 750 microg, 12 microg to 300 microg, 12 microg to 120 microlog, 0.01 microg to 100 mg, 0.01 microg to 100 microg, about. Claims 34-42, selected from the group consisting of 0.5-about 100 microg, about 1-about 80 microg, about 1.5-about 60 microg, about 1.8-about 60 microg, or about 1.8-about 40 microg. The method according to any one of the above. Claim that the dose of the antiviral composition is administered twice daily or about every 12 hours and the amount of oleandrin in the dose is about 0.25 to about 50 micrologs or about 0.9 to 5 micrologs. The method according to any one of 34 to 43. The dose of the antiviral composition is about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg, based on the unit amount of the antiviral composition per kg body weight of the subject per day. / Kg / day, about 0.05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0 .5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0. The method of any one of claims 34-44, comprising 05-0.3 microg / kg / day. a) The daily dose of oleandrin in the antiviral composition is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day or about 30 microg / day. And / or b) The daily dose of oleandrin in the antiviral composition is at least about 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day. The method according to any one of claims 34 to 45, which is about 2 microg / day or about 5 microg / day. The method of any one of claims 1-46, wherein the cardiac glycoside is administered in at least two dosing phases: a loading phase and a maintenance phase. 47. The method of claim 47, wherein the cardiac glycoside is digoxin and the dosing regimen for the loading and maintenance phases is as follows.

The antiviral composition is a) oleandrin; b) a combination of oleandrin, oleanolic acid (free acid, salt, or prodrug) and ursolic acid (free acid, salt, or prodrug); c) ole. Combination of andrin, oleanolic acid (free acid, salt, or prodrug) and bethulinic acid (free acid, salt, or prodrug); d) oleandrin, oleanolic acid (free acid, salt, or prodrug), Combination of ursolic acid (free acid, salt, or prodrug) and bethulinic acid (free acid, salt, or prodrug); e) oleandrin, oleanolic acid (free acid or salt thereof), ursolic acid (free acid or Salt), and a combination of bethulinic acid (free acid or); or f) oleandrin, and oleanolic acid (free acid, salt, or prodrug), ursolic acid (free acid, salt, or prodrug), bethulinic acid. The method of any one of claims 1-48, comprising a combination of at least two triterpenes selected from the group consisting of (free acid, salt, or prodrug). 49. The method of claim 49, wherein the molar ratio of oleandrin to the triterpenes at a dose of antiviral composition is selected from any of the following:

The molar ratio of total triterpene content (oleanolic acid + ursolic acid + bethulinic acid) to oleandrin is 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 49. The method of claim 49, which is in the range of about 10: 1. The molar ratio of individual triterpenes (oleanolic acid (OA): ursolic acid (UA): bethuric acid (BA)) to oleandrin (OL) is in the following range: 2-8 (OA): 2-8. (UA): 0.1 to 1 (BA): 0.5 to 1.5 (OL); or 3 to 6 (OA): 3 to 6 (UA): 0.3 to 8 (BA): 0. 7 to 1.2 (OL); or 4 to 5 (OA): 4 to 5 (UA): 0.4 to 0.7 (BA): 0.9 to 1.1 (OL); 4.6 ( OA): 4.4 (UA): 0.6 (BA): 1 (OL); about 9-12 (OA): up to about 2 (UA): up to about 2, or about 10 (OA): about 1 (UA): about 1, or about 9-12 (OA): about 0.1-2 (UA): about 0.1-2 (BA), or about 9-11 (OA): about 0.5- 1.5 (UA): about 0.5 to 1.5 (BA), or about 9.5 to 10.5 (OA): about 0.75 to 1.25 (UA): about 0.75 to 1. .25 (BA), or about 9.5 to 10.5 (OA): about 0.8 to 1.2 (UA): about 0.8 to 1.2 (BA), or about 9.75 to 10. .5 (OA): Approximately 0.9 to 1.1 (UA): Approximately 0.9 to 1.1 (BA), the method according to claim 49. The method according to any one of claims 1 to 52, wherein the antiviral composition comprises an extract of biomass. 