TW202137991A - Method and compositions for treating coronavirus infection - Google Patents

Abstract

A method of treating viral infection, such as viral infection caused by a virus of the Coronaviridae family, is provided. A composition having at least oleandrin is used to treat viral infection.

Description

Method and composition for treating coronavirus infection

The present invention relates to an antiviral composition and its use in the treatment of arenaviridae infections, Bunyaviridae infections, Flaviviridae infections, and Togaviridae infections in mammals , Paramyxoviridae infection, Retroviridae infection, Coronaviridae infection or Filoviridae infection. Some embodiments relate to the treatment of hemorrhagic viral infections.

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

Traditionally, boiling water, cold water, supercritical fluid or organic solvents are used to extract plant components from Apocynaceae species.

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

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

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

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

It is known that triterpenes have various therapeutic activities. Some known triterpenes include oleanolic acid, ursolic acid, betulinic acid, bardosol, behenic acid and others. The therapeutic activities of these triterpenes are mainly evaluated individually, rather than in a combination of triterpenes.

Oleanolic acid belongs to a class of triterpenoids represented by compounds such as bardoxolone. It has been shown to be an effective activator of innate cell phase 2 detoxification pathways. The activation of the transcription factor Nrf2 results in transcription containing antioxidants. The transcription program of downstream antioxidant genes of response element (ARE) is increased. Bardoxolone itself has been extensively studied in clinical trials under inflammatory conditions; however, due to several adverse events, the Phase 3 clinical trial of chronic kidney disease was terminated. The reason for the bed test may be related to the cytotoxicity of some triterpenoids containing bardoxolone at high concentrations.

Compositions containing triterpenes combined with other therapeutic ingredients are found as plant extracts. Fumiko et al. (Biol. Pharm. Bull (2002), 25(11), 1485-1487) disclosed that a methanol extract of rosemary (Rosmarimus officinalis L.) was used in the evaluation of the treatment of trypanosomiasis. Addington et al. (US 8481086, US 9220778, US 9358293, US 20160243143 A1) disclosed a supercritical fluid extract of oleander (SCF; PBI-05204) containing oleandrin and triterpenes for the treatment of neurological conditions. Addington et al. (US 9011937, US 20150283191 A1) disclosed the triterpene-containing fraction (PBI-04711) of the supercritical fluid extract of oleander containing oleandrin and triterpenes, which is used for the treatment of neurological disorders. Jäger et al. (Molecules (2009), 14, 2016-2031) disclosed various plant extracts containing mixtures of oleanolic acid, ursolic acid, betulinic acid and other components. Mishra et al. (PLoS One 201625; 11(7): e0159430.Epub July 25, 2016) disclosed a birch ( Betula utilis ) containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components Bark extract. Wang et al. (Molecules (2016), 21, 139) disclosed an extract of Alstonia scholaris containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components. Le Silva et al. (Molecules (2012), 17, 12197) disclosed an Eriope blanchetti extract containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components. 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, betulinic acid and other components . Ayatollahi et al. (Iran. J. Pharm. Res. (2011), 10(2), 287-294) disclosed a Euphorbia microsciadia extract containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components. Wu et al. (Molecules (2011), 16, 1-15) disclosed extracts of Ligustrum species (Ligustrum species) containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components. Lee et al. (Biol. Pharm. Bull (2010), 33(2), 330) disclosed a Forsythia viridissima extract containing a mixture of oleanolic acid, ursolic acid, betulinic acid and other components.

Oleanolic acid (O or OA), ursolic acid (U or UA) and betulinic acid (B or BA) are in PBI-05204 (PBI-23; supercritical fluid extract of oleander) and PBI-04711 (PBI The three main triterpene components found in the triterpene-containing fraction of -05204 (0-4). By comparing the neuroprotective activity of triterpenes in a brain slice glyco-oxygen deprivation (OGD) model test at similar concentrations, we (two of the inventors) previously reported (Van Kanegan et al., in Nature Scientific Reports (2016) May), 6: 25626. doi: 10.1038/srep25626) the contribution of triterpenes to the neuroprotective activity. We found that PBI-05204 (PBI) and PBI-04711 (fractions 0-4) provide neuroprotective activity.

It is known that the extracts of Apocynaceae species contain many different kinds of compounds: cardiac glycosides, glycosides, steroids, triterpenes, polysaccharides and others. Specific compounds include oleandrin; oleandroside; ordonoside; oleanolic acid; ursolic acid; betulinic acid; oleandrin; oleandrin A; betulin (urso-12-ene-3β,28- Diol) (urs-12-ene-3β,28-diol); 28-norurs-12-en-3β-ol (28-norurs-12-en-3β-ol); urs-12- En-3β-alcohol; 3β,3β-hydroxy-12-oleanen-28-oic acid (3β,3β-hydroxy-12-oleanen-28-oic acid); 3β,20α-dihydroxyurso-21- En-28-acid (3β,20α-dihydroxyurs-21-en-28-oic acid); 3β,27-dihydroxyurs-21-en-28-oic acid (3β,27-dihydroxy-12-ursen-28 -oic acid); 3β,13β-dihydroxyurs-11-en-28-oic acid (3β,13β-dihydroxyurs-11-en-28-oic acid); 3β,12α-dihydroxyursol-28 ,13β-lactone (3β,12α-dihydroxyoleanan-28,13β-olide); 3β,27-dihydroxy-12-oleanan-28-acid (3β,27-dihydroxy-12-oleanan-28-oic acid ); and other components.

Viral hemorrhagic fever (VHF) can be caused by 5 different virus families: arenaviridae, bunyaviridae, filoviridae, flaviviridae and paramyxoviridae. For example, filoviruses such as Ebola virus (EBOV) and Marburg virus (MARV) are the most pathogenic viruses known to humans, and are the pathogens of viral hemorrhagic fever outbreaks with a mortality rate of up to 90%. Each virion contains A molecule of antisense single-stranded RNA. Except for supportive care or symptomatic treatment, there are no commercially available therapeutic and preventive drugs that can be used to treat EBOV (Ebola virus) and MARV (Marburg virus) infections (ie filovirus infections). Five Ebola virus species have been identified: Taï Forest (formerly known as Ivory Coast), Sudan, Zaire, and Reston And Bundibugyo (Bundibugyo).

Antisense single-stranded enveloped RNA virus ((-)-(ss)-envRNAV) includes arenaviridae, bunyaviridae (buniaviridae), filoviridae, orthomyxoviridae, and paramucus Viruses in the Viridae and Rhabdoviridae. Antisense virus RNA is complementary to mRNA, and must be converted into sense RNA by RNA polymerase before translation; therefore, the purified RNA of antisense virus itself is not infectious because it must be converted into sense RNA for replication. Exemplary viruses and infections from the arenaviridae family include Lyssa virus, aseptic meningitis, Guanarito virus, Junin virus, Lujo virus, and Horse Qiubo virus (Machupo virus), Sabia virus (Sabia virus) and Whitewater Arroyo virus (Whitewater Arroyo virus). Exemplary viruses and infections from Bunyaviridae include Hantavirus, Crimean-Congo Haemorrhagic Fever Oronerovirus. Exemplary viruses and infections of the Paramyxoviridae family include mumps virus, Nipah virus, Hendra virus, respiratory fusion cell virus (RSV), human parainfluenza virus (HPIV) and NDV. Exemplary viruses and infections from the Orthomyxoviridae family include influenza virus (A to C), salmon anemia virus (Isavirus), Thogotovirus (Thogotovirus), Quaranja virus (Quaranjavirus), H1N1, H2N2, H3N2, H1N2 , Spanish flu, Asian flu, Hong Kong flu, Russian flu. Exemplary viruses and infections of the Rhabdoviridae family include rabies virus, vesicular virus, Lisavirus, intracellular rice yellow dwarf virus.

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

Coronavirus (CoV) is the common name of the coronavirus family. In humans, respiratory infections caused by CoV are usually mild, but they are fatal in rare forms such as SARS (Severe Acute Respiratory Syndrome)-CoV, MERS (Middle East Respiratory Syndrome Coronavirus Infection)-CoV and COVID-19 . CoV has a spirally symmetrical nucleocapsid, and the genome size ranges from about 26 to about 32 kilobases. Other exemplary human CoVs include CoV 229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU20. The envelope of CoV carries three glycoproteins: S-thorn protein: receptor binding, cell fusion, major antigen; E-envelope protein: small, envelope-related protein; and M-membrane protein: transmembrane-sprouting and envelope form. Among a few CoV types, there is a fourth glycoprotein: HE-hemagglutinin esterase. The genome has 5'methylated end caps and 3'polyadenylic acid tails, and functions directly as mRNA. CoV enters human cells through endocytosis and membrane fusion; and replicates in the cell cytoplasm. CoV is spread by aerosol, fecal-oral route, and mechanical transmission of respiratory secretions. Most viruses grow in epithelial cells. Occasionally, it can infect the liver, kidney, heart or eyes, as well as other cell types such as macrophages. In cold-type respiratory infections, growth appears to be confined to the upper respiratory epithelium. Coronavirus infection is very common and occurs all over the world. Its infection rate is highly seasonal. Children have the highest infection rate in winter, and adult infections are relatively rare. The number of coronavirus serotypes and the degree of antigenic variation are unknown. The possibility of re-infection in life means that there are multiple serotypes (at least four known) and/or antigenic variants of the coronavirus. Therefore, the possibility of immunizing all serotypes with a single vaccine is extremely low. SARS is a type of viral pneumonia. Symptoms include fever, dry cough, dyspnea (shortness of breath), headache, and hypoxemia (Low blood oxygen concentration). Typical laboratory results include lymphopenia (decrease in the number of lymphocytes) and mildly elevated levels of aminotransferase (indicating liver damage). Progressive respiratory failure caused by alveolar damage can lead to death. The typical clinical course of SARS involves improvement of symptoms in the first week of infection, followed by deterioration in the second week. There is still a large demand for treatments (compositions and methods) that can effectively resist the human coronavirus.

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

Oleandrin has shown anti-HIV activity, but has not been evaluated against multiple viruses. The triterpene oleanolic acid, betulinic acid, and ursolic acid have been reported to exhibit different levels of antiviral activity, but have not been evaluated against multiple viruses. Betulinic acid has shown partial antiviral activity against HSV-1 1C strain, influenza A H7N1, ECHO 6 and HIV-1. Oleanolic acid has shown partial antiviral activity against HIV-1, HEP C and HCV H strain NS5B. Ursolic acid has been shown to be partially antiviral against HIV-1, HEP C, HCV H strains NS5B, HSV-1, HSV-2, ADV-3, ADV-8, ADV-11, HEP B, ENTV CVB1 and ENTV EV71 active. In terms of efficacy against specific viruses, the antiviral activities of oleandrin, oleanolic acid, ursolic acid and betulinic acid are unpredictable. The presence of viruses for which oleandrin, oleanolic acid, ursolic acid and/or betulinic acid have little antiviral activity means that it is impossible to predict oleandrin, oleanolic acid, ursolic acid and/or betulinic acid a priori Whether to show antiviral activity to a specific virus genus.

Barrows et al. ("A screen of FDA-approved drugs for inhibitors of Zikavirus infection" in Cell Host Microbe (2016), 20, 259-270) reported that digitonin showed antiviral activity against Zika virus, but the dose was too high. High and may be toxic. Cheung et al. ("Antiviral activity of lanatoside C against dengue virus infection" in Antiviral Res. (2014) 111, 93-99) reported that lanatoside C showed antiviral activity against dengue virus.

Human T Lymphocyte Cell Virus Type 1 (HTLV-1) is a retrovirus, belonging to the Retroviridae family and the delta retrovirus genus. It has a sense RNA genome that can be reverse transcribed into DNA and then integrated into cell DNA. Once integrated, HTLV-1 can only survive as a proviral form that spreads between cells via viral synapses. Even if there are free virions, only a small amount is produced. Although the virus is present in the genital secretions, there is usually no detectable virus in the plasma. HTLV-1 mainly infects CD4 ⁺ T lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL)-a rare but aggressive hematological malignancy that contains infectious skin except for certain autoimmune/inflammatory conditions It also has high treatment resistance rate and usually poor clinical results. The clinical features of HAM/TSP are chronic progressive spastic paresis, urinary incontinence and mild sensory disturbance. Although ATLL is etiologically related to the incubation period of the virus, carcinogenic transformation, and the selection and expansion of HTLV-1 infected cells, inflammatory diseases such as HTLV-1-related myelopathy/tropical spasmodic paresis (HAM/TSP) are caused by Caused by autoimmunity and/or immunopathological response to proviral replication and viral antigen expression. HAM/TSP is a progressive neuroinflammatory disease that causes degeneration and demyelination of the lower spinal cord. HTLV-1 infected circulating T cells invade the central nervous system (CNS) and cause an immunopathological response to the virus and possible CNS components. Nerve damage and subsequent degeneration can cause severe disability in HAM/TSP patients. The persistence of proviral replication and the proliferation of HTLV-1-infected cells in the CNS lead to cytotoxic T cell responses to viral antigens, which may be the cause of autoimmune destruction of nervous tissue.

Although it has been proven that cardiac glycosides exhibit partial antiviral activity against a few viruses, the level of antiviral activity of specific compounds against different viruses is very different, which means that when the same disease is When the virus (one or more) is evaluated, some will show very poor antiviral activity, and some will show good antiviral activity.

Pharmaceutical compositions containing oleandrin, oleanolic acid, ursolic acid, betulinic acid, or any combination thereof that have therapeutic activity against specific viral infections still need to be improved.

【Prior Technical Literature】 None

The present invention provides a pharmaceutical composition and method for treating and/or preventing viral infection in mammalian subjects. The present invention also provides a pharmaceutical composition and method for treating viral infections such as viral hemorrhagic fever (VHF) infection in mammalian subjects. The present invention also provides a method for treating viral infections in mammals by administering the aforementioned pharmaceutical composition. The inventors have successfully prepared an antiviral composition, which exhibits sufficient antiviral activity to prove its use in the treatment of viral infections in humans and animals. The inventors have developed corresponding treatment methods using specific dosing schedules. The present invention also provides a preventive method for treating a subject at risk of viral infection, the method comprising long-term administration of one or more to the subject in a repeated manner before the subject is infected with the virus. The antiviral composition at a dose is used to prevent the subject from being infected with the virus; wherein the aforementioned antiviral composition contains oleandrin.

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

In some embodiments, the viral infection is caused by any of the following virus families: arenaviridae, arteritis virus, bunyaviridae, filoviridae, flaviviridae, orthomyxoviridae, paramyxoviridae, Rhabdoviridae, Retroviridae (especially delta retrovirus), Coronaviridae or Togaviridae. In some embodiments, the viral infection is caused by (+)-ss-envRNAV or (-)-ss-envRNAV.

Some embodiments of the present invention relate to compositions and methods for treating filovirus infection, flavivirus infection, Henney virus infection, alphavirus infection or togavirus infection. Viral infections that can be treated include at least Ebola virus, Marburg virus, alpha virus, flavivirus, yellow fever, dengue fever, Japanese encephalitis, West Nile virus, Zika virus, Venezuelan equine encephalomyelitis (encephalitis) (VEE ) Virus, Qu Gong virus, western equine encephalomyelitis (encephalitis) (WEE) virus, eastern equine encephalomyelitis (encephalitis) (EEE) virus, tick-borne encephalitis, Kjeldahl forest disease, Alkhurma disease, Omu Hemorrhagic fever, Hendra virus, Nipah virus, delta retrovirus genus, HTLV-1 virus and their species.

Some embodiments of the present invention relate to compositions and methods for the treatment of viral infections from the following viruses: arenaviridae, arteriviridae, bunyaviridae, filoviridae, flaviviridae (flaviviridae) Genus), Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae (delta retroviral genus), Coronaviridae, (+)-ss-envRNAV, (-)-ss-envRNAV or Togaviridae.

Part embodiment of the invention relates to the treatment of a virus from a genus Henney, genus Ebola virus, flavivirus, Marburg virus genus, genus [delta] retrovirus, coronavirus (CoV) viral or α genus of viruses The composition and method of infection.

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

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 tissues. In some embodiments, the coronavirus is selected from the group consisting of: SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1 and CoV HKU20 .

In some embodiments, (+)-ss-envRNAV is selected from the following components Groups of viruses: flavivirus, yellow fever virus, dengue fever virus, Japanese encephalitis virus, West Nile virus, Zika virus, tick-borne encephalitis virus, Kjeldahl forest disease virus, Alkhurma disease virus, Omsk hemorrhagic fever Virus and Powassen virus.