53. The method of claim 53, wherein the extract is prepared by hot water extraction, cold water extraction, organic solvent extraction, supercritical fluid extraction, subcritical liquid extraction, or a combination thereof. The method of claim 53 or 54, wherein the biomass is a biomass of plant material from a Nerium species, or Agrobacterium species. The method of any one of claims 53-55, wherein the extract comprises a combination of oleandrin and one or more compounds extracted from the biomass. 13. Method. The extracts are oleandrin, as well as strong worried sugars, glycones, aglycons, steroids, triterpenes, polysaccharides, sugars, alkaloids, fats, proteins, neritarosides, odrosides, oleanolic acids, ursoleic acids, bethuric acids, oleandrigenins. , Oleacid A, Betulin (Ursa-12-en-3β, 28-diol), 28-Noruursa-12-en-3β-ol, Ursa-12-en-3β-ol, 3β, 3β-hydroxy-12-oleanen -28-acid, 3β, 20α-dihydroxyursa-21-en-28-acid, 3β, 27-dihydroxy-12-ursen-28-acid, 3β, 13β-dihydroxyursa-11-en-28-acid, 3β , 12α-dihydroxyoleanan-28,13β-oride, 3β,27-dihydroxy-12-oleanan-28-acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, coffee acid, L-quinic acid, 4 -Kumaloyl-CoA, 3-O-cafe oil quinic acid, 5-O-cafe oil quinic acid, cardenolide B-1, cardenolide B-2, oleagenin, nerididinenoside, nerisoside, odroside-H, galacturonic acid, ramnorth, arabinose, 3-Beta-O- (D-diginosyl) -5-beta, 14 beta-dihydroxy-cardo-20 (22) -enolidepectinic polysaccharide composed of xylose and galactose, MW in the range 17,000 to 120,000 D , Or a polysaccharide having a MW of about 35000D, about 3000D, about 5500D, or about 12000D, cardenolide monoglycoside, cardenolide N-1, cardenolide N-2, cardenolide N-3, cardenolide N-4, pregnane, 4, , 6-Diene-3,12,20-Trione, 20R-Hydroxypregna-4,6-Diene-3,12-Dion, 16 Beta, 17 Beta-Epoxy-12 Beta-Hydroxypregna-4,6- Diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A), 20S, 21-dihydroxypregna-4,6-diene-3,12 -Dione (neridienone B), neriukumalic acid, isoneriukumalic acid, oleanderonic acid, oleanderene, 8alpha-methoxylabdan-18-acid, 12-ursen, caneroside, neriumoside, 3β-O- (D-diginosyl) -2α -Hydroxy-8,14β-Epoxy-5β-Carda-16: 17, 20:22-Dienolide, 3β-O- (D-diginosyl) -2α, 14β-Dihydroxy-5β-Carda-16: 17, 20:22 -Dienolide, 3β, 27-dihydroxy-Ursa-18-en-13,28-oride, 3β, 22α, 28-trihydroxy-25-norlurp-1 (10), 20 (29) -dien-2- On, cis-calenin (3β-hydroxy-28-Z-z-p-kumaloyloxy-ursa-12-ene-27-acid), trans-calenin (3-β-hydroxy-28-E-p-kumaroyloxy) -Ursa-12-en-27-acid), 3beta-hydroxy-5alpha-carda-14 (15), 20 (22) -dienolide (beta-anhydroepidigitoxygenin), 3beta-O- ( D-Digitarocil) -21-Hydroxy-5 Beta-Carda-8,14,16,20 (22) -Tetraenolide (neriumogenin-A-3 beta-D-digitaroside), proceragenin, neridienone A, 3 beta, 27-dihydroxy -12-Ursen-28-acid, 3beta, 13 beta-dihydroxyursa-11-en-28-acid, 3beta-hydroxyursa-12-en-28-aldehyde, 28-olursa-12-en-3 beta -Oll, Ursa-12-En-3 Beta-Ol, Ursa-12-En-3 Beta, 28-diol, 3beta, 27-Dihydroxy-12-oleanen-28-acid, (20S, 24R) -Epoxydan Malang-3 Beta, 25-diol, 20 Beta, 28-Epoxy-28 Alpha-methoxytalaxasteran-3 Beta-Ol, 20 Beta, 28-Epoxy Taraxaster-21-En-3 Beta-Ol, 28- Nor-Ursa-12-En-3 Beta, 17 Beta-Dolyl, 3 Beta-Hydroxy Ursa-12-En-28-aldehyde, Alpha-Neriursate, Beta-Neriursate, 3Alpha-Acetphenoxy-Ursa-12-En- 28-acid, 3beta-acetophenoxy-ursa-12-ene-28-acid, oleanderolic acid, canelology, 3β-p-hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursen-28-acid, 28-Hydroxy-20 (29) -Lupen-3,7-Dione, Canerosin, 3Alpha-Hydroxy-Urser- 18,20-dien-28-acid, D-salmentose, D-diginose, nerididinoside, nerisoside, isolicinoleic acid, gentiobiosylnerigoside, gentiobiosylvomontside, gentiobiosyloleandrin, holineline, 12β-hydroxy-5β-carda-8,14,16,20 (22) -tetraenolide, 8β-hydroxy-digitoxygenin, Δ16-8β-hydroxy-digitoxygenin, Δ16-neriagenin, uvaol, ursolealdehyde, 27 (p-kumaroyl). Oxy) Ursolic acid, oleanderol, 16-anhydro-deacetyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, azigoside, aginerin, alpha-amyrin, beta-citosterol, campesterol, kauchuk, capric acid, Caprylic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl-nerigoside, horiandrin, pseudoclamin, kercetin, kercetin-3-ramnoglucoside, cercitrin, rosaginine, rutin, stearic acid, stigmasterol, strospeside, urechtxin, The method of any one of claims 53-57, comprising one or more compounds selected from the group consisting of and usaligenin. Claims 53-58, wherein the daily dose of the extract is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microg / day or about 30 microg / day. The method according to any one of the above. Claims that the daily dose of the extract is at least 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg / day, or about 5 microg / day. Item 5. The method according to any one of Items 52 to 58. The method of any one of claims 1-60, wherein the viral titer in the blood or plasma of the subject is reduced or not increased as a result of the treatment. The method according to any one of claims 1 to 61, wherein the administration is systemic, parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, oral, oral, or a combination thereof. The method according to any one of claims 1 to 62, wherein one or more doses are administered at a daily, weekly, or monthly frequency. Any one of claims 1 to 63, wherein the antiviral composition is administered immediately after infection, at any time within 1 to 5 days after infection, or at the earliest time after a definitive diagnosis of viral infection. The method described in. The method according to any one of claims 1 to 64, wherein the antiviral composition is administered as a primary antiviral therapy, an adjuvant antiviral therapy, or a combined antiviral therapy. The administration comprises separate or co-administration with at least one other antiviral composition of the antiviral composition, or at least one other composition for treating symptoms associated with the viral infection. , The method according to any one of claims 1 to 65. 66. Method. A sublingual form containing oleandrin, digoxin or a combination thereof. The dosage form of claim 68, further comprising at least one liquid carrier. It contains oleandrin, Nerium sp. Or Thevetia sp. Extracts prepared by subcritical liquid extraction of biomass in The extract according to claim 70, wherein the subcritical liquid contains carbon dioxide and may further contain alcohol. A method for preparing an extract containing oleandrin, which is sufficient to extract oleandrin from the biomass using subcritical liquid carbon dioxide which may further contain alcohol in the oleandrin-containing biomass. A method that involves performing a period extraction. A method of preparing an extract containing oleandrin,
Extracting the oleandrin-containing biomass with an extract containing subcritical liquid carbon dioxide, which may further contain alcohol, for a period sufficient to extract oleandrin from the biomass.
A method comprising separating the biomass from the extract while retaining the extract and then removing the extract to form the extract. A method for producing a combination of oleandrin-containing extracts.
The first oleandrin-containing biomass was subjected to organic solvent extraction to obtain an oleandrin-containing organic solvent extract.
Subcritical liquid (SbCL) extraction (SbCLE) was performed on the second oleandrin-containing biomass to obtain an oleandrin-containing SbCL extract.