In some embodiments, (+)-ss-envRNAV is a togaviridae virus selected from the group consisting of: arborvirus (arborvirus), eastern equine encephalomyelitis virus (EEEV), western equine brain and spinal cord Inflammatory virus (WEEV), Venezuelan equine encephalomyelitis virus (VEEV), Tragonia virus (CHIKV), Anionion virus (ONNV), Pogosta disease virus, Sindbis virus, Ross River fever virus (RRV) And Semliki Forest Virus.

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

In some embodiments, the arenaviridae virus is selected from the group consisting of: Lyssa virus, aseptic meningitis, Guanarito virus, Junin virus, Luyo virus, Matjupo virus, Sabia virus Virus and White Water River Virus.

In some embodiments, the Bunyaviridae virus is selected from the group consisting of: Hantavirus, Crimean-Congo Haemorrhagic Fever, Oronerovirus.

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

In some embodiments, the Orthomyxoviridae virus is selected from the group consisting of influenza virus (A to C), salmon anemia virus, togotu virus, quaranza virus, H1N1 virus, H2N2 virus, H3N2 virus , H1N2 virus, Spanish influenza virus, Asian influenza virus, Hong Kong influenza virus and Russian influenza virus.

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

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

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

In some embodiments, the present invention provides an antiviral composition containing the following (essentially consisting of): a) a specific cardiac glycoside (one or more); b) multiple triterpenes; or c) a specific cardiac glycoside A combination of glycosides (one or more) and multiple triterpenes.

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

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

The present invention also provides a method for treating viral infections for subjects in need The aforementioned methods include:

Determine whether the subject has a viral infection;

Instruct the administration of the antiviral composition;

Administer the initial dose of the antiviral composition to the subject for a period of time according to the prescribed initial dosing schedule;

Regularly determine the subject's clinical response and/or the appropriateness of the treatment response to treatment with the antiviral composition; and

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

If the clinical response and/or treatment response of the subject under the initial dose and initial dosing regimen is inappropriate, the dose is increased or decreased until the desired clinical response and/or treatment response is achieved in the subject.

Continue to administer the required antiviral composition to the subject for treatment, and adjust the dose or dosing schedule as needed until the patient reaches the desired clinical endpoint (one or more), such as reducing or alleviating specific symptoms related to viral infection . The appropriateness of clinical response and/or treatment response can be determined by clinicians familiar with viral infections.

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

The present invention provides alternative embodiments for all the embodiments described in the specification, in which oleandrin is substituted for oleandrin, or oleandrin is used in combination with digitonin. The method of the present invention can use oleandrin, digitonin or a combination of oleandrin and digitonin. Therefore, oleandrin, digitonin, a composition containing oleandrin, a composition containing oleandrin, or a composition containing oleandrin and digitonin can be used in the method of the present invention. Cardiac glycosides can be considered as oleandrin, digitonin or Its combination. The composition containing cardiac glycosides includes oleandrin, digitonin or a combination thereof.

The present invention also provides a method for the treatment of coronavirus infections, especially infections with coronaviruses that are pathogenic to humans (such as SARS-CoV-2 infection), the foregoing method comprising long-term administration to subjects suffering from the foregoing infections A therapeutically effective dose of cardiac glycosides (a composition containing cardiac glycosides).

The present invention also provides a two-way method for the treatment of coronavirus infections, especially coronavirus infections that are pathogenic to humans, such as SARS-CoV-2 infections, the foregoing method comprising long-term administration of treatment to subjects suffering from the foregoing infections An effective dose of cardiac glycosides (a composition containing cardiac glycosides) can inhibit the viral replication of the aforementioned coronaviruses and reduce the infectivity of the progeny viruses of the aforementioned coronaviruses.

The present invention also provides a method for the treatment of coronavirus infections, especially SARS-CoV-2 infections, by repeating administration (by any of the administration methods discussed in this specification) to subjects suffering from the aforementioned infections. A therapeutically effective dose of cardiac glycosides (a composition containing cardiac glycosides). One or more doses can be administered daily on one or more days of the week, any one or more weeks per month, and any one month or more months per year.

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

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

In some embodiments, the concentration of oleandrin and/or digitonin is about 10 μg/mL or less, about 5 μg/mL or less, about 2.5 μg/mL or less, about 2 μg/mL or less, or about 1 μg/mL or less. In some embodiments, the concentration of oleandrin and/or digitonin in the plasma of subjects suffering from coronavirus infection is about 0.0001 μg/mL or more, about 0.0005 μg/mL or more, or about 0.001 μg/mL or more, about 0.0015 μg/mL or more, about 0.01 μg/mL or more, about 0.015 μg/mL or more, about 0.1 μg/mL or more, about 0.15 μg/mL or more, about 0.05 μg/mL or more, or about 0.075 Above μg/mL. In some embodiments, the concentration of oleandrin and/or digitonin in the plasma of subjects treated for infection is about 10 μg/mL to about 0.0001 μg/mL, about 5 μg/mL to about 0.0005 μg/mL mL, about 1μg/mL to about 0.001μg/mL, about 0.5μg/mL to about 0.001μg/mL, about 0.1μg/mL to about 0.001μg/mL, about 0.05μg/mL to about 0.001μg/mL, about 0.01 μg/mL to about 0.001 μg/mL, about 0.005 μg/mL to about 0.001 μg/mL. The present invention includes all combinations and options of the plasma concentration range described in this specification.

The antiviral composition can be administered for a long period of time, that is, in a repeated manner, such as every day, every other day, every two days, every three days, every four days, every five days, every six days, every week, every other week, every two weeks , Every three weeks, every month, every two months (bimonthly), every half month, every other month, every two months, every quarter, every other quarter, every three months, seasonally, every It is applied half a year and/or every year. The treatment period is one week or more, one month or several months, one quarter or several quarters, and/or one year or multiple years. An effective dose of cardiac glycosides (composition containing cardiac glycosides) is administered one or more times a day.

In some embodiments, 140 μg of cardiac glycoside is administered to the subject daily to 315μg. In some embodiments, the dose contains 20 μg to 750 μg, 12 μg to 300 μg, or 12 μg to 120 μg of cardiac glycosides. The daily dose of cardiac glycoside may be 20 μg to 750 μg, 0.01 μg to 100 mg, or 0.01 μg to 100 μg of cardiac glycoside per day. The recommended daily dose of oleandrin present in the SCF extract is generally about 0.25 to about 50 μg twice daily, or about 0.9 to 5 μg twice daily or about every 12 hours. The dosage may be about 0.5 to about 100 μg/day, about 1 to about 80 μg/day, about 1.5 to about 60 μg/day, about 1.8 to about 60 μg/day, about 1.8 to about 40 μg/day. The maximum tolerated dose may be about 100μg/day, about 80μg/day, about 60μg/day, about 40μg/day, about 38.4μg/day or about 30μg/day of oleander extract containing oleandrin, and the minimum effective dose may be About 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day, or about 5 μg/day. A suitable dosage containing cardiac glycosides and triterpenes can be about 0.05~0.5mg/kg/day, about 0.05~0.35mg/kg/day, about 0.05~0.22mg/kg/day, about 0.05~0.4mg/kg/day , About 0.05~0.3mg/kg/day, about 0.05~0.5μg/kg/day, about 0.05~0.35μg/kg/day, about 0.05~0.22μg/kg/day, about 0.05~0.4μg/kg/day , Or about 0.05~0.3μg/kg/day. In some embodiments, the dosage of oleandrin is about 1 mg to about 0.05 mg, about 0.9 mg to about 0.07 mg, about 0.7 mg to about 0.1 mg, about 0.5 mg to about 0.1 mg, about 0.4 mg to about 0.1 mg, About 0.3mg to about 0.1mg, about 0.2mg. The present invention includes all combinations of the dosages described in this specification.

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

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

The aforementioned antiviral composition can be administered systemically. The method of systemic administration includes non-digestive Chemical path, oral and buccal, intestinal, intramuscular, subcutaneous, sublingual, oral, pulmonary or oral. The aforementioned composition can also be administered by injection or intravenously. The composition can also be administered to the same subject by two or more routes. In some embodiments, the composition is administered by a combination of any two or more modes of administration selected from the group consisting of: parenteral tract, oral buccal, intestinal, intramuscular, subcutaneous, sublingual , Oral, Lung and Oral.

The present invention also provides a sublingual dosage form, which comprises oleandrin and a liquid carrier. The present invention also provides a method for the treatment of viral infections, particularly coronavirus infections (such as defined in this specification), the foregoing method comprising sublingually administering a plurality of oleandrin (containing long leaf hairs) to subjects suffering from the foregoing viral infections Rehmannia Glucoside) composition dosage. One or more doses can be administered per day on two or more days a week and within one or more weeks of each month, optionally within one month or multiple months per year.

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

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

Some embodiments of the present invention include such pharmaceutical compositions containing at least one pharmaceutical excipient and an antiviral composition. In some embodiments, the antiviral composition includes: a) at least one cardiac glycoside and at least one triterpene; b) at least one cardiac glycoside and at least two triterpenes; c) at least one cardiac glycoside and at least three triterpenes; d) at least two triterpenes and no cardiac glycosides; e) at least three triterpenes and no cardiac glycosides; or f) at least one cardiac glycoside, such as oleandrin and digitonin. As used in this specification, unless otherwise indicated, the general terms triterpene and cardiac glycoside also include their salts and derivatives.

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

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

Embodiments of the present invention include Nerium sp. biomass (plant material) containing oleandrin: Nerium oleander ( Nerium oleander L) ( Nerium oleander), Nerium odourum , White oleander , Pink oleander, yellow oleander species ( Thevetia sp.), yellow oleander ( Thevetia peruviana ), yellow oleander, yellow oleander (Thevetia nerifolia) , Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), cell cultures (cell clusters) of any of the foregoing species ) Or a combination thereof. In some embodiments, the biomass includes leaves, stems, flowers, bark, fruits, seeds, sap, and/or pods.

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

In some embodiments, the extract contains one or more cardiac glycosides and one or more cardiac glycoside precursors (such as cardiac steroids, cardadienolides and cardatrienolides), all of which are cardiac glycosides The aglycone component of, for example, digoxigenin, acetyl-digitoxin, digitontoxin, digitonin, aceto-digitonin, long-leaf hair Rehmannia aglycone, digitonin, cephalosporin, cephalin, ouabain or cephalosporin). The extract may further include one or more glycoside components of the cardiac glycoside (for example, glucoside, fructoside and/or glucuronide) as a cardiac glycoside precursor. Therefore, the antiviral composition may include one or more cardiac glycosides and two or more cardiac glycoside precursors selected from the group consisting of one or more aglycone components and one or more glycosomal components. The extract may also contain one or more other non-cardiac glycoside therapeutically effective agents obtained from plant materials of Apocynaceae species or Apocynaceae species.

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

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

In some embodiments, the molar ratio of a single triterpene to oleandrin ranges from about 2 to 8 (OA): about 2 to 8 (UA): about 0.1 to 1 (BA): about 0.5 to 1.5 ( OL); or about 3~6(OA): about 3~6(UA): about 0.3~8(BA): about 0.7~1.2(OL); or about 4~5(OA): about 4~5( UA): about 0.4 to 0.7 (BA): about 0.9 to 1.1 (OL); or about 4.6 (OA): about 4.4 (UA): about 0.6 (BA): about 1 (OL).

In some embodiments, other therapeutic agents, such as those obtained by extraction of plant materials from Apocynaceae species or Apocynaceae species, are not polysaccharides obtained during the preparation of the extract, which means that they are not acidic homogalactose. Uronic acid (homopolygalacturonan) or arabinogalacturonan (arabinogalaturonan). In some embodiments, the extract does not contain other therapeutic agents and/or does not contain homogalacturonic acid or arabinogalacturonic acid obtained during the preparation of the extract.

In some embodiments, other therapeutic agents, such as those obtained by extraction of plant materials from Apocynaceae species or Apocynaceae species, are polysaccharides obtained during the preparation of the extract, such as acidic homopolygalacturonic acid. Or arabinogalacturonic acid. In some embodiments, the extract contains another therapeutic agent and/or the acidic homogalacturonic acid or arabinogalacturonic acid obtained when the extract is prepared from the aforementioned plant material.

In some embodiments, the extract comprises oleandrin and at least one other compound selected from the group consisting of cardiac glycosides, glycosides, aglycones, steroids, triterpenes, polysaccharides, sugars, alkaloids, fats , Protein, oleandroside, ordonoside, oleanolic acid, ursolic acid, betulinic acid, oleandrin, oleandrin A, betulin (Urso-12-ene-3β, 28-diol) , 28-Norsol-12-ene-3β-ol, Urso-12-ene-3β-ol, 3β,3β-hydroxy-12-oleanolic-28-acid, 3β,20α-dihydroxy Urso-21-ene-28-acid, 3β,27-dihydroxy-12-ursoene-28-acid, 3β,13β-dihydroxyurso-11-ene-28-acid, 3β,12α-di Hydroxyoleanane-28,13β-lactone, 3β,27-dihydroxy-12-oleanolic-28-acid, homogalacturonic acid, arabinogalacturonic acid, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumarol coenzyme A, 3-O-caffeine quinic acid, 5-O-caffeine quinic acid, cardiac glycoside B-1, cardiac glycoside B-2, oleandrin (oleagenin), gangliosides (neridiginoside), nerizoside (nerizoside), ordonoside-H, 3-β-β- composed of galacturonic acid, rhamnose, arabinose, xylose and galactose O-(D-Glyoside)-5-β,14β-Dihydroxy-cardonosta-20(22)-nonolactone pectin polysaccharide, MW is in the range of 17000~120000D or MW is about 35000D, about 3000D , About 5500D or about 12000D polysaccharides, cardenolide monoglycoside (cardenolide monoglycoside), cardiac glycoside N-1, cardiac glycoside N-2, cardiac glycoside N-3, cardiac glycoside N-4, pregnane, 4,6-di Ene-3,12,20-trione, 20R-hydroxypregna-4,6-diene-3,12-dione, 16β,17β-epoxy-12β-hydroxypregna-4,6-diene -3,20-dione, 12β-hydroxyprogesterone-4,6,16-triene-3,20-dione (Ouyidienone A), 20S,21-dihydroxyprogester-4,6 -Diene-3,12-dione (Ouyi Dienone B), neriucoumaric acid, isneriucoumaric acid, oleanderoic acid, oleanderen , 8α-Methoxy half-day flower-18-acid, 12-ursoene, mannoside (kaneroside), neriumoside, 3β-O-(D-diquat glycoside)-2α-hydroxy-8,14β-epoxy -5β-Cardiotoner-16:17,20:22-Dienolactone, 3β-O-(D-dipyridine glycoside) -2α,14β-Dihydroxy-5β-Cardiotoner-16:17,20:22-dienolactone, 3β,27-dihydroxy-Usol-18-ene-13,28-lactone, 3β,22α , 28-trihydroxy-25-nor-lupin-1(10),20(29)-dien-2-one, cis -karenin (3β-hydroxy-28-Z-p-couma Oxy-Urso-12-ene -27-acid), trans -Carrinin (3-β-hydroxy-28-E-p-coumaroloxy-Urso-12-ene-27-acid) , 3β-hydroxy-5α-cardiotoner-14(15), 20(22)-dienolactone (β-anhydrourosaglycone), 3β-O-(D-digitonin)-21-hydroxyl -5β-Cardiotonin-8,14,16,20(22)-tetranolactone (neriumogenin-A-3β-D-digitaloside (neriumogenin-A-3β-D-digitaloside)), horn Proceragenin, Ouyi Dienone A, 3β,27-dihydroxy-12-Ursole-28-acid, 3β,13β-Dihydroxyursol-11-ene-28-acid, 3β- Hydroxy Urso-12-ene-28-aldehyde, 28-Methyl Urso-12-ene-3β-alcohol, Urso-12-ene-3β-alcohol, Urso-12-ene-3β,28-di Alcohol, 3β,27-dihydroxy-12-oleanolene-28-acid, (20S,24R)-epoxydammarane-3β,25-diol, 20β,28-epoxy-28α-methyl Oxyl dandelion ster-3β-alcohol, 20β, 28-epoxy dandelion ster-21-en-3β-alcohol, 28-nor-urso-12-ene-3β, 17β-diol, 3β-hydroxyl 12-ene-28-aldehyde, α-neriursate, β-neriursate, 3α-acetoxyphenoxy-urso-12-ene-28-acid, oleanolic acid, kanerodione, 3β- P-Hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursene-28-acid, 28-hydroxy-20(29)-luene-3,7-dione, kanerocin, 3α-hydroxy -Urso-18,20-diene-28-acid, D-salmonose, D-digrainose, ganglioside, nerolin, isoricinoleic acid, gentioside (gentiobiglycoside nerve Glycoside), gentiobiosylbeaumontoside, gentiobiosyloleandrin, folinerin, 12β-hydroxy-5β-carda-8, 14, 16, 20 (22 )-Tetraenolactone, 8β-hydroxy-digitoxin, △16-8β-hydroxy-digitoxin, △16-neriagenin, black Alcohol, ursaldehyde, 27 (p-coumarol) ursolic acid, oleanol, 16-anhydro-deacetyl-ganglioside, 9-D-hydroxy-cis-12-octadecanoic acid, Adi Fruit glycosides (adigoside), oleandrin B, α-Aromatics, β-sitosterol, campesterol, rubber (caoutchouc), capric acid, caprylic acid, choline, cornerin, cortenerin, deacetyl oleandrin, Diacetyl-ganglioside, oleandrin, pseudocuramine, quercetin, quercetin-3-rhamnoside, quercetin, rosaginin, rutin, stearic acid, Stigmasterol, digitoxin, urehitoxin and ursaglycin. The other components present in the extract are disclosed by Gupta et al. (IJPSR (2010(, 1(3), 21-27, the entire disclosure of which is incorporated into this specification by reference)).