A method comprising combining the oleandrin-containing organic solvent extract and the oleandrin-containing SbCL extract to obtain the combined oleandrin-containing extract. An improved oleandrin-containing composition comprising a portion of the oleandrin-containing organic solvent extract as defined herein and a portion of the oleandrin-containing SbCL extract as defined herein. A method of treating a coronavirus infection, which comprises administering to subjects in need thereof multiple doses of oleandrin per day over a treatment period of at least about 5 days to about 1 month. A method in which phosphorus is present in a pharmaceutical composition comprising at least one extract obtained from oleandrin-containing biomass. The total daily dose of oleandrin is about 1 microg to about 180 microg, about 1 microg to about 120 microg, about 5 microg to about 100 microg, about 10 microlog to about 80 microlog, about 10 microlog to about 75 microg, about 15 microg to about 120 microg. ~ About 100 microg, about 15 microg ~ about 80 microg, about 30 microg ~ about 120 microg, about 30 microlog ~ about 100 microlog, about 30 microlog ~ about 90 microlog, about 40 microlog ~ about 70 microlog, about 1 microlog, about 5 microg, about 10 microg, about 10 microg 40 microg, about 50 microg, about 60 microg, about 70 microg, about 80 microlog, about 90 microg, about 100 microlog, about 110 microlog, about 120 microlog, about 130 microlog, about 140 microlog, about 150 microlog, about 160 microlog, about 170 microg, about 160 microg, about 170 microg 76. The method of claim 76. The multiple doses of oleandrin per day are 2 to 10 times per day, 2 to 8 times per day, 2 to 6 times per day, 2 to 4 times per day, and 2 times per day. 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or 10 times a day The method of claim 76 or 77. The method of any one of claims 76-78, wherein the pharmaceutical composition comprises at least two extracts obtained from the Nerium species. 39. The method of claim 79, wherein the extract is independently selected from the group consisting of hot water extract, solvent extract, supercritical fluid extract, and subcritical liquid extract at each outbreak. A method of treating a viral infection described herein. The compositions described herein. The antiviral composition described herein. The oleandrin-containing compositions described herein. A method of treating a coronavirus infection, which comprises administering to a subject in need thereof a dose of oleandrin at least once a day for a treatment period of at least about 5 days to about 1 month or more. , The method by which the coronavirus infection is pathogenic to humans. 85. The method described in. The total daily dose of oleandrin is about 1 microg to about 180 microg, about 1 microg to about 120 microg, about 5 microg to about 100 microg, about 10 microlog to about 80 microlog, about 10 microlog to about 75 microg, about 15 microg to about 120 microg. ~ About 100 microg, about 15 microg ~ about 80 microg, about 30 microg ~ about 120 microg, about 30 microlog ~ about 100 microlog, about 30 microlog ~ about 90 microlog, about 40 microlog ~ about 70 microlog, about 1 microlog, about 5 microg, about 10 microg, about 10 microg 40 microg, about 50 microg, about 60 microg, about 70 microg, about 80 microg, about 90 microg, about 100 microlog, about 110 microlog, about 120 microlog, about 130 microlog, about 140 microlog, about 150 microg, about 160 microg, about 170 microg, about 170 microg About 20 micrologs to about 750 micrologs, about 12 micrologs to about 300 micrologs, about 12 micrologs to about 120 micrologs, about 0.01 micrologs to about 100 mg, about 0.01 micrologs to about 100 micrologs, about 0.5 to about 100 micrologs, about 1 to about 80 micrologs, about 1 The method of claim 85, wherein each outbreak is independently selected from .5 to about 60 microg, about 1.8 to about 60 microg, and about 1.8 to about 40 microg. Dosages were about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg / kg / day, about 0. 05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day 85. The method of claim 85, comprising day. a) The daily dose of oleandrin is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microlog / day or about 30 microg / day and / or. b) The daily dose of oleandrin is a minimum of 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg / day, or about 5 microg / day. , The method of claim 85. 