Oleandrin can also be obtained from an extract derived from a suspension culture of callus transformed by Agrobacterium tumefaciens. According to the present invention, hot water, organic solvents, aqueous organic solvents or supercritical fluid extracts of Agrobacterium can be used.

Oleandrin can also be obtained from the extract of the in vitro microculture of Oleander, thus The stem segment culture can be started from seedlings and/or stem tips of oleander species such as Splendens Giganteum, Revanche or Alsace or other varieties. According to the present invention, hot water, organic solvent, aqueous organic solvent or supercritical fluid extract of micro-cultured oleander can be used.

The extract can also be obtained by extracting cell masses (for example, present in cell culture) of any of the aforementioned plant species.

The present invention also provides the use of cardiac glycosides in the preparation of medicaments for treating viral infections in subjects. In some embodiments, the preparation of the aforementioned drug includes: providing one or more antiviral compounds of the present invention; including one dose of the antiviral compound(s) in a pharmaceutical dosage form; and packaging the pharmaceutical dosage form. In some embodiments, it can be prepared as described in PCT International Application No. PCT/US06/29061. The preparation may also include one or more additional steps, such as: delivering the packaged dosage form to a supplier (retailer, wholesaler, and/or distributor); selling or otherwise providing the packaged dosage form to subjects with viral infections ; Contains the drug label and package insert, which provide instructions on the use of the dosage form, dosage regimen, administration, content and toxicological profile. In some embodiments, the treatment of viral infection includes: determining that the subject has a viral infection; administering the subject's pharmaceutical dosage form according to the dosing schedule; administering one or more pharmaceutical dosage forms to the subject, wherein according to The dosage regimen administers one or more of the aforementioned pharmaceutical dosage forms.

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

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

The pharmaceutical composition optionally further comprises: 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) a combination thereof .

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

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

The following drawings form part of this specification and describe exemplary embodiments of the claimed invention. Based on these drawings and the description of this specification, those with ordinary knowledge in the field can implement the present invention without undue experimentation.

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

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

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

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

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

Figure 8 is a chart describing the summary of the in vitro dose-response antiviral activities of various components (oleandrin, digitonin and PBI-05204) against Marburg virus in Vero E6 cells.

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

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

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

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

Figures 13A and 13B summarize the effects of various components (oleandrin, digitonin and PBI-05204) on Venezuelan equine encephalomyelitis virus (Figure 13A) and western equine encephalomyelitis virus (Figure 13B) in Vero E6 cells. ) In vitro dose-response graph of antiviral activity.

Figure 14 is a description and summary of the vector control (vehicle control), oleandrin or European oleander extract determined by the quantification of ^{HTLV-1 p19 Gag on the release of HTLV-1 replication or newly synthesized virus particles (see implementation} Example 19 and 20) chart. Untreated (UT) cells are shown for comparison. Any data represents at least three independent experiments. The data represents the mean ± standard deviation of the experiment (error bars).

Figure 15 is a chart describing the summary of the relative cytotoxicity of the carrier control group, oleandrin, and European oleander extract to the HTLV-1+SLB1 lymphoma T cell line. Any data representative At least three independent experiments. The data represents the mean ± standard deviation of the experiment (error bars).

Figures 16A-16F are representative micrographs depicting the results of phospholipid binding protein V-FITC (green) and PI (red) staining displayed by DIC phase difference in the merged image. Also provides individual phospholipid binding protein V-FITC and PI fluorescence channel images. Scale bar, 20μm.

Figure 17 is a graph describing the summary of the release effects of vector control group, oleandrin or Oleander extracts on HTLV-1 replication or newly synthesized virus particles from oleandrin-treated HTLV-1+ lymphoma T cells.

Figure 18 is a graph depicting a summary of the relative cytotoxicity of the vehicle control group, oleandrin or European oleander extract to treated huPBMC.

Figure 19 is a graph depicting a summary of the relative inhibition of HTLV-1 transmission in a huPBMC co-cultivation test of a carrier control group containing huPBMC and an extract of oleandrin and European oleander.

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

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

Figure 22 is a graph depicting the average data with standard errors (error bars) quantified from the microscopic image of Figure 21.

Figures 23A to 23D describe the results of VERO E6 cells infected with SARS-CoV-2 virus and treated with oleandrin (red bar) or control vector (medium) (black bar) 24 hours and 48 hours after "treatment" A graph of the logarithm of SARS-CoV-2 virus titer (PFU/mL) versus time (hour) (Example 28). The cells were pre-treated with oleandrin before infection. After the first 2 hours of culture after infection, the infected cells were washed to remove extracellular viruses and oleandrin. Next, the recovered infected cells were processed as follows. Treat the infected cells with oleandrin (Figure 23A: 1 μg/mL as an aqueous component in a 0.1% DMSO aqueous solution with RPMI 1640 medium; Figure 23C: 0.1 μg/mL in a 0.01% DMSO aqueous solution with RPMI 1640) or Only the control vehicle (Figure 23B: 0.1% DMSO aqueous solution with RPMI 1640; Figure 23D: 0.01% DMSO aqueous solution with RPMI 1640), and the virus titer was measured.

Figure 24A depicts virus replication (Y1, left axis) and Vero-E6 cell count (Y2, right axis: the expression of oleandrin against the potential cytotoxicity of the aforementioned cells) in the culture medium 24 hours after infection relative to the concentration of oleandrin (μg/mL) dual y-axis plot of percent inhibition (Example 29). Figure 24B is the culture used in Figure 24A, but obtained 48 hours after infection.

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

Figures 26A~26B describe the 24 hours (Figure 26A) and 48 hours (Figure 26B) after "treatment", for infection with SARS-CoV-2 virus followed by oleandrin (blue circle) or control vector (medium) (Red squares) VERO CCL-81 cells (normal halophilic monkey kidney cells; https://www.atcc.org/products/all/CCL-81.aspx) treated with SARS-CoV- 2 Graph of the logarithm of virus titer (PFU/mL) versus the concentration of oleandrin (Example 31).

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

Figures 27A-27D describe the VERO E6 cells infected with SARS-CoV-2 virus and treated with oleandrin (blue circle) or control vector (medium) (red square) 24 hours and 48 hours after "treatment" , Graph of logarithm of SARS-CoV-2 virus titer (PFU/mL) versus time (hour) (Example 28). The cells were pre-treated with oleandrin before infection. exist After the first 2 hours of culture after infection, the infected cells were washed to remove extracellular viruses and oleandrin. Next, the recovered infected cells were processed as follows. The infected cells were treated with oleandrin (Figure 27A: 0.005μg/mL in DMSO (0.005%) aqueous solution with RPMI 1640 medium as the aqueous component; Figure 27B: 0.01 in DMSO (0.01%) aqueous solution with RPMI 1640 μg/mL); Figure 27C: 0.05 μg/mL in DMSO (0.05%) aqueous solution with RPMI 1640; Figure 27D: 0.1 μg/mL in DMSO (0.1%) aqueous solution with RPMI 1640), and measured Virus titer.

Figures 28A and 28B depict the 24 hours (Figure 28A) and 48 hours (Figure 28B) after the "treatment", in the case of SARS-CoV-2 virus infection, followed by oleandrin (dark blue circle (Example 2) and Light blue circle (Example 3)) or control vehicle (medium) (dark red square (Example 2) and light red square (Example 3)) treated VERO 81 cells, SARS-CoV- 2 Graph of the logarithm of virus titer (PFU/mL) versus the concentration of oleandrin. Example 2 and Example 3 are merely repeated executions of this measurement.

Figures 29A and 29B are bar graphs depicting the virus titer in the culture medium relative to the concentration of oleandrin, where the virus titer was measured at 24 hours (Figure 29A) and 48 hours (Figure 29B) after infection. For some samples, before and after infection (2 hours), cells were treated with oleandrin (blue solid bars) or a control vehicle containing only DMSO (red solid bars). For other samples, use oleandrin (blue dashed bar: 12 hours after infection; open blue bar: 24 hours after infection) or control vector containing only DMSO (red dashed bar: 12 hours after infection; open red bar: infection After 24 hours) the cells were treated.

Figures 30A and 30B are graphs describing the anti-COVID-19 activity used to evaluate the dual extract composition (PBI-A). In Figure 30B, the text description of mg/ml (oleandrin concentration) means that PBI-A is provided as a 1 mg/ml (oleandrin concentration) solution. Determine the virus titer (Log ₁₀ (PFU/mL)) relative to the Log ₁₀ dilution factor (Figure 30A) or relative to the Log ₁₀ concentration of oleandrin (Figure 30B). FIG. 30A is for the data processing of the pre-infection measurement in Example 31, and FIG. 30B is for the processing of the post-infection measurement in Example 34.

The present invention provides for the treatment of subjects by long-term administration or acute administration of one or more effective doses of antiviral composition (or pharmaceutical composition comprising antiviral composition and at least one pharmaceutical excipient) to the subject Methods of virus infection. The composition is administered according to the dosage regimen most suitable for the subject, and the suitability of the clinical dosage and dosage regimen is determined according to routine clinical trials and clinical treatment endpoints of viral infections.

As used in this specification, the term "subject" means warm-blooded animals such as mammals, for example, cats, dogs, mice, guinea pigs, horses, cows, sheep, and humans.

As used in this manual, subjects who are at risk of viral infection are: a) Live in mosquitoes such as Aedes species ( Aedes egypti , Aedes albopictus ), etc. Subjects in the geographical area of life; b) Subjects living with or near a person or group of people with a virus infection; c) Subjects having a sexual relationship with a person with a virus infection; d) Living in Particularly, subjects in the geographic area where ticks live, such as Ixodes species (species: Ixodes marx , Ixodes scapularis , or Ixodes cooke); e) live in Subjects in the geographic area where the fruit bats live; f) Subjects living in tropical areas; g) Subjects living in Africa; h) Subjects contacting 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 female, a female capable of becoming pregnant, or a pregnant female.

Subjects treated in accordance with the present invention will exhibit a therapeutic response. "Therapeutic response" means that, as a result of treatment with cardiac glycosides, subjects suffering from viral infections will enjoy at least one of the following clinical benefits: reduction of active viral titer in the subject’s blood or plasma, and subject Blood or Eradication of active virus in plasma, improvement of infection, reduction in the occurrence of infection-related symptoms, partial or complete remission of infection or increase in infection progression time, and/or reduction in the infectivity of the virus that causes the aforementioned viral infection. The treatment response can be all or part of the treatment response.

As used in this manual, the "progression time" is the period, length or duration after the virus infection is diagnosed (or treated) until the infection begins to worsen. This term refers to a period of time when the infection level is flat without further deterioration, and this period ends when the infection starts to progress again. The progression of the disease is determined by "stages" the infected subjects before or at the beginning of treatment. For example, the health of the subject is determined before or at the beginning of treatment. The subject is then treated with the antiviral composition and the virus titer is monitored regularly. At a later point in time, the symptoms of the infection may worsen, thereby marking the progression of the infection and the end of the "progress time". The period during which the infection does not progress or the level or severity of the infection does not worsen during the period is the "progress time".

The dosing regimen includes one or more cardiac glycosides and/or triterpenes (one or more) in a treatment-related dose (or effective dose) administered according to the dosing plan. Therefore, the treatment-related dose is the therapeutic response observed when the antiviral composition is administered to treat a viral infection, and the antiviral composition can be administered to the subject without excessive undesirable or harmful side effects of the therapeutic dose. The treatment-related dose is non-lethal to the subject, although it may cause some side effects in the patient. The level of clinical benefit of the administration of the treatment-related dose to the subject of the antiviral composition will exceed the level of harmful side effects experienced by the subject due to the administration of the antiviral composition or its component(s). According to various established principles of pharmacology, pharmacodynamics and pharmacokinetics, treatment-related doses are different in different subjects. However, the treatment-related dose (for example, relative to oleandrin) is usually about 25 μg, about 100 μg, about 250 μg, about 500 μg, or about 750 μg of cardiac glycosides per day, or it may be in the range of about 25 to 750 μg of cardiac glycosides per dose. Alternatively, it may not exceed about 25 μg, about 100 μg, about 250 μg, about 500 μg, or about 750 μg of cardiac glycosides per day. Another example of a treatment-related dose (e.g., relative triterpenes alone or together) is generally about 0.1 μg to 100 μg, about 0.1 mg to about 500 mg, about 100 to about 1000 mg per kg body weight, about 15 to about 25 mg/kg, about 25 to about 50 mg/kg, about 50 to about 100 mg/kg, about 100 to about 200 mg/kg, about 200 to The range is about 500 mg/kg, about 10 to about 750 mg/kg, about 16 to about 640 mg/kg, about 15 to about 750 mg/kg, about 15 to about 700 mg/kg, or about 15 to about 650 mg/kg of body weight. According to the basic principles of pharmaceutics, it is known in the art that the actual amount of antiviral composition required to provide a subject's target treatment result is different in different subjects.

The digitonin treatment can be performed in two or more administration phases: the implementation phase and the maintenance phase. The following dosage regimen can be used in the implementation phase until the steady-state plasma level of digitonin is reached. After the implementation phase is completed, the following dosage regimen can be used in the maintenance phase.

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

Example 15 provides a detailed description of the in vitro test. The foregoing test was used to evaluate the ^{extraction of oleandrin (as the only active), Anvirzel TM} (hot water extract of oleander), and PBI-05204 (supercritical fluid (SCF) of oleander) Ebola virus (Figures 1~2) and Marburg virus (Figures 3~4), both of which are filoviruses.

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

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

Example 6 provides a detailed description of the in vitro test used to evaluate the efficacy of cardiac glycosides in treating Zika virus (a flavivirus) infection. In the presence of oleandrin (Figure 5) or digitonin (Figure 6), Vero E6 cells were infected with Zika virus (ZIKV PRVABC59 strain) at an MOI of 0.2. The cells were incubated with virus and cardiac glycosides for 1 hour, and then the inoculum and unabsorbed cardiac glycosides (if present) were removed. The cells were immersed in fresh culture medium and cultured for 48 hours, then the cells were fixed with formalin and stained for ZIKV infection. The data indicate that the two cardiac glycosides have antiviral activity against Zika virus; however, oleandrin exhibits a higher (almost 8 times larger) antiviral activity than digitonin.

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

Figure 7 is a graph summarizing the in vitro dose-response antiviral activities of various components (oleandrin, digitonin and PBI-05204) against Ebola virus (EBOV) in Vero E6 cells. Figure 8 is a description and summary of various components (oleandrin, digitonin and PBI-05204) Graph of in vitro dose response antiviral activity against Marburg virus (MARV) in Vero E6 cells. Figure 9 is a graph describing and summarizing the in vitro cell viability of Vero E6 cells in the presence of various components (oleandrin, digitonin and PBI-05204). For Figures 7-8, the host cells were exposed to the composition before infection with the virus. In the presence of oleandrin, digitonin or PBI-05204 (a plant extract containing oleandrin), use EBOV/Kik (Figure 7, MOI=1) or MARV/Ci67 (Figure 8, MOI =1) Infect Vero E6 cells. After 1 hour, the inoculum and compound were removed and fresh culture medium was added to the cells. After 48 hours, the cells were fixed and immunostained to detect cells infected with EBOV or MARV. Use Operetta to count infected cells.

To ensure that no false positives are observed in terms of antiviral activity, the cell survival rate in the presence of the composition is tested. For the data in Figure 9, Vero E6 cells were treated with the above compounds. ATP level was measured by CellTiter-Glo as a measure of cell viability. It has been determined that oleandrin, digitonin and PBI-05204 do not reduce cell viability, which means that the antiviral activity detailed in other figures in this specification is not caused by false positives caused by the cytotoxicity of a single compound .

Therefore, the present invention provides a method for treating viral infections in mammals or host cells, the foregoing method comprising: administering an antiviral composition to the mammals or host cells before being infected with the foregoing viral infections, so that the foregoing mammals or host cells When a cell is infected with a virus, the antiviral composition reduces the virus titer and improves, reduces or eliminates the virus infection.

The antiviral composition and method of the present invention are also useful in treating viral infections that occur before the administration of the antiviral composition. Vero E6 cells were infected with EBOV (Figure 10A, 10B) or MARV (Figure 11A, 11B). 2 hours after infection (Figure 10A, 11A) or 24 hours after infection (Figure 10B, 11B), oleandrin or PBI-05204 was added to the cells for 1 hour, then discarded, and the cells were cultured in the culture medium.

Figures 10A and 10B are graphs describing and summarizing the ability of the composition (oleandrin and PBI-05204) to inhibit Ebola virus in Vero E6 cells shortly after exposure to the virus: Figure 10A-2 hours after infection; Figure 10B-After infection 24 hours. When the antiviral composition is administered within 2 hours (or within up to 12 hours) after viral infection, the viral titer antiviral composition provides effective treatment and reduces the EBOV virus titer. Even after 24 hours, the viral composition is effective; however, its efficacy decreases as the time after initial viral infection increases. The same assessment is performed for MARV. Figures 11A and 11B are graphs describing and summarizing the ability of the composition (oleandrin and PBI-05204) to inhibit Marburg virus in Vero E6 cells shortly after exposure to the virus: Figure 11A-2 hours after infection; Figure 11B-After infection 24 hours. When the antiviral composition is administered within 2 hours (or within up to 12 hours) after viral infection, the viral titer antiviral composition provides effective treatment and reduces MARV viral titer. Even after 24 hours, the viral composition is effective; however, its efficacy decreases as the time after initial viral infection increases.

Considering that the antiviral activity of the composition in this specification is reduced for the single generation of virus-infected cells, for example, within 24 hours after infection, we assessed whether the antiviral composition can inhibit virus reproduction, which means whether it inhibits the production of infectious offspring . In the presence of oleandrin or PBI-05204, Vero E6 cells were infected with EBOV or MARV and cultured for 48 hours. The supernatant from the infected cell culture was transferred to fresh Vero E6 cells for culture, cultured for 1 hour, and then discarded. The cells containing the transferred supernatant were cultured for 48 hours. As described in this specification, evaluate cells infected with EBOV (B) or MARV (C). The control infection rate of EBOV was 66%, and the control infection rate of MARV was 67%. The antiviral composition of the present invention inhibits the production of infectious offspring.

Therefore, the antiviral composition of the present invention: a) can be administered prophylactically before viral infection to inhibit viral infection after exposure to the virus; b) can be administered after viral infection to inhibit or reduce viral replication and infection of offspring Produce; or c) that is a combination of a) and b).

The VEE virus and WEE virus in Vero E6 cells were used to evaluate the antiviral activity of the antiviral composition against Alphavirus of the Togaviridae family. Figures 13A and 13B describe and summarize the effects of various components (oleandrin, digitonin and PBI-05204) on Venezuelan equine encephalomyelitis virus (Figure 13A) and western equine encephalomyelitis virus (Figure 13B) in Vero E6 cells. ) In vitro dose-response graph of antiviral activity. In the presence or absence of the indicator compound, Vero E6 cells were infected with Venezuelan equine encephalitis virus (Figure 13A, MOI=0.01) or western equine encephalitis virus (Figure 13B, MOI=0.1) for 18 hours. The infected cells were detected and counted on Operetta as previously described. It has been found that the antiviral composition of the present invention is effective.

Therefore, the present invention provides a method for treating a virus infection in a subject or host cell caused by: arenaviridae, filoviridae, flaviviridae (flavivirus), retroviridae Viruses, delta retroviruses, coronaviruses, paramyxoviridae, or togaviridae viruses, the foregoing method comprises administering an effective amount of an antiviral composition, thereby exposing the virus to the antiviral composition and treating the foregoing Viral infection.

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

Figure 14 is a description of the oleandrin compound or the extract of European oleander (Examples 19 and 20) with a control carrier (1.5μL, 7.5μL or 15μL), or an increased concentration (10μg/mL, 50μg/mL and 100μg/mL). ) Quantitative data ^{of HTLV-1 p19 Gag} expressed by the HTLV-1+SLB1 lymphoma T cell line treated for 72 hours. Anti-HTLV-1 p19 ^Gag ELISA (Zeptometrix) was performed to quantify virus replication and extracellular particles released into the culture supernatant. Oleandrin did not significantly inhibit HTLV-1 replication or the release of newly synthesized virus particles. We determined that neither the extract nor the oleandrin will significantly inhibit virus replication or the ^{release of p19 Gag-} containing particles into the culture supernatant. Therefore, we expected no further antiviral activity; however, we unexpectedly found that virus particles collected from the treated cells showed reduced infectivity to primary human peripheral blood mononuclear cells (huPBMC). Unlike HIV-1, extracellular HTLV-1 particles are less infectious, and virus transmission usually occurs through direct cell-to-cell interaction across viral synapses.

The present invention therefore provides a method for producing HTLV-1 virus particles with reduced infectivity. The aforementioned method comprises treating the HTLV-1 virus particles with the antiviral composition of the present invention to provide HTLV-1 virus particles with reduced infectivity.

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

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

Next, we investigated whether oleandrin or European oleander extract can inhibit the transmission of the virus from the HTLV-1+ lymphoma T cell line expressing green fluorescent protein (GFP) to huPBMC in a co-culture experiment (Example 20). For these studies, HTLV-1+SLB1 lymphoma T cells were treated with increasing concentrations of oleandrin compounds or European oleander extract, or a control vehicle in a 96-well microtiter plate for 72 hours, and then the virus-containing supernatant was collected And directly used to infect primary cultured human peripheral blood mononuclear cells (huPBMC) in vitro. ^{After 72 hours, the relative level of extracellular p19 Gag-} containing virus particles released into the culture supernatant as a result of direct infection was quantified by anti-HTLV-1 p19 ^Gag ELISA.

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

The data (Figure 17) shows that compared with the same amount of control vehicle, even at the lowest concentration (10μg/ml), both oleandrin and European oleander extract can inhibit the release to the culture supernatant of the treated cells The newly synthesized ^{virus particles containing p19 Gag} in the liquid are infectious. Both oleandrin and crude extract inhibit the formation of viral synapses and the spread of HTLV-1 in vitro. Extracellular virus particles produced by HTLV-1+ lymphoma T cells treated with oleandrin showed reduced infectivity to primary huPBMC. Importantly, oleandrin exhibits antiviral activity against enveloped viruses by reducing the incorporation of envelope glycoproteins into mature particles, which represents a unique stage of the retroviral infection cycle.

In order to ensure that the observed antiviral activity is not an artifact caused by the potential cytotoxicity of the antiviral composition to the treated huPBMC, we also studied (Example 21), compared with the carrier negative control group, in the treated huPBMC The cytotoxicity of purified oleandrin and European oleander extract. The primary buffy coat huPBMC were isolated, stimulated with phytohemagglutinin (PHA), and cultured in the presence of recombinant human interleukin-2 (hIL-2). The cells were then treated with an increased concentration of oleandrin or European oleander extract, or with an increased volume of the carrier for 72 hours. Subsequently, the sample was stained with Annexin V-FITC and PI, and the count was repeated three times by a conjugated fluorescence microscope to quantify the relative percentage of apoptotic (ie, Annexin V-FITC and/or PI positive) cells per field.

By treating the primary huPBMC for 72 hours, the cytotoxic effects of the control vehicle, European oleander extract, and oleandrin compounds were evaluated, and then the cultures were stained with AnnexinV-FITC and PI. Quantify the relative percentage of apoptotic (ie, AnnexinV-FITC and/or PI positive) cells by using a fluorescent microscope and counting a field of view repeated three times using a 20x objective lens. Determine the total number of cells using a DIC phase contrast microscope. Cyclophosphamide (50 μM) treated cells were included as a positive control group for apoptosis. NA indicates that the number of cells in this sample is too low because it is highly toxic and cannot be accurately assessed.

The data (Figure 18) indicated that oleandrin showed moderate cytotoxicity in huPBMC compared to the control vehicle (for example, 35~37% at the lowest concentration). On the contrary, European oleander extract has significant cytotoxicity and can induce high levels of programmed cell death even at the lowest concentration. Compared with HTLV-1+SLB1 lymphoblasts, huPBMC is more sensitive to purified oleandrin. However, huPBMC is more sensitive to crude European oleander extract, which The extract also contains other cytotoxic compounds, such as the triterpenes described in this specification.

We further studied (Example 22) whether oleandrin or European oleander extract can interfere with the transmission of HTLV-1 particles to the target huPBMC in the co-culture experiment. For these studies, the virus-producing HTLV-1+SLB1 T cell line was treated with mitomycin C followed by increasing amounts of oleandrin, European oleander extract, or a control vector for 15 minutes or 3 hours. Wash SLB1 cells twice with serum-free culture medium, then add the same amount of huPBMC to each well, and incubate the sample in a complete culture medium _{at 37°C and 10% CO 2 in a humidified incubator for 72 hours.} . The relative cell-to-cell transmission of HTLV-1 was evaluated by performing an anti-HTLV-1 p19 ^{Gag ELISA to determine the level of extracellular virus released into the culture supernatant.}

The primary generation huPBMC and mitomycin C-treated HTLV-1+SLB1 lymphoma T cells were co-cultured, and the control vector was used to increase the concentration (10μg/mL, 50μg/mL and 100μg/mL) of European oleander extract or oleander Glycoside compounds pretreated HTLV-1+SLB1 lymphoma T cells for 15 minutes or 3 hours. The control group carrier, extract and compound were also present in the co-culture solution. After 72 hours, the supernatant was collected, and the amount of extracellular virus particles released ^{by anti-HTLV-1 p19 Gag ELISA was quantified.}

The results described in Figure 19 indicate that compared with the control vehicle, both oleandrin and European oleander extract inhibited the spread of HTLV-1; however, no observation was observed between 15 minutes and 3 hours of pretreatment of HTLV-1+SLB1 cells difference.

We also investigated whether oleandrin inhibits the formation of viral synapses and the spread of HTLV-1 in the co-culture experiment (Example 22). By transducing SLB1 lymphoma T cells with pLenti-6.2/V5-DEST-GFP vector, and using blasticidin (5μg/mL; Life Technologies) for two weeks of screening, HTLV-1 expressing GFP was produced +SLB1 T cell line. GFP-positive colonies were screened by fluorescence microscopy (upper panel in Figure 20) and immunoblotting (lower panel in Figure 20), and then amplified and repeated. Provide DIC phase difference images for comparison.

Visualize the formation of viral synapses between huPBMC and mitomycin C-treated HTLV-1+SLB1/pLenti-GFP lymphoblasts (green cells) by fluorescence microscope, the aforementioned HTLV-1+SLB1/pLenti-GFP lymph Mother cells have been pretreated with control vehicle or increased amounts (10 μg/mL, 50 μg/mL, and 100 μg/mL) of European Oleander extract or Oleandrin compounds for 3 hours (Figure 21). The relative percentage of infected (i.e., HTLV-1 gp21-positive, red) huPBMC (GFP-negative) in 20 fields of view (n =20) was quantified by using a fluorescent microscope with a 20-fold objective lens to assess virus transmission (see The arrow in the control vector diagram). Measure the data obtained by the fluorescence microscope (Figure 22). It can be confirmed by the data that the antiviral composition inhibited the formation of viral synapses and the spread of HTLV-1 in the co-culture test.

Therefore, the present invention also provides a method for inhibiting (reducing) the infectivity of HTLV-1 particles released into the supernatant of the treated cell culture and reducing the spread of HTLV-1 cells by inhibiting the Env-dependent formation of viral synapses. Method, the aforementioned method comprises treating virus-infected cells (in vitro or in vivo) with an effective amount of antiviral composition.

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

As detailed in Example 26, CoV infection can be treated in vivo, where the antiviral composition is administered to the subject as a monotherapy or a combination therapy. According to Example 27, the efficacy of oleandrin against CoV was established in vivo. Mixed in a small portion of orange juice, 0.25 ml of recombinant ANVIRZEL ^{TM is} administered to children, and then 0.5 ml of recombinant ANVIRZEL ^{TM is} administered to children every 12 hours for about 2 to 3 days. The baby recovers from COVID-19 infection.

According to Example 28, further evidence of the efficacy of oleandrin (a composition containing oleandrin) against coronaviruses such as SARS-CoV-2 (COVID-19) was obtained by in vitro evaluation, in which Vero cells were pretreated with oleandrin, and then After infection with SARS-CoV-2, cells were infected with extracellular virus and oleandrin, and then the infected cells were treated with oleandrin (Figure 23A: oleandrin was 1 μg/mL in 0.1% v/v aqueous DMSO; 23C: Oleandrin in 0.01% v/v aqueous DMSO is 0.1 μg/mL) or only aqueous DMSO is used as the control carrier (Figure 23B: 0.1% v/v aqueous DMSO; Figure 23D: 0.01% v/v aqueous DMSO ). The results showed that a) Oleandrin pretreatment caused a 1368-fold reduction in viral load at 24 hours and a 369-fold reduction at 48 hours; b) Oleandrin was effective in the entire concentration range of about 0.1 to about 1.0 μg/mL The high dose is slightly better than the low dose, so oleandrin is likely to be effective at even lower concentrations, such as 0.01 to 0.1 μg/mL; c) oleandrin should be administered repeatedly, because a single dose is not enough to stop completely Virus replication; and d) Pre-culturing Vero cells with only oleandrin for 30 minutes is only slightly effective in reducing the initial viral infection, and does not seem to affect the infectivity of the offspring virions. The results also showed that oleandrin at concentrations of 0.1 and 1.0 μg/mL was not excessively toxic to Vero cells. The results further show that oleandrin inhibits the infectivity of offspring viruses by: a) about 1 log ₁₀ without continuous drug treatment; and b) about >3 log ₁₀ continuous drug treatment (non-toxic).

In order to determine whether oleandrin directly inhibits virus replication, according to Example 29, the SARS-CoV-2 virus was used to infect Vero-E6 cells and treated with different concentrations of oleandrin. The results are depicted in Figures 24A and 24B. 24 hour time point (FIG. 24A), only in the hole of oleandrin treated only in the absorption stage (pre-data), the observed antiviral activity, IC ₅₀ of approximately 0.625μg / mL. In the wells treated with oleandrin, for the test duration (duration data), even in the presence of a large amount of inoculated virus, oleandrin significantly restricted virus entry and/or virus replication. At the 48 hour time point (Figure 24B), in the wells treated with oleandrin, only during the absorption phase (pretreatment data), the lowest antiviral activity was observed at the end of the time period. For the test duration (duration data) in the wells treated with oleandrin, oleandrin significantly inhibited viral infection. Possible methods of action include inhibition of virus replication, assembly and/or release.

In order to ensure that the observed antiviral activity of oleandrin against SARS-CoV-2 was not caused by the extracellular toxicity of oleandrin against Vero-E6 cells, it was determined at 24 hours (Figure 24A) and 48 hours (Figure 24B). Cell titer. When the concentration of oleandrin is above 1.0μg/mL, cytotoxicity appears and potentially interferes with the determination; however, when the concentration of oleandrin is below 0.625μg/mL, it significantly reduces the interference of cytotoxicity, thus confirming that even at very low levels The strong antiviral activity of oleandrin at a concentration of 100%. Additional evidence of the degree of toxicity of oleandrin against Vero-E6 cells was observed in the assay of Example 30 (Figure 25). When the concentration of oleandrin was 0.625μg/mL, at the 24-hour time point, about 80% of Vero cells could survive, and the toxicity observed at lower concentrations was even lower. It should be understood that the toxicity of oleandrin to Vero-E6 cells does not indicate that oleandrin is toxic to humans. When measuring antiviral activity, this toxicity test is only used to determine the potential impact of background cell death.

Therefore, oleandrin has at least a dual mechanism (approach) for the treatment of viral infections, especially coronavirus infections, such as SARS-CoV-2 infection: a) directly inhibiting virus replication; and b) reducing the infectivity of offspring viruses.