85. The method of claim 85, wherein the dose is administered twice daily or about every 12 hours, and the amount of oleandrin in said dose is about 0.25 to about 50 micrologs or about 0.9 to 15 microrogs. The above-mentioned dose of oleandrin at least once a day is 2 to 10 times a day, 2 to 8 times a day, 2 to 6 times a day, 2 to 4 times a day, and a day. 2 times, 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or 10 times a day 25. The method of claim 85, which is a time. 85. The method of claim 85, wherein oleandrin is present in the composition comprising at least one extract obtained from oleandrin-containing biomass. 29. The method of claim 92, wherein the extract is independently selected from the group consisting of hot water extract, solvent extract, and supercritical fluid extract at each occurrence. After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng 85. The method of claim 85, which is in the range of / mL, or about 0.05 to about 2.5 ng / mL. A method of treating a coronavirus infection, comprising administering to the subject having the infection a plurality of therapeutically effective doses of oleandrin or an oleandrin-containing composition. 95. The method of claim 95, wherein the plurality of therapeutically effective doses are administered at least once per day over a period of 2 days or more per week. The method according to claim 96, wherein the administration is continued for one week or more per month. The method according to claim 97, wherein the administration is continued for one month or more per year. The method of claim 95, wherein the coronavirus is pathogenic to humans. 99. 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 HKU 20. The method described in. 95. The method of claim 95, wherein oleandrin is present in the composition comprising at least one extract obtained from oleandrin-containing biomass. 10. The method of claim 101, wherein the extract is independently selected from the group consisting of hot water extract, solvent extract, and supercritical fluid extract at each occurrence. 10. The method of claim 101, wherein the extract comprises a combination of oleandrin and one or more compounds extracted from the biomass. 10. The method of claim 103, wherein the one or more compounds comprises one or more cardiac glycoside precursors, one or more cardiac glycoside constituents of cardiac glycosides, or combinations thereof. 95. The method of claim 95, wherein the administration is systemic, parenteral, buccal, enteral, intramuscular, subcutaneous, sublingual, oral, oral, or a combination thereof. 19. The method of claim 95, wherein the antiviral composition is administered immediately after infection, at any time within 1-5 days after infection, or at the earliest time after a definitive diagnosis of viral infection. The antiviral composition is administered as primary antiviral therapy, adjuvant antiviral therapy, or combined antiviral therapy, or said administration to at least one other antiviral composition of said composition, or said virus infection. 95. The method of claim 95, comprising separate or co-administration with at least one other composition for treating the associated symptom. The total daily dose of oleandrin is about 1 microg to about 180 microg, about 1 microg to about 120 microg, about 5 microg to about 100 microg, about 10 microlog to about 80 microlog, about 10 microlog to about 75 microg, about 15 microg to about 120 microg. ~ About 100 microg, about 15 microg ~ about 80 microg, about 30 microg ~ about 120 microg, about 30 microlog ~ about 100 microlog, about 30 microlog ~ about 90 microlog, about 40 microlog ~ about 70 microlog, about 1 microlog, about 5 microg, about 10 microg, about 10 microg 40 microg, about 50 microg, about 60 microg, about 70 microg, about 80 microg, about 90 microg, about 100 microlog, about 110 microlog, about 120 microlog, about 130 microlog, about 140 microlog, about 150 microg, about 160 microg, about 170 microg, about 170 microg About 20 micrologs to about 750 micrologs, about 12 micrologs to about 300 micrologs, about 12 micrologs to about 120 micrologs, about 0.01 micrologs to about 100 mg, about 0.01 micrologs to about 100 micrologs, about 0.5 to about 100 micrologs, about 1 to about 80 micrologs, about 1 The method of claim 95, wherein each outbreak is independently selected from .5 to about 60 microg, from about 1.8 to about 60 microg, and from about 1.8 to about 40 microg. Dosages were about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg / kg / day, about 0. 05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, or about 0.05-0.3 microg / kg / day 95. The method of claim 95, comprising a day. a) The daily dose of oleandrin is up to about 100 microg / day, about 80 microg / day, about 60 microg / day, about 40 microg / day, about 38.4 microlog / day or about 30 microg / day and / or. b) The daily dose of oleandrin is a minimum of 0.5 microg / day, about 1 microg / day, about 1.5 microg / day, about 1.8 microg / day, about 2 microg / day, or about 5 microg / day. , The method of claim 95. The method of claim 95, wherein the dose is administered twice daily or about every 12 hours, and the amount of oleandrin in said dose is about 0.25 to about 50 micrologs or about 0.9 to 15 microrogs. The above-mentioned dose of oleandrin at least once a day is 2 to 10 times a day, 2 to 8 times a day, 2 to 6 times a day, 2 to 4 times a day, and a day. 2 times, 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, or 10 times a day The method of claim 95, which is a time. After administration of the one or more doses, the plasma concentration of oleandrin in the subject is about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng as the amount of oleandrin per 1 mL of plasma. / ML, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng 95. The method of claim 95, which is in the range of / mL, or about 0.05 to about 2.5 ng / mL. The extracts are oleandrin, as well as strong worried sugars, glycones, aglycons, steroids, triterpenes, polysaccharides, sugars, alkaloids, fats, proteins, neritarosides, odrosides, oleanolic acids, ursoleic acids, bethuric acids, oleandrigenins. , Oleacid A, Betulin (Ursa-12-en-3β, 28-diol), 28-Noruursa-12-en-3β-ol, Ursa-12-en-3β-ol, 3β, 3β-hydroxy-12-oleanen -28-acid, 3β, 20α-dihydroxyursa-21-en-28-acid, 3β, 27-dihydroxy-12-ursen-28-acid, 3β, 13β-dihydroxyursa-11-en-28-acid, 3β , 12α-dihydroxyoleanan-28,13β-oride, 3β,27-dihydroxy-12-oleanan-28-acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, coffee acid, L-quinic acid, 4 -Kumaloyl-CoA, 3-O-cafe oil quinic acid, 5-O-cafe oil quinic acid, cardenolide B-1, cardenolide B-2, oleagenin, nerididinenoside, nerisoside, odroside-H, galacturonic acid, ramnorth, arabinose, 3-Beta-O- (D-diginosyl) -5-beta, 14 beta-dihydroxy-cardo-20 (22) -enolidepectinic polysaccharide composed of xylose and galactose, MW in the range 17,000 to 120,000 D , Or a polysaccharide having a MW of about 35000D, about 3000D, about 5500D, or about 12000D, cardenolide monoglycoside, cardenolide N-1, cardenolide N-2, cardenolide N-3, cardenolide N-4, pregnane, 4, , 6-Diene-3,12,20-Trione, 20R-Hydroxypregna-4,6-Diene-3,12-Dion, 16 Beta, 17 Beta-Epoxy-12 Beta-Hydroxypregna-4,6- Diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A), 20S, 21-dihydroxypregna-4,6-diene-3,12 -Dione (neridienone B), neriukumalic acid, isoneriukumalic acid, oleanderonic acid, oleanderene, 8alpha-methoxylabdan-18-acid, 12-ursen, caneroside, neriumoside, 3β-O- (D-diginosyl) -2α -Hydroxy-8,14β-Epoxy-5β-Carda-16: 17, 20:22-Dienolide, 3β-O- (D-diginosyl) -2α, 14β-Dihydroxy-5β-Carda-16: 17, 20:22 -Dienolide, 3β, 27-dihydroxy-Ursa-18-en-13,28-oride, 3β, 22α, 28-trihydroxy-25-norlurp-1 (10), 20 (29) -dien-2- On, cis-calenin (3β-hydroxy-28-Z-z-p-kumaloyloxy-ursa-12-ene-27-acid), trans-calenin (3-β-hydroxy-28-E-p-kumaroyloxy) -Ursa-12-en-27-acid), 3beta-hydroxy-5alpha-carda-14 (15), 20 (22) -dienolide (beta-anhydroepidigitoxygenin), 3beta-O- ( D-Digitarocil) -21-Hydroxy-5 Beta-Carda-8,14,16,20 (22) -Tetraenolide (neriumogenin-A-3 beta-D-digitaroside), proceragenin, neridienone A, 3 beta, 27-dihydroxy -12-Ursen-28-acid, 3beta, 13 beta-dihydroxyursa-11-en-28-acid, 3beta-hydroxyursa-12-en-28-aldehyde, 28-olursa-12-en-3 beta -Oll, Ursa-12-En-3 Beta-Ol, Ursa-12-En-3 Beta, 28-diol, 3 Beta, 27-Dihydroxy-12-oleannen-28-Acid, (20S, 24R) -Epoxydan Malang-3 Beta, 25-diol, 20 Beta, 28-Epoxy-28 Alpha-methoxytalaxasteran-3 Beta-Ol, 20 Beta, 28-Epoxy Taraxaster-21-En-3 Beta-Ol, 28- Nor-Ursa-12-En-3 Beta, 17 Beta-Dolyl, 3 Beta-Hydroxy Ursa-12-En-28-aldehyde, Alpha-Neriursate, Beta-Neriursate, 3Alpha-Acetphenoxy-Ursa-12-En- 28-acid, 3beta-acetophenoxy-ursa-12-ene-28-acid, oleanderolic acid, canelology, 3β-p-hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursen-28-acid, 28-Hydroxy-20 (29) -Lupen-3,7-Dione, Canerosin, 3Alpha-Hydroxy-Urser- 18,20-dien-28-acid, D-salmentose, D-diginose, nerididinoside, nerisoside, isolicinoleic acid, gentiobiosylnerigoside, gentiobiosylvomontside, gentiobiosyloleandrin, holineline, 12β-hydroxy-5β-carda-8,14,16,20 (22) -tetraenolide, 8β-hydroxy-digitoxygenin, Δ16-8β-hydroxy-digitoxygenin, Δ16-neriagenin, uvaol, ursolealdehyde, 27 (p-kumaroyl). Oxy) Ursolic acid, oleanderol, 16-anhydro-deacetyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, azigoside, aginerin, alpha-amyrin, beta-citosterol, campesterol, kauchuk, capric acid, Caprylic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl-nerigoside, horiandrin, pseudoclamin, kercetin, kercetin-3-ramnoglucoside, cercitrin, rosaginine, rutin, stearic acid, stigmasterol, strospeside, urechtxin, The method of claim 113, comprising one or more compounds selected from the group consisting of and usaligenin.

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