In addition, oleandrin has antiviral activity even at very low doses, and oleandrin exhibits substantial preventive effects. This is described according to Example 31, where SARS-CoV-2 is used to infect VERO CCL-81 cells. The cells were pre-treated with oleandrin before infection. After an initial culture for 2 hours after infection, the infected cells were washed to remove extracellular viruses and oleandrin. Next, the recovered infected cells were processed as follows. Treat the infected cells with oleandrin (in various concentrations of oleandrin as a component of the aqueous solution in a DMSO aqueous solution with RPMI 1640 medium) or only the control vehicle (a DMSO aqueous solution with RPMI 1640), and after "treatment" Virus titer was measured at 24 hours (Figure 26A) and 48 hours (Figure 26B). In the absence of oleandrin, SARS-CoV-2 reached a high _{titer (about 6 log 10} plaque forming units (pfu)/mL) at a 24-hour time point, and maintained that titer at subsequent time points: It always remains at or below the detection limit of the determination. Oleandrin at a concentration of 1 μg/mL to 0.05 μg/mL provides a greatly reduced virus titer even in exactly 24 hours. The two higher doses basically reduced the virus titer to equal to or below the detection limit, and no cytotoxicity was observed at any tested oleandrin concentration. The calculated virus titer of these samples showed a multiple reduction. The fold reduction in virus titer was observed at the 48-hour time point (FIGS. 26C and 26D) ranging from about 1,000-fold to about 40,000-fold, and at the 24-hour time point, it was observed from about 1,000-fold to about 20,000-fold. Although the 10 ng/mL dose did not have a significant effect compared to its DMSO control group at 24 hours after infection, it resulted in a significant decrease in titer at 48 hours after infection. It is worth noting that the highest value of the virus titer reduction factor caused by oleandrin was not observed at the 24th hour, but at the 48th hour. The increase in the preventive efficacy of oleandrin over time (24 hours and 48 hours) is reflected in its EC ₅₀ value, which is calculated as 11.98 ng/ml at 24 hours after infection and 7.07 ng/ml at 48 hours after infection.

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

Additional studies were performed to determine the dose response of oleandrin to COVID-19 infection at 24 and 48 hours after infection (Figures 27A-27B). A dose-specific response was observed, that is, increasing the concentration of oleandrin in the culture solution greatly reduces the virus titer. Even the lowest concentration in the test (0.05μg/mL) will cause the titer to decrease 24 hours after infection, and it will be 48 hours after infection. Hours even cause a greater titer reduction. The highest dose resulted in a more than 1,000-fold reduction in infectious SARS-CoV-2 titer, with 0.5μg/mL and 100ng/mL doses resulting in a reduction greater than 100-fold, and 50ng/ml dose resulting in a 78-fold reduction.

Figures 28A and 28B depict the results of repeated studies. Each study was repeated three times to determine the dose response of COVID-19 to different concentrations of oleandrin in the culture medium (0.005 to 1 μg/mL). At a concentration greater than 0.01 μg/mL, in Vero 81 cells, a large amount of antiviral activity can be observed even 24 hours and 48 hours after infection. Even at a very low concentration of 0.05 μg/mL, a significant decrease in virus titer is observed.

In order to determine the antiviral efficacy of oleandrin after infection, a study according to Example 34 was carried out. Vero 81 cells were not pretreated with oleandrin before infection. Instead, the cells were infected with the COVID-19 virus, followed by treatment with oleandrin (according to the specified concentration) 12 hours and 24 hours after infection. The virus titer was then measured 24 hours (Figure 29A) and 48 hours (Figure 29B) after infection. The data shows that even with only a single treatment, oleandrin can exhibit antiviral activity at least 12 hours, at least 24 hours, or at least 36 hours after infection. It is worth noting that compared with the human virus infection process, the measurement is compressed in time. The 24-hour time point is equivalent to about 5 to 7 days after human infection, and the 48-hour time point is equivalent to about 10 to about 10 days after human infection. 14 days.

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

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

It is also important to observe that the concentration of oleandrin evaluated in the assay is clinically relevant in terms of dose and plasma concentration.

Evidence of the safety of the composition containing oleandrin is further provided by in vitro cell assays for determining the release of lactate dehydrogenase after the aforementioned cells are exposed to solutions containing different concentrations of oleandrin. It has been determined that at a concentration of up to 1 μg/mL, there is no other toxicity compared with the control vehicle.

The efficacy of oleandrin (a composition containing oleandrin, an extract containing oleandrin) in the treatment of COVID-19 virus infection was further based on Example 35 under the FDA's Expanded Access program of the FDA. The test subjects administered a sublingual dosage form containing oleandrin (Example 32 or 37) to establish it. Four 15 μg doses of oleandrin (as a dual extract composition) per day at about 6 hour intervals or three 15 mg doses per day at about 8 hour intervals were administered to subjects aged 18 to 78 years old. Before starting treatment, observe the subject's clinical status and/or virus titer. Some subjects received palliative care or hospice care. During one to two weeks, ten to fourteen days of treatment, the clinical status and/or virus titer is determined regularly. The following results were observed after starting the treatment.

Under the FDA's expanded acquisition program, another in vivo study was conducted on the second group of subjects who showed varying degrees of COVID-19-related symptoms. Before starting treatment, determine the subject's clinical status and/or virus titer to confirm COVID-19 infection. Some subjects showed moderate to severe symptoms. Four groups of 15 μg doses of oleandrin (as a dual extract composition) were administered to subjects of different ages at about 6 hour intervals every day. During one to two weeks, ten to fourteen days of treatment, the clinical status and/or virus titer is determined regularly. Within five to twelve days after starting treatment, all subjects completely recovered from COVID-19 infection.

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

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

Therefore, the present invention provides a method for the treatment of coronavirus infections, especially those that are pathogenic to humans, such as SARS-CoV-2. The foregoing method comprises long-term administration of a therapeutically effective dose of tonic to subjects with the foregoing infections. Glycosides (compositions containing cardiac glycosides). Long-term administration can be achieved by repeatedly administering one or more (plural) therapeutically effective doses of cardiac glycosides (a composition containing cardiac glycosides). It is possible to administer more than one dose per day for more than one day per week, arbitrarily for more than one week per month, and arbitrarily for more than one month per year.

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

PBI-05204 (such as this manual and Addington's US 8187644 B2 (Announcement on May 29, 2012), Addington's US 7402325 B2 (Announcement on July 22, 2008), Addington et al.'s US 8394434 B2 (March 2013) The 12th announcement), the entire disclosure of which is incorporated into this specification by reference) includes cardiac glycosides (oleandrin, OL) and triterpenes (oleanolic acid (OA), ursolic acid (UA) and betulinic acid) (BA)) as the main pharmacologically active component. The molar ratio of OL to all triterpenes is about 1: (10~96). The molar ratio of OA:UA:BA is approximately 7.8:7.4:1. When compared based on isomolar OL, the combination of OA, UA and BA in PBI-05204 increased the antiviral activity of oleandrin. PBI-04711 is a fraction of PBI-05204, but it does not contain cardiac glycosides (OL). The molar ratio of OA:UA:BA in PBI-04711 is about 3:2.2:1. PBI-04711 also has anti-virus active. Therefore, based on an equal molar content of OL, an antiviral composition containing OL, OA, UA, and BA is more effective than a composition containing OL as the sole active ingredient. In some embodiments, the molar ratio of a single triterpene to oleandrin ranges from about 2 to 8 (OA): about 2 to 8 (UA): about 0.1 to 1 (BA): about 0.5 to 1.5 (OL ); or about 3~6(OA): about 3~6(UA): about 0.3~8(BA): about 0.7~1.2(OL); or about 4~5(OA): about 4~5(UA ): about 0.4 to 0.7 (BA): about 0.9 to 1.1 (OL); or about 4.6 (OA): about 4.4 (UA): about 0.6 (BA): about 1 (OL).

An antiviral composition containing oleandrin as the sole antiviral agent is within the scope of the present invention. An antiviral composition containing digitonin as the sole antiviral agent is within the scope of the present invention.

An antiviral composition containing oleandrin and various triterpenes as antiviral agents is within the scope of the present invention. In some embodiments, the antiviral composition contains oleandrin, oleanolic acid (the free acid, salt, derivative or prodrug thereof), ursolic acid (the free acid, salt, derivative or prodrug thereof), and birch Acid (its free acid, salt, derivative or prodrug). The molar ratio of the compound is described in this specification.

Antiviral compositions containing multiple triterpenes as main active ingredients (meaning that they do not contain steroids, cardiac glycosides and pharmacologically active ingredients) are also within the scope of the present invention. As described above, PBI-04711 contains OA, UA and BA as the main active ingredients, and it exhibits antiviral activity. In some embodiments, the triterpene-based antiviral composition includes OA, UA, and BA, each of which is independently selected from its free acid form, salt form, deuterated form, and derivative form at each occurrence.

PBI-01011 is a triterpene-based modified antiviral composition containing OA, UA and BA. The molar ratio of OA:UA:BA is about 9-12: up to about 2: up to about 2; or about 10: About 1: about 1; or about 9~12: about 0.1~2: about 0.1~2; or about 9-11: about 0.5~1.5: about 0.5~1.5; or about 9.5~10.5: about 0.75~1.25: about 0.75~1.25; or about 9.5~10.5: about 0.8~1.2: about 0.8~1.2; or about 9.75~10.5: about 0.9~1.1: about 0.9~1.1.

In some embodiments, the antiviral composition contains at least oleanolic acid (including its free acid, salt, derivative or prodrug) and ursolic acid ( Including its free acid, salt, derivative or prodrug). The molar amount of OA is greater than that of UA.

In some embodiments, the antiviral composition contains at least oleanolic acid (including its free acid, salt, derivative or prodrug) and betulinic acid (containing Its free acid, salt, derivative or prodrug). The molar amount of OA is greater than that of BA.

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

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

Generally, treatments with arenaviridae infections, arteritis virus infections, filoviridae infections, flaviviridae infections (flaviviruses), delta retroviruses, coronaviridae, paramyxoviridae, Subjects infected with Orthomyxoviridae or Togaviridae. The subject is evaluated to determine whether the aforementioned subject is infected by the aforementioned virus, the administration of the antiviral composition is instructed, and the initial dose of the antiviral composition is administered to the subject for a period of time (a treatment period) as in the indicated dosage regimen. Regularly determine the subject’s clinical response and treatment response level. If the treatment response level is too low at a dose, increase the dose according to a pre-determined dose escalation plan until the subject reaches the desired level of treatment response, as needed Continue to treat the subject with the antiviral composition. The dosage or dosing regimen can be adjusted as needed until the patient reaches the desired one or more clinical trials. Bed end points, such as the cessation of the infection itself, the reduction of infection-related symptoms, and/or the reduction of the progression of the infection.

If the clinician intends to use a combination of antiviral composition and one or more other therapeutic agents to treat a subject with a viral infection, and it is known that the viral infection of the subject has at least Part of the treatment response, the method of the invention comprises: administering a treatment-related dose of the antiviral composition and the treatment-related dose of one or more of the aforementioned other therapeutic agents to a subject in need, wherein the antiviral is administered as in the first dosing regimen The composition and one or more other therapeutic agents are administered as in the second dosing regimen. In some embodiments, the first and second dosing regimens are the same. In some embodiments, the first and second dosing regimens are different.

The antiviral composition(s) of the present invention can be administered as primary antiviral therapy, auxiliary antiviral therapy, or combined antiviral therapy. The method of the present invention comprises the separate administration or co-administration of the antiviral composition and at least one other known antiviral composition, which means that the antiviral composition of the present invention can be used in the known antiviral composition (one or more compounds). Or before, during or after the administration of the composition for the treatment of symptoms associated with viral infections. For example, drugs for the treatment of inflammation, vomiting, nausea, headache, fever, diarrhea, urticaria, conjunctivitis, physical malaise, muscle pain, arthralgia, epilepsy or paralysis can be administered together or separately with the antiviral composition of the present invention .

One or more other therapeutic agents can be administered at a therapeutically effective dose and a baseline dosing schedule recognized by the clinician, or at a dose lower than the therapeutically effective dose recognized by the clinician. The clinical benefit and/or therapeutic effect provided by the administration of the combination of the antiviral composition and one or more other therapeutic agents can be additive or synergistic, and the degree of such benefit or effect can be compared to the combined administration , And the separate antiviral composition component (one or more), and one or more other therapeutic agents to determine the administration. You can use the U.S. Food and Drug Administration, World Health Recommended or described by organizations, European Medicines Agency (EMEA), Drug Administration (TGA, Australia), Pan American Health Organization (PAHO), Medicines and Medical Device Safety Administration (Medsafe, New Zealand) or various world health agencies Dosage and baseline dosing regimen to administer one or more other therapeutic agents.

The antiviral composition of the present invention includes exemplary other therapeutic agents that can be used to treat viral infections, including antiretroviral agents, alpha-interferon (IFN-a), zidovudine, lamiv Lamivudine, cyclosporine A, CHOP with arsenic trioxide, sodium valproate, methotrexate, azathioprine, one or more symptom relief drugs, steroid sparing drugs, Corticosteroids, cyclophosphamide, immunosuppressive agents, anti-inflammatory agents, Janus kinase inhibitors, tofacitinib, calcineurin inhibitors, tacrolimus, mTOR inhibitors, siroline Sirolimus, everolimus, IMDH inhibitor, azathioprine, leflunomide, mycophenolate, biologics, abatacept, adalim Monoclonal antibody (adalimumab), anakinra (anakinra), certolizumab (certolizumab), etanercept (etanercept), golimumab (golimumab), infliximab (infliximab), Iraq Ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, Vedolizumab, monoclonal antibody, basiliximab, daclizumab, multiple antibodies, nucleoside analogs, reverse transcriptase inhibitors, emtricitabine ( emtricitabine, telbivudine, abacavir, adefovir, didanosine, emtricitabine, entecavir, stata Stavudine, tenofovir, azithromycin, macrolide-type antibiotic, protease inhibitor, interferon, immune response modifier, mRNA synthesis inhibitor, protein Synthesis, inhibitor, thiol Thiazolides, CYP3A4 inhibitors, heterocyclic biguanides, CCR5 receptor inhibitors and combinations thereof. The therapies studied also include plasma exchange and/or radiation. Antibodies against specific viruses can also be administered to subjects treated with the antiviral composition of the present invention. The plasma obtained from the blood of a survivor of the first viral infection can be administered to other subjects with the same type of viral infection, and the aforementioned other subjects are also administered the antiviral composition of the present invention. For example, plasma from a survivor of COVID-19 infection can be administered to another subject suffering from COVID-19 infection, and the aforementioned other subject is also administered the antiviral composition of the present invention.

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

The antiviral compounds (such as one or more triterpenes, one or more cardiac glycosides, etc...) present in the pharmaceutical composition can exist in their unmodified form, salt form, derivative form or a combination thereof. As used in this specification, the term "derivative" refers to: a) a chemical substance structurally related to the first chemical substance and theoretically derived from it; b) a compound produced from a similar first compound; or imaginable because One atom of the first compound is replaced by another atom or group of atoms, resulting in a compound; c) a compound derived or obtained from the parent compound and containing the basic elements of the parent compound; or d) from a similar compound in one or more steps The first compound of the structure produces a chemical compound. For example, the derivative may comprise its deuterated form, oxidized form, dehydrated, unsaturated, polymer conjugated or its glycosylated form, or may comprise its ester, amide, lactone, homologue, ether, Thioether, cyano, amine, alkylamino, sulfhydryl, heterocycle, fused heterocycle, polymerization, pegylation, benzylidene, triazolyl, piperazinyl or deuterated forms.

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

The supercritical fluid (SCF) extract can be prepared as described in US 7,402,325, US 8394434, US 8187644 or PCT International Publication No. WP 2007/016176 A2, the entire disclosure of which is incorporated herein by reference. The extraction can be performed with supercritical carbon dioxide in the presence or absence of a modifier (organic solvent) such as ethanol.

Other extracts containing cardiac glycosides, especially oleandrin, can be prepared by various different processes. The extract can be prepared by a process developed by Dr. Huseyin Ziya Ozel (US Patent No. 5,135,745), which describes the steps used to prepare the hot water extract. It is reported that the aqueous extract contains a variety of polysaccharides, oleandrin, oleandrin, ordonoside, and oleandroside with molecular weights ranging from 2KD to 30KD. It has been reported that polysaccharides contain acidic homogalacturonic acid or arabinogalacturonic acid. US Patent No. 5,869,060 to Selvaraj et al. discloses a hot water extract of Nerium species and its production method, such as Example 2. The resulting extract is then lyophilized to produce a powder. US Patent No. 6,565,897 (US Pre-Grant Publication No. 20020114852 and PCT International Publication No. WO 2000/016793 to Selvaraj et al.) discloses a hot water extraction process for preparing substantially sterile extracts. Erdemoglu et al. ( J. Ethnopharmacol. (2003) Nov. 89(1), 123-129) disclosed the comparison results of aqueous and ethanol extracts of plants containing oleander based on its analgesic and anti-inflammatory activities. Adome et al. ( Afr. Health Sci. (2003) August. 3(2), 77-86; ethanol extract), el-Shazly et al. ( J. Egypt Soc. Parasitol. (1996), August. 26 (2), 461-473; ethanol extract), Begum et al. ( Phytochemistry (1999) Feb. 50(3), 435-438; methanol extract), Zia et al. ( J. Ethnolpharmacol. (1995) ten Jan. 49(1), 33-39; methanol extract) and Vlasenko et al. ( Farmatsiia. (1972) September-October. 21(5), 46-47; alcohol extract) revealed the Organic solvent extract. US Pre-Grant Patent Application Publication No. 20040247660 by Singh et al. discloses the preparation of a protein stabilized liposomal formulation of oleandrin for cancer treatment. US Pre-Grant Patent Application Publication No. 20050026849 by Singh et al. discloses a water-soluble formulation of oleandrin containing cyclodextrin. US pre-grant patent application publication No. 20040082521 by Singh et al. discloses the preparation of a protein-stabilized nanoparticle formulation of oleandrin derived from hot water extracts.

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

Oleandrin can also be obtained from the extracts of oleander in vitro microcultures, so that it can be cultivated from the seedlings and/or stem tips of oleander species such as Splendens Giganteum, Revanche, Alsace or other varieties (Vila et al., "Micropropagation of Oleander (Nerium oleander L.)" in Hort Science (2010), 45(1), 98-102, the entire disclosure of which is incorporated into this specification by reference). According to the present invention, hot water, organic solvent, aqueous organic solvent or supercritical fluid extract of micro-cultured oleander can be used.

The polysaccharide and carbohydrate content of the extract is also different. Compared with the standard curve made with glucose, the hot water extract contains 407.3 glucose equivalent units of carbohydrates, and the analysis of the SCF CO ₂ extract found that the content of carbohydrates is far below the minimum level required for quantification. However, the amount of carbohydrates in the hot water extract of oleander is at least 100 times higher than the amount _{of carbohydrates in the SCF CO 2 extract.} The polysaccharide content of the SCF extract can be 0% by weight, <0.5% by weight, <0.1% by weight, <0.05% by weight, or <0.01% by weight. In some embodiments, the SCF extract does not contain polysaccharides obtained during the extraction of the plant material.

DART TOF-MS (Direct Analysis in Real Time Time of Flight Spectrometry) on the JEOL AccuTOF-DART mass spectrometer (JEOL USA, Peabody, MA, USA) was used to determine the SCF CO ₂ extract and Part of the composition of the hot water extract.

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

The SCF extract contains oleandrin and triterpene oleanolic acid, betulinic acid and ursolic acid, and any other components described in this specification. The content of oleandrin and triterpene can vary from batch to batch; however, the degree of variation must not be too large. For example, analyzing these four components for a batch of SCF extract (PBI-05204), it is found that each contains the following approximate amounts.

WRT表示「相對於」。

WRT stands for "relative to".

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

Oleandrin, oleanolic acid, ursolic acid, betulinic acid and their derivatives can also be purchased from Sigma-Aldrich ( www.sigmaaldrich.com ; St. Louis, MO, USA). The digitonin can be obtained from HIKMA Pharmaceuticals International LTD (NDA N012648, elixirs, 0.05 mg/mL; tablets, 0.125 mg, 0.25 mg), VistaPharm Inc. (NDA A213000, elixirs, 0.05 mg/mL), Sandoz Inc. (NDA A040481, injection, 0.25mg/mL), West-Ward Pharmaceuticals International LTD (NDA A083391, injection, 0.25mg/mL), Covis Pharma BV (NDA N009330, 0.1mg/mL, 0.25mg/mL) mL), Impax Laboratories (NDA A078556, tablets, 0.125mg, 0.25mg), Jerome Stevens Pharmaceuticals Inc. (NDA A076268, tablets, 0.125mg, 0.25mg), 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, 0.5mg, LANOXIN), GlaxoSmithKline LLC (NDA 018118, capsules, 0.05mg, 0.1mg, 0.15mg, 0.2mg, LANOXICAPS).

As used in this specification, a separately named triterpene can be independently selected from its natural (unmodified, free acid) form, its salt form, derivative form, prodrug form, or a combination thereof at each occurrence. The composition containing the deuterated form of triterpene and the method of using the deuterated form of triterpene are also within the scope of the present invention.

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

Ursolic acid derivatives, prodrugs and salts are disclosed in the following documents: Gribble et al. US 20150011627 A1 (published on January 8, 2015), Gribble et al. 20130303607 A1 (published on November 14, 2013), US 20150218206 A1 by Yoon et al. (published on August 6, 2015), US 6824811 by Fritsche et al. (authorized on November 30, 2004), US by Ochiai et al. 7718635 (authorized on May 8, 2010), Lin et al.'s US 8729055 (authorized on May 20, 2014), and Yoon et al.'s US 9120839 (authorized on September 1, 2015), the entire disclosure of which is based on Reference is incorporated into this manual.

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

The antiviral composition can be formulated in any suitable, pharmaceutically acceptable dosage form. Non-digestive tract, ear, eye, nose, inhalable, buccal, sublingual, enteral, topical, oral, oral, and injectable dosage forms are particularly useful. Specific dosage forms include solid or liquid dosage forms. Exemplary suitable dosage forms include tablets, capsules, pills, caplets, lozenges, granules, solutions, suspensions, dispersions, vials, bags, bottles, injectable liquids, iv (intravenous), im (intramuscular ) Or ip (intraperitoneal) administrable liquid, and other such dosage forms known to those with ordinary knowledge in the field of pharmacy.

Because viral infections can affect multiple organs at the same time and cause multiple organ failure, it is advantageous to administer the composition by more than one route. For example, COVID-19 is known to affect the lungs, heart, gastrointestinal tract, and brain. Therefore, the composition containing cardiac glycosides can be advantageously administered as an inhalable composition and an oral composition; a sublingual composition and an oral composition; an inhalable composition and a sublingual composition; an inhalable composition and a non-digestible composition Tract composition; sublingual composition and non-digestive tract composition; oral composition and non-digestive tract composition, or other such combinations.

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

It can also be based on Addington's US 8187644 B2 (authorized on May 29, 2012), Addington's US 7402325 B2 (authorized on July 22, 2008), and Addington et al.'s US 8394434 B2 (authorized on March 12, 2013) A suitable dosage form is prepared, the entire contents of which are incorporated herein by reference. Suitable dosage forms can also be prepared as described in Examples 13-15.

Special attention should be paid to the effective amount or therapeutically relevant amount of the antiviral compound (cardiac glycoside, triterpene or a combination thereof). The term "effective amount" should be understood as the expected pharmaceutically effective amount. A pharmaceutically effective amount refers to the amount or quantity of the active ingredient sufficient to achieve a desired or desired therapeutic response; or in other words, an amount sufficient to produce a noticeable biological response when administered to a patient. Perceivable biological reactions can be produced as a result of the administration of a single or multiple doses of the active substance. A dose may contain one or more dosage forms. It should be understood that the specific dosage level for any patient will depend on a variety of factors, including the indication for treatment, the severity of the indication, the patient’s health, age, sex, weight, diet, pharmacological response, the specific dosage form used, and other such factors. Class factors.

The desired dose for oral administration is at most 5 dosage forms, although as few as 1 and as many as 10 dosage forms can still be administered as a single dose. An exemplary dosage form may contain 0.01 to 100 mg or 0.01 to 100 μg of the antiviral composition per dosage form, for a total of 0.1 to 500 mg per dose (1 to 10 dose level). The dose will be administered according to a dosage regimen that can be predetermined and/or customized to achieve a specific therapeutic response or clinical benefit in the subject.

Cardiac glycosides may be present in the dosage form in an initial dose of about 20 to about 100 μg, about 12 μg to about 300 μg, or about 12 μg to about 120 μg of oleandrin in an amount sufficient to provide the subject. The dosage form may contain about 20 to about 100 μg of oleandrin, about 0.01 μg to about 100 mg or about 0.01μg to about 100μg oleandrin, oleandrin extract, or oleandrin containing oleandrin.

Antiviral agents may be included in oral dosage forms. Part of the embodiment of the dosage form is that it has no enteric coating and releases the antiviral composition it carries in 0.5 to 1 hour or less. Part of the embodiment of the dosage form is enteric-coated and releases the antiviral composition carried by it, for example, from the downstream of the stomach such as jejunum, ileum, small intestine, and/or large intestine (colon). The enteric-coated dosage form will release the antiviral composition into the systemic circulation within 1 to 10 hours after oral administration.

The antiviral composition can be contained in a fast-release, immediate-release, controlled-release, sustained-release, extended-release, delayed-release, burst-release, continuous-release, slow-release, or pulsatile-release dosage form, or in two types that exhibit these release types. One or more dosage forms. The release profile of the antiviral composition from the dosage form can be a zero-order, pseudo-zero-order, first-order, pseudo-first-order, or S-type release curve. The plasma concentration curve of triterpenes in subjects in which the antiviral composition is administered may show one or more maxima.

Based on human clinical data, it is expected that the administered dose of 50% to 75% oleandrin will be available through oral administration, thus providing about 10 to about 20 μg, about 20 to about 40 μg, about 30 to about 50 μg, about 40 to about 40 μg per dosage form. 60 μg, about 50 to about 75 μg, or about 75 to about 100 μg of oleandrin. Considering that the average blood volume of an adult is 5 liters, the expected plasma concentration of oleandrin will be about 0.05 to about 2ng/ml, about 0.005 to about 10ng/ml, about 0.005 to about 8ng/ml, about 0.01 to about 7ng. /ml, about 0.02 to about 7ng/ml, about 0.03 to about 6ng/ml, about 0.04 to about 5ng/ml, or about 0.05 to about 2.5ng/ml. The recommended daily dose of oleandrin present in the SCF extract is usually about 0.2 μg to about 4.5 μg/kg body weight, twice a day. The dosage of oleandrin may be about 0.2 μg to about 1 μg/kg body weight/day, about 0.5 to about 1.0 μg/kg body weight/day, about 0.75 to about 1.5 μg/kg body weight/day, about 1.5 to about 2.52 μg/kg Body weight/day, about 2.5 to about 3.0 μg/kg body weight/day, about 3.0 to about 4.0 μg/kg body weight/day, or about 3.5 to 4.5 μg oleandrin /kg body weight/day. The maximum tolerated dose of oleandrin may be about 3.5 μg/kg body weight/day to about 4.0 μg/kg body weight/day. The minimum effective dose may be about 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day, or about 5 μg/day.

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

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

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

The liquid composition may contain one or more pharmaceutically acceptable liquid carriers. The liquid carrier can be an aqueous, non-aqueous, polar, non-polar, and/or organic vehicle. The liquid carrier contains- For example, but not limited to-water miscible solvents, water immiscible solvents, water, buffers and mixtures thereof.

As used in this specification, the interchangeable terms "water-soluble solvent" or "water-miscible solvent" refer to an organic liquid that does not form a two-phase mixture with water, or is sufficiently soluble in water to provide a solvent containing at least 5% and no Liquid phase separation of organic liquids from aqueous solvent mixtures. The solvent is suitable for administration to humans or animals. Exemplary water-soluble solvents include-such as but not limited to-PEG (poly(ethylene glycol)), PEG 400 (poly(ethylene glycol) having a molecular weight of about 400), ethanol, acetone, alkanol, alcohol, ether, propylene glycol , Glycerol, triacetin, poly(propylene glycol), PVP (poly(vinylpyrrolidone)), dimethyl sulfide, N,N-dimethylformamide, formamide, N,N- Dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluhor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

As used in this specification, the interchangeable terms "water-insoluble solvent" or "water-immiscible solvent" refer to an organic liquid that forms a two-phase mixture with water, or phase separation occurs when the concentration of the solvent in water exceeds 5% Organic liquid. The solvent is suitable for administration to humans or animals. Exemplary water-insoluble solvents include-for example, but not limited to-medium/long chain triglycerides, oil, castor oil, corn oil, vitamin E, vitamin E derivatives, oleic acid, fatty acids, olive oil, softisan 645 (diglyceride) Acid ester/capric acid ester/stearic acid ester/hydroxystearate adipate), miglyol, captex (Captex 350: glycerol tricaprylate/capric acid ester/lauric acid triglyceride; Captex 355: glycerin Tricaprylate/Capric Triglyceride; Captex 355 EP/NF: Caprylic Triglyceride/Capric Medium Chain Triglyceride).

Listed in "International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance for industry Q3C Impurities: Residual Solvents: Residual Solvents )" (1997) To find a suitable solvent, this suggests how much residual solvent in the drug should be considered safe. Exemplary solvents are listed as type 2 or type 3 solvents. Type 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 Ester, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol Alcohol, pentane, 1-pentanol, 1-propanol, 2-propanol, or propyl acetate.

Other materials that can be used as water-immiscible solvents in the present invention include: Captex 100: Propylene Glycol Dicaprylate; Captex 200: Propylene Glycol Dicaprylate/Dicaprate; Captex 200P: Propylene Glycol Dicaprylate/Dicaprylate Capric acid ester; Propylene Glycol Dicaprylocaprate (Propylene Glycol Dicaprylocaprate); Captex 300: glycerol tricaprylate/caprate; Captex 300 EP/NF: glycerol tricaprylate/capric acid medium chain triglyceride; Captex 350: glyceryl tricaprylate/caprate/laurate; Captex 355: glyceryl tricaprylate/caprate; Captex 355 EP/NF: glyceryl tricaprylate/capric acid medium chain triglyceride; Captex 500: glyceryl triacetate; Captex 500 P: glyceryl triacetate (pharmaceutical grade); Captex 800: propylene glycol di(2-ethylhexanoate); Captex 810 D: glyceryl tricaprylate/caprate/ Oleate; Captex 1000: glycerol tricaprate; Captex CA: medium chain triglyceride; Captex MCT-170: medium chain triglyceride; Capmul GMO: glycerol monooleate; Capmul GMO-50 EP/NF: Glycerol monooleate; Capmul MCM: Medium Chain Mono- &Diglycerides; Capmul MCM C8: Glycerol monocaprylate; Capmul MCM C10: Glycerol monocaprate; Capmul PG-8: propylene glycol monocaprylate; Capmul PG-12: propylene glycol monolaurate; Caprol 10G10O: decaglyceryl decaoleate; Caprol 3GO: triglycerol monooleate; Caprol ET: polyglycerol of mixed fatty acids Glycerides; Caprol MPGO: hexaglycerol dioleate; Caprol PGE 860: Decaglycerol Mono-, Dioleate.

As used in this specification, "surfactant" refers to a compound containing a polar or charged hydrophilic part and a non-polar hydrophobic (lipophilic) part; that is, a surfactant is two Pro-sexual. The term surfactant can refer to one compound or a mixture of multiple compounds. The surfactant can be a solubilizer, emulsifier or dispersant, and the surfactant can be hydrophilic or hydrophobic.

Hydrophilic surfactants can be any hydrophilic surfactants suitable for pharmaceutical compositions. The aforementioned surfactants can be anionic, cationic, zwitterionic or nonionic, although they are currently active as nonionic hydrophilic surfactants. The agent is better. As noted above, these non-ionic hydrophilic surfactants will generally have an HLB value greater than about 10. Mixtures of hydrophilic surfactants are also within the scope of the present invention.

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

Examples of other suitable solubilizers include: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butylene glycol and its isomers, glycerol, pentaerythritol, sorbitol , Mannitol, transcutol, isosorbide dimethyl ether, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrin and cyclodextrin Dextrin derivatives; ethers of polyethylene glycol having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol polyethylene glycol ether (tetraethylene glycol, commercially available from BASF, trade name Tetraglycol) Or methoxy polyethylene glycol (Union Carbide); amides, such as 2-pyrrolidone, 2-piperidone, caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkyl Piperidone, N-alkylcaprolactam, dimethylacetamide, and polyvinylpyrrolidone; esters, such as ethyl propionate, tributyl citrate, acetyl triethyl citrate, acetamide Tributyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, glycerol triacetate, propylene glycol monoacetate, propylene glycol diacetate, caprolactone and its isomers Valerolactone and its isomers, butyrolactone and its isomers; and other solubilizers known in the art, such as dimethylacetamide, isosorbide Alcohol dimethyl ether (Arlasolve DMI (ICI)), N-methylpyrrolidone (Pharmasolve (ISP)), glyceryl monocaprylate, diethylene glycol monoethyl ether (available from Gattefosse, trade name Transcutol), and water. Mixtures of solubilizers are also within the scope of the present invention.

Unless otherwise indicated, the compounds mentioned in this specification can be easily obtained from standard commercial sources.

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

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

It should be understood that compounds used in the field of pharmaceutical preparations generally have multiple functions or purposes. Therefore, if the compound named in this specification is mentioned only once or used to define more than one term in this specification, its purpose or function should not be interpreted as being limited to the claimed purpose or multiple functions, or one or more functions .

One or more components of the formulation may exist in the form of its free base, free acid, or pharmaceutically or analytically acceptable salt. As used in this specification, "pharmaceutically or analytically acceptable salt" refers to a compound that has been modified as needed by reacting it with an acid to form an ionic bond pair. Examples of acceptable salts include conventional non-toxic salts formed from, for example, non-toxic inorganic or organic acids. Suitable non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, sulfamic acid, phosphoric acid, nitric acid, etc., and other salts known to those with ordinary knowledge in the art. Salts prepared from organic acids, and other salts known to those with ordinary knowledge in the art. The aforementioned organic acids are, for example, amino acid, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, and malic acid. , Tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamine acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid , Fumaric acid, Toluenesulfonic acid, Methanesulfonic acid, Ethane disulfonic acid, Oxalic acid, Isethionic acid. On the other hand, in the case where the pharmaceutically active ingredient has an acidic functional group, a pharmaceutically acceptable base is added to form a pharmaceutically acceptable salt. In Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA , 1985, p.1418 Other suitable salts found in the list, the relevant disclosure of which is incorporated herein by reference.

The term "pharmaceutically acceptable" used in this manual refers to within the scope of reasonable medical judgment, suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or any other problems or complications, and reasonable The compound, material, composition and/or dosage form that is commensurate with the benefit/risk ratio.

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

Conventional packaging equipment and materials can be used to package the dosage form. It can be contained in packaging, bottles, vials, bags, syringes, envelopes, packets, blister packs, boxes, safety Ampoules or other similar containers.

The composition of the present invention can be contained in any dosage form. Specific dosage forms include solid or liquid dosage forms. Exemplary suitable dosage forms include tablets, capsules, pills, caplets, lozenges, granules, and other similar dosage forms known to those with ordinary knowledge in the pharmaceutical field.

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

Vero CCL81 cells are used in prevention and treatment trials (ATCC, Manassas, VA). The plaque assay was performed in Vero E6 cells gifted by Vineet Menachery (UTMB, Galveston, TX). The cells are maintained in a 37°C incubator _{with 5% CO 2.} The cells were cultured using Dulbecco's modified Eagle medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin/streptomycin (Gibco, NY). The maintenance medium reduces FBS to 2%, but otherwise the same. SARS-CoV-2, USA_WA1/2020 strain (Genbank accession number MT020880) is provided by the World Reference Center for Emerging Viruses and Arboviruses. All studies used the Vero 4th generation reserve sequenced by NextGen of SARS-CoV-2.

[Examples]

Example 1

Supercritical fluid extraction of powdered oleander leaves

Method A. Use carbon dioxide

Harvest, wash and dry the oleander leaf material, and then use, for example, US Patent No. 5,236,132, The crushing and dehydrating equipment described in 5,598,979, 6,517,015, and 6,715,705 are used to prepare powdered oleander leaves. The weight of the starting material used was 3.94 kg.

_{In the extractor device, the starting material is combined with pure CO 2} 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 achieve a solvent to raw material ratio of 50:1. Then, _{the mixture of CO 2} and raw materials is sent to the separation device, and the pressure and temperature of the mixture are changed to separate the extract and carbon dioxide.

A brown, sticky, and viscous extract (65 g) with good fragrance was obtained. The color may be caused by chlorophyll and other residual color compounds. In order to determine the exact yield, rinse the tube and separator with acetone, and evaporate the acetone to obtain another 9 g of extract. The total extraction volume is 74g. Based on the weight of the starting material, the yield of the extract was 1.88%. The content of oleandrin in the extract calculated using high pressure liquid chromatography and mass spectrometry was 560.1 mg, or a yield of 0.76%.

Method B. Use a mixture of carbon dioxide and ethanol

The oleander leaf material is harvested, washed, and dried, and then powdered oleander leaves are prepared by the crushing and dehydrating equipment described in, for example, US Patent Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. The weight of the starting material used was 3.85 kg.

_{In the extractor device, the starting material was combined with pure CO 2} and 5% ethanol as a modifier at a pressure of 280 bar (28 MPa, 4061 psi) and a temperature of 50° C. (122° F). A total of 160 _{kg of CO 2} and 8 kg of ethanol were used to achieve a solvent to raw material ratio of 43.6:1. Then, _{the mixture of CO 2} , ethanol and raw materials is sent to the separation device, and the pressure and temperature of the mixture are changed to separate the extract and carbon dioxide.

After removing the ethanol, a dark green, sticky, sticky lumpy extract (207g) was obtained, which obviously contained part of chlorophyll. Based on the weight of the starting material, the yield of the extract was 5.38%. The content of oleandrin in the extract calculated using high pressure liquid chromatography and mass spectrometry is 1.89g, or Yield of 0.91%.

Example 2

Hot water extract of powdered oleander leaves.

(Comparative example)

Hot water extraction is usually used to extract oleandrin and other active components from oleander leaves. Examples of hot water extraction processes can be found in U.S. Patent Nos. 5,135,745 and 5,869,060.

Use 5g of powdered oleander leaves for hot water extraction. To the powdered oleander leaves, 10 volumes of boiling water (based on the weight of the oleander starting material) were added, and the mixture was continuously stirred for 6 hours. Then the mixture was filtered and the leaf residues were collected and extracted again under the same conditions. The filtrates were combined and lyophilized. The appearance of the extract is brown. The weight of the dried extract material was about 1.44 g. 34.21mg of the extract material was dissolved in water and analyzed for oleandrin content using high pressure liquid chromatography and mass spectrometry. The amount of oleandrin was determined to be 3.68 mg. Based on the amount of extract, the yield of oleandrin was calculated to be 0.26%.

Example 3

Preparation of pharmaceutical composition.

Method A. Cremophor-based drug delivery system

The following ingredients are provided in the amounts indicated.

Carrier #1 (LN2005-055-035)

The excipients were dispensed into jars and shaken in a New Brunswick Scientific C24KC refrigerated incubator shaker (Refrigerated Incubator shaker) at 60°C for 24 hours to ensure uniformity. Then take out the sample and visually check for dissolution. After 24 hours, in all preparations, the excipients and antiviral composition were all dissolved.

Method B. GMO/Cremophor-based drug delivery system

The following ingredients are provided in the amounts indicated.

Carrier #2 (LN2005-055-045)

Follow the steps of Method A.

Method C. Labrasol-based drug delivery system

The following ingredients are provided in the amounts indicated.

Carrier #3 (LN2005-055-047)

Follow the steps of Method A.

Method D. Micelle formation system based on vitamin E-TPGS

The following ingredients are provided in the amounts indicated.

Follow the steps of Method A.

Method E. Multi-component drug delivery system

The following ingredients are provided in the amounts indicated.

Follow the steps of Method A.

Method F. Multi-component drug delivery system

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

Follow the steps of Method A.

Example 4

Preparation of enteric-coated capsules

Step I: Preparation of liquid-filled capsules

Fill hard gelatin capsules (50 pieces, 00 size) with the liquid composition of Example 3. These capsules were manually filled with 800 mg of preparation, and then manually sealed with 50% ethanol/50% aqueous solution. Then use the indicated amount of 22% gelatin solution containing the following ingredients to manually bind these capsules.

Mix the gelatin solution thoroughly and swell for 1 to 2 hours. After the swelling period, the solution was tightly covered and placed in an oven at 55°C to liquefy. Once all the gelatin solution becomes liquid, banding is performed.

Use a pointed round head 3/0 paint brush to apply the sol solution on the capsule. Use the strapping tool provided by Shionogi. After the strapping, the capsules were kept under ambient conditions for 12 hours to allow the strapping to solidify.

Step II: Coating of liquid-filled capsules

The coating dispersion was prepared from the ingredients listed in the table below.

[Table 13]

If the capsules bundled as in step I are used, the dispersion is applied to the capsules at a coating level of ^{20.0 mg/cm 2.} The following conditions are used to coat the capsules.

Example 5

Treatment of Zika virus in subjects

Method A. Antiviral composition therapy

An antiviral composition is administered to a subject exhibiting Zika virus infection, and a therapeutically relevant dose is administered to the subject for a period of time as specified in the dosing schedule. Periodically determine the subject's treatment response level. The treatment response can be determined by determining the Zika virus titer in the subject's blood or plasma level. If the level of therapeutic response at a dose is too low, the dose will be increased according to a pre-determined dose escalation plan until the desired level of therapeutic response is reached in the subject. Subjects continue to be treated with the antiviral composition as needed, and the dosage or dosing regimen can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination therapy: antiviral composition and other agents

In addition to instructing and administering to the subject one or more other therapeutic agents for the treatment of Zika virus infection or its symptoms, method A described above is followed. Thus, one or more other therapeutic agents can be administered before, after, or together with the antiviral composition. It is also possible to increase (or decrease) the dose of one or more other therapeutic agents.

Example 6

In vitro evaluation of antiviral activity against Zika virus infection

Method A. Pure compound

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

Method B. Compounds in the form of extracts

In addition to normalizing the amount of the extract to the amount of the target compound in the extract, the extract containing the target compound to be detected is evaluated as detailed in Method A. For example, an extract containing 2% by weight of oleandrin contains 20 μg of oleandrin/1 mg of extract. Therefore, if the The expected amount of oleandrin is 20 μg, and 1 mg of the extract will be used for the determination.

Example 7

Preparation of tablets containing antiviral composition

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

Extra-granular addition

Final production mix:

Abbreviated

[Table 17]

Final production mix:

detailed

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

Example 8

HPLC analysis of solutions containing oleandrin

Analyze the samples in HPLC (Waters) using the following conditions (oleandrin standard, SCF extract and hot water extract): Symmetry C18 column (5.0μm, 150×4.6mm I.D.; Waters); MeOH: water = 54: 46 (v/v) mobile phase and a flow rate of 1.0 ml/min. The detection wavelength is set to 217nm. The sample is prepared by dissolving the compound or extract in a fixed amount of HPLC solvent to achieve an approximate target concentration of oleandrin. Determine the retention time of oleandrin by using an internal standard. The concentration of oleandrin can be determined/corrected by forming a signal response curve using internal standards.

Example 9

Preparation of pharmaceutical composition

The pharmaceutical composition of the present invention can be prepared by any of the following methods. The mixing can be carried out under wet or dry conditions. During the preparation, the pharmaceutical composition can be compressed, dried, or compressed and dried. The pharmaceutical composition can be dispensed into a dosage form.

Method A.

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

Method B.

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

Method C.

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

Method D

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

Method E.

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

Method F.

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

Example 10

Preparation of triterpene mixture

The following composition was prepared by mixing the specified triterpenes at the approximate molar ratio shown.

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

Example 11

Preparation of antiviral composition

The antiviral composition can be prepared by mixing the individual triterpene components to form a mixture. The triterpene mixture prepared above and providing acceptable antiviral activity can be formulated into an antiviral composition.

Antiviral composition with oleanolic acid and ursolic acid

According to the predetermined molar ratio of the components as defined in this specification, known amounts of oleanolic acid and ursolic acid are mixed. The components are mixed in solid form or mixed in the following solvents (one or more): for example, methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfide (DMSO), dimethylformamide (DMF), two Methylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains the relative molar ratio of components as described in this specification.

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

Antiviral composition with oleanolic acid and betulinic acid

According to the predetermined molar ratio of the components as defined in this specification, known amounts of oleanolic acid and betulinic acid are mixed. The components are mixed in solid form or mixed in the following solvents (one or more): for example, methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfide (DMSO), dimethylformamide (DMF), two Methylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains the relative molar ratio of components as described in this specification.

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

Antiviral composition with oleanolic acid, ursolic acid and betulinic acid

According to the predetermined molar ratio of the components defined in this specification, known amounts of oleanolic acid, ursolic acid and betulinic acid are mixed. The components are mixed in solid form or mixed in the following solvents (one or more): for example, methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfide (DMSO), dimethylformamide (DMF), two Methylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains the relative molar ratio of components as described in this specification.

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

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

According to the predetermined molar ratio of the components as defined in this specification, known amounts of oleandrin, oleanolic acid, ursolic acid and betulinic acid are mixed. The components are mixed in solid form or mixed in the following solvents (one or more): for example, methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfide (DMSO), dimethylformamide (DMF), two Methylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains the relative molar ratio of components as described in this specification.

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

Example 12

Treatment of filovirus infection in subjects

Exemplary filovirus infections include Ebola virus and Marburg virus.

Method A. Antiviral composition therapy

An antiviral composition is administered to a subject exhibiting a filovirus infection, and in addition, a therapeutically relevant dose is administered to the subject for a period of time as specified in the dosing schedule. Periodically determine the subject's treatment response level. The level of treatment response can be determined by determining the filovirus titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose will be escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is reached. Subjects continue to be treated with the antiviral composition as needed, and in addition, the dosage or dosing regimen can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination therapy: antiviral composition and other agents

In addition to instructing and administering to the subject one or more other therapeutic agents for the treatment of filovirus infection or its symptoms, the above method A is followed. Thus, one or more other therapeutic agents can be administered before, after, or together with the antiviral composition. It is also possible to increase (or decrease) the dose of one or more other therapeutic agents.

Example 13

Treatment of flavivirus infections in subjects

Exemplary flavivirus infections include yellow fever, dengue fever, Japanese encephalitis, West Nile virus, Zika virus, tick-borne encephalitis, Kjeldahl forest disease, Alkhurma's disease, Trakong virus, Omsk hemorrhagic fever and Powassen Viral infection.

Method A. Antiviral composition therapy

An antiviral composition is administered to a subject exhibiting a flavivirus infection, and in addition, a treatment-related dose is administered to the subject for a period of time as specified in the dosing schedule. Regularly determine the subject’s response to treatment level. The level of treatment response can be determined by determining the flavivirus titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose will be escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is reached. Subjects continue to be treated with the antiviral composition as needed, and in addition, the dosage or dosing regimen can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination therapy: antiviral composition and other agents

In addition to instructing and administering to the subject one or more other therapeutic agents for treating flavivirus infection or its symptoms, the above method A is followed. Thus, one or more other therapeutic agents can be administered before, after, or together with the antiviral composition. It is also possible to increase (or decrease) the dose of one or more other therapeutic agents.

Example 14

Evaluation of antiviral activity against Zika virus and dengue virus

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

Adherent cell lines are used to assess the antiviral activity of the composition against a group of viruses. Before adding the virus to the cells, the composition and the target cells are pre-cultured for 30 minutes. Incubation period It exists in the cell culture medium. For each infection test, use the same concentration of the composition (in duplicate) to establish a survival rate test in parallel to determine the cytotoxic effect of the composition in the absence of virus.

The antiviral activity of the test composition is determined by comparing the infection level (test based on immunostaining) or survival rate (test based on CPE) of the cells under the test conditions with the infection level or survival rate of uninfected cells. By comparing the survival rate in the presence of the inhibitor with the survival rate of sham-treated cells, the cytotoxic effect was evaluated in uninfected cells. The XTT survival rate test is used to determine the cytotoxicity. The aforementioned XTT survival rate test is performed at the same time point as the reading of the corresponding infection test.

The test composition was dissolved in 100% methanol. A composition of 8 concentrations (in duplicate) was generated by performing an 8-fold dilution, starting with 50 μM as the highest test concentration. The highest test concentration of the composition (50 μM) resulted in a final concentration of methanol in the culture solution of 0.25% (v/v%). Each test disk contains an 8-fold dilution series of methanol carriers, the concentration of which reflects the final concentration of methanol in the test conditions of each composition. Where possible, use GraphPad Prism software to determine the EC50 and CC50 of each test composition.

The antiviral activity was evaluated by the degree of protection against virus-induced cytopathic effect (CPE). In the presence of different concentrations of controls or compositions, the cells are attacked with viruses. After 6 days of infection (ZIKV, Zika virus) or 7 days (DENV, Dengue virus), by quantifying the cell survival rate under different test conditions and comparing the value with untreated cells and only using vector (infection medium) ) The treated cells are compared to monitor the degree of protection against CPE.

The quality control of the neutralization test is performed on each disk to determine: i) the signal versus background (S/B) value; ii) the inhibition by known inhibitors; and iii) the changes in the test, which are determined by all data points The coefficient of variation (CV) is determined. The overall change in the infection test ranged from 3.4% to 9.5%, and the overall change in the survival rate test ranged from 1.4% to 3.2%, calculated as the average of all C.V. values. Infect The signal-to-background (S/B) range of the test is 2.9 to 11.0, and the signal-to-background (S/B) range of the survival test is 6.5 to 29.9.

DENV2-induced cytopathic effect (CPE) is protected with neutral red readings: For the DENV2 antiviral test, the 08-10381 Montserrat strain was used. Produce virus stock in C6/36 insect cells. Vero cells (epithelial kidney cells derived from green monkey (Cercopithecus aethiops )) were cultured in MEM (MEM5) with 5% FBS. For both infection and survival rate tests, cells were seeded at 10,000 cells/well in a 96-well transparent flat bottom plate, and maintained in MEM5 at 37°C for 24 hours. On the day of infection, the samples were diluted 8-fold in a U-chassis using MEM with 1% bovine serum albumin (BSA). Prepare the test material dilution solution at a final concentration of 1.25X, and take 40 μl and incubate the target cells at 37°C for 30 minutes. After pre-incubation of the test material, 10 μl of virus dilution prepared in MEM with 1% BSA was added to each well (50 μl final volume/well) and the plate was placed in a humidified incubator _{with 5% CO 2} Incubate at 37°C for 3 hours. The volume of the virus used in the test is predetermined to generate a signal in the linear range inhibited by Ribavirin and compound A3 (a known inhibitor of DENV2). After infection and culture, the cells were washed with PBS and then with MEM (MEM2) containing 2% FBS to remove unbound virus. Thereafter, 50 μl of the culture solution containing the inhibitor dilution prepared in MEM2 at a concentration of 1X was added to each well. In an incubator (5% CO ₂ ), a 96-well transparent flat plate was cultured at 37°C for 7 days. The test plate contains a virus-free control group ("sham infection"), infected cells cultured only with culture medium, infected cells cultured with carrier (methanol) only, and cell-free wells (to determine the background). The test disc also contained control wells containing 50 μM ribavirin and 0.5 μM compound A3. After 7 days of infection, the cells were stained with neutral red to monitor cell viability. The test materials were evaluated in duplicate using serial 8-fold dilutions in the infection culture medium. The control group contained cells cultured without virus ("sham infection"), infected cells cultured only with culture medium, or infected cells in the presence of ribavirin (0.5 μM) or A3 (0.5 μM). The complete repetitive inhibition curve with only the methanol carrier was included on the same test plate.

The cytopathic effect (CPE) induced by ZIKV is protected with neutral red readings: For the ZIKV antiviral test, the PLCal_ZV strain was used. Vero cells (epithelial kidney cells derived from green monkeys) were cultured in MEM (MEM5) with 5% FBS. For both infection and survival rate tests, the cells were seeded at 10,000 cells/well in a 96-well transparent flat pan, and maintained in MEM5 at 37°C for 24 hours. On the day of infection, the samples were diluted 8-fold in a U-chassis using MEM with 1% bovine serum albumin (BSA). Prepare the test material dilution solution at a final concentration of 1.25X, and take 40 μl and incubate the target cells at 37°C for 30 minutes. After the test material was pre-incubated, 10 μl of virus dilution prepared in MEM with 1% BSA was added to each well (50 μl final volume per well) and the plate was placed in a humidified incubator _{with 5% CO 2} Incubate at 37°C for 3 hours. After infection and culture, the cells were washed with PBS, and then washed with MEM (MEM2) containing 2% FBS to remove unbound virus. Thereafter, 50 μl of the culture solution containing the inhibitor dilution prepared in MEM2 at a concentration of 1X was added to each well. The dish was cultured at 37°C for 6 days in an incubator (5% CO _{2 ).} The test plate contains a virus-free control group ("sham infection"), infected cells cultured only with culture medium, infected cells cultured with carrier (methanol) only, and cell-free wells (to determine the background). After 6 days of infection, the cells were stained with neutral red to monitor cell viability. The test material was evaluated in duplicate using an 8-fold dilution of the sequence in the infection medium. The control group contained cells cultured without virus ("sham infection"), infected cells cultured only with culture medium, or infected cells with A3 (0.5 μM). The complete repetitive inhibition curve with only the methanol carrier was included on the same test plate.

Analysis of survival rate data based on CPE: For the neutral red test, the cell survival rate is determined by monitoring the absorbance at 490nm. The average signal obtained in the cell-free wells is subtracted from all samples. Next, all data points were calculated as the percentage of the average signal observed in 8 wells of fake (uninfected) cells on the same test plate. Infected cells treated with culture medium alone reduced the signal to an average of 4.2% (for HRV), 26.9% (for DENV), and 5.1% (for ZIKV) of the signal observed in uninfected cells. The signal-to-background (S/B) of this test is 2.9 (for DENV) And 7.2 (for ZIKV), which is determined as the percentage of survival in "pseudo-infected" cells compared to the percentage of survival of infected cells treated with vector only.

Survival test (XTT) for evaluating compound-induced cytotoxicity: Using the same experimental settings and inhibitor concentration as used in the corresponding infection test, sham-infected cells are cultured with inhibitor dilution (or only culture medium). The culture temperature and duration of the culture period are the same as the conditions of the corresponding infection test. The XTT method was used to evaluate the cell survival rate. The XTT test measures mitochondrial activity and is based on the cleavage of the yellow tetrazolium salt (XTT), which forms an orange formazan dye. The aforementioned reaction only occurs in living cells with active mitochondria. A scanning multiwell spectrophotometer was used to directly quantify the formazan dye. Subtract the background level obtained from the cell-free wells from all data points. The survival rate test plate contains a control group (7 concentrations, which are the same as the final percentage of methanol in each oleandrin test well) that only uses methanol as a carrier. The viability was monitored by measuring the absorbance at 490nm.

Analysis of cytotoxicity data: For the XTT experiment, the cell survival rate was determined by monitoring the absorbance at 490nm. The average signal obtained in the cell-free wells is subtracted from all samples. Then, all data points are calculated as the percentage of the average signal observed in 8 wells of fake (uninfected) cells on the same test plate. The signal to background (S/B) of this test is 29.9 (for IVA), 8.7 (for HRV), 6.5 (for DENV) and 6.7 (for ZIKV), which are determined to be compared with the signal observed in cell-free wells Percentage of survival rate among "pseudo-infected" cells.

Example 15

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

Method A.

In the presence of oleandrin, digitonin or PBI-05204 (plant extract containing oleandrin), use EBOV/Kik (A, MOI=1) or MARV/Ci67 (B, MOI=1) Infect Vero E6 cells. After 1 hour, the inoculum and compound were removed and fresh culture medium was added to the cells. After 48 hours, the cells were fixed and immunostained to detect cells infected with EBOV or MARV. Use Operetta to count infected cells. C) Treat Vero E6 cells with the above compounds. ATP level was 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). 2 hours after infection (A, C) or 24 hours after infection (B, D), oleandrin or PBI-05204 were added to the cells for 1 hour, then discarded and fed back to the culture medium. 48 hours after infection, the analysis of infected cells is shown in Figure 1.

Method C.

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

Example 16

Evaluation of antiviral activity against Togaviridae viruses

(Alphavirus: VEEV and WEEV)

In the presence or absence of the indicator compound, Vero E6 cells were infected with Venezuelan equine encephalitis virus (A, MOI=0.01) or western equine encephalitis virus (B, MOI=0.1) for 18 hours. Detect infected cells and count on Operetta as described in this manual.

Example 17

Treatment of Paramyxoviridae Infection in Subjects

Exemplary Paramyxoviridae virus infections include Hennivirus infections, Nipah virus infections, or Hendra virus infections.

Method A. Antiviral composition therapy

The antiviral composition is administered to a subject exhibiting a Paramyxoviridae infection, and in addition, a treatment-related dose is administered to the subject for a period of time as specified in the dosing schedule. Periodically determine the subject's treatment response level. The level of treatment response can be determined by determining the virus titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose will be escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is reached. Subjects continue to be treated with the antiviral composition as needed, and in addition, the dosage or dosing regimen can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination therapy: antiviral composition and other agents

In addition to instructing the subject and administering one or more other therapeutic agents for the treatment of Paramyxoviridae infection or its symptoms, the above method A is followed. Thus, one or more other therapeutic agents can be administered before, after, or together with the antiviral composition. It is also possible to increase (or decrease) the dose of one or more other therapeutic agents.

Example 18

Cell line and isolation of primary huPBMC

In a humidified incubator at 37°C and 10% CO ₂ supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biowest), 100U/mL penicillin, 100μg/mL streptomycin sulfate, and 20μg/mL sodium sulfate Iscove's modified Dulbecco's culture medium (IMDM; ATCC No. 30-2005) produced by Damycin (Life Technologies) was cultured in the HTLV-1 transformed (HTLV-1+) SLB1 lymphoma T cell line (Arnold et al. , 2008; courtesy of P. Green, The Ohio State University-Comprehensive Cancer Center).

Isolation of primary human peripheral blood mononuclear cells (huPBMC) from whole blood samples. The aforementioned samples were provided by SMU Memorial Health Center in accordance with an agreement approved by the SMU Institutional Review Board. Sample without identification code and in compliance with the Declaration of Helsinki principles. In short, mix 2ml of whole blood with an equal volume of sterile phosphate buffered saline (PBS) pH7.4 in a polypropylene conical tube (Corning), and then gently spread the sample on 3ml of lymphocyte separation Medium (MP Biomedicals). The samples at room temperature tilting rotor centrifuged for 30 minutes at 400x g and subsequently huPBMC buffy coat aspirated, washed twice with the culture solution (ATCC No.30-2001) in RPMI-1640, and to 260x Centrifuge the pellet at g for 7 minutes. Resuspend the cells in supplemented with 20% FBS, 100U/ml penicillin, 100μg/ml streptomycin sulfate, 20μg/ml gentamicin sulfate and 50U/ml recombinant human interleukin-2 (hu-IL-2; Roche Applied Science) RPMI-1640 culture medium, and stimulated with 10ng/ml phytohemagglutinin (PHA; Sigma-Aldrich) for 24 hours, and grow _{in a humidified incubator at 37°C and 10% CO 2.} The next day, the cells were centrifuged 7 minutes at 260x g to pellet the cells, and washed with RPMI-1640 cultured twice to remove the PHA, supplemented with antibiotics and then complete medium 50U / ml hu-IL-2 were resuspended in And cultivate.

Example 19

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

In order to produce GFP-expressing HTLV-1+SLB1 T cell clones, 2x10 ^{6 SLB1 cells were plated in a 60mm 2} tissue culture dish (Corning) supplemented with 10% heat-inactivated FBS and antibiotic IMDM, and then used with pLenti -6.2/V5-DEST green fluorescent protein expression vector, and lentiviral particles carrying blasticidin resistance gene for transduction. After 6 hours, the transduced cells were pelleted by centrifugation at 260 x g for 7 minutes at room temperature, washed twice with serum-free IMDM, and then resuspended in complete medium supplemented with 5 μg/mL blasticidin (Life Technologies ), and add aliquots to 96-well microtiter plates (Corning). In a humidified incubator at 37°C and 10% CO ₂ , blasticidin was used to screen and maintain the culture for two weeks. The lymphoblasts expressing GFP were screened by fluorescence microscope, and then plated with limiting dilution in 96-well microtiter plates to obtain homogenous cell clones expressing GFP. The HTLV-1+SLB1/pLenti-GFP T lymphocyte clone obtained was expanded and passaged repeatedly; the expression of GFP was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and rabbit anti- GFP(FL) multiple antibodies were confirmed by immunoblotting (Santa Cruz Biotechnology).

Example 20

Quantitative virus production and particle infectivity by anti-HTLV-1 p19 ^{Gag ELISA}

In order to determine the effect of oleandrin or European oleander extract on the replication of HTLV-1 provirus and the release of newly synthesized extracellular virus particles, the HTLV-1+SLB1 lymphoma T cell line was plated in 96 wells ^{at 2x10 4 cells/well} In the microtiter plate, 300 μl of complete culture medium supplemented with antibiotics were incubated at 37°C and 10% CO ₂ . The purified oleandrin compound and European oleander extract (Phoenix Biotechnology; see Singh et al., 2013) were resuspended in a carrier solution (20% v/v dimethyl sulfoxide, DMSO, in MilliQ distillation/deionized H ₂ O), the concentration of the stock solution was 2mg/ml, and then it was sterilized using a luer-lock 0.2μm syringe filter (Millipore). The HTLV-1+SLB1 cells were treated with oleandrin or European oleander extract at concentrations of 10, 50, and 100 μg/ml, or with an increase (1.5, 7.5, and 15 μl) of the carrier control group for 72 hours. Then use Eppendorf A-2-DWP at room temperature in a pendulum rotor at 260x g to centrifuge the 96-well microtiter plate for 7 minutes to pellet the cells, and perform a colorimetric anti-p19 ^Gag enzyme-linked immunosorbent assay (ELISA ; Zeptometrix), quantitatively released into the culture supernatant, extracellular HTLV-1 particles ^{containing p19 Gag} , relative to the level of the ^{p19 Gag protein standard.} The samples were analyzed in three replicates in absorbance mode at 450 nm on the Berthold Tristar LB 941 multi-mode microplate reader.

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

Example 21

Measure apoptosis

In order to evaluate the relative cytotoxicity of oleandrin compounds, European oleander extracts or control carriers in the treated cell culture, 2 ^{×10 4} HTLV-1+SLB1 lymphoma T cells or activated/cultured huPBMC were plated in 300 μl In a complete medium supplemented with antibiotics, and maintained in a humidified incubator at 37°C and 10% CO ₂ . Increasing concentrations (10, 50 and 100 μg/ml) of oleandrin or European oleander extract, or control vehicle (1.5, 7.5 and 15ml) were cultured for 72 hours. Cyclophosphamide (50 μM; Sigma-Aldrich) treated cells were included as a positive control group for apoptosis. Then the cells were aspirated and plated on Permanox 8-chamber tissue culture slides (Nalge), the slides had been used 0.01% Poly- _L -lysine and concanavalin A (1mg/mL; Sigma-Aldrich) Pretreatment with sterile solution. Subsequently, the sample was stained with a microscope apoptosis detection kit, and phospholipid binding protein V was conjugated with fluorescein isothiocyanate (phospholipid binding protein V-FITC) and propidium iodide (PI; BD-Pharmingen), and The relative percentage of apoptotic (ie, phospholipid binding protein V-FITC and/or PI-positive) cells in each field was quantified three times by using a conjugated focus fluorescence microscope with a 20x objective lens. The total number of cells in each field of view was quantified by a microscope using a DIC phase difference filter.

Example 22

Transmission of HTLV-1 and viral synapse formation in co-culture experiment

Because the transmission of HTLV-1 usually occurs through direct contact between infected cells and uninfected target cells across viral synapses (Igakura et al., 2003; Pais-Correia et al., 2010; Gross et al., 2016 ; Omsland et al., 2018; Majorovits et al., 2008), we tested whether oleandrin, European oleander extract or control vector can affect the formation of viral synapses and/or infectious HTLV-1 particles in vitro by intercellular The spread of interaction. For these experiments, 2x10 ⁴ virus-producing HTLV-1+SLB1 T cells were plated in a 96-well microtiter plate, and mitomycin was used in 300 μl of complete culture medium _{at 37°C and 10% CO 2} C (100 μg/mL) was treated for 2 hours (Bryja et al., 2006). Then remove the culture medium, wash the cells twice with serum-free IMDM, and treat the cells with increasing amounts (10, 50, and 100 μg/ml) of oleandrin or European oleander extract, or the control vehicle (1.5, 7.5, and 15 μl) 15 Minutes or 3 hours. Optionally, 2x10 ⁴ GFP-expressing HTLV-1+SLB1/pLenti-GFP T cells were plated on 8-chamber tissue culture glass slides in 300 μl complete culture medium, and treated with mitomycin C, and serum-free IMDM was washed twice, and then treated with oleandrin, European oleander extract, or control carrier as described in the conjugate focus microscope experiment. Subsequently, we aspirated and medium with serum-free culture washings were HTLV-1 + SLB1 cells twice, and added 2x10 ⁴ th huPBMC to have 20% FBS, antibiotics and 50U / ml hu-IL-2 in 300μl of complement In each well of the RPMI-1640 medium, the cells were co-cultured in a humidified incubator at 37°C and 10% CO ₂ for 72 hours (the cells were co-cultured for 6 hours, using SLB1/pLenti-GFP lymphoblasts Observe the formation of virus synapses and virus spread by conjugate focus microscope). As a negative control group, huPBMC was cultured alone without virus-producing cells. Maintain oleandrin, European oleander extract and carrier in the co-culture medium. An anti-HTLV-1 p19 ^Gag ELISA was performed to quantify the relative level of ^{extracellular p19 Gag} -containing HTLV-1 particles released into the supernatant of the co-culture due to virus transmission between cells. Use an immunofluorescence conjugate focus microscope to observe the relationship between GFP-positive HTLV-1+SLB/pLenti-GFP cells and huPBMC by staining the fixed sample with anti-HTLV-1 gp21 ^{Env primary antibody and rhodamine red-conjugated secondary antibody} The formation of viral synapses. Diamidino-2-phenylindole, dihydrochloride (DAPI; molecular probe) nuclear staining was included for comparison and observation of uninfected (ie, HTLV-1 negative) cells. By using a 20x objective lens to count ^{the relative percentage of HTLV-1 gp21 Env} positive (and GFP negative) huPBMC in 20 fields of view, the transmission of HTLV-1 to huPBMC in the co-culture experiment was quantified.

Example 23

microscope

Use Plan-Apochromat 20x/0.8 objective lens and Zeiss ZEN system software (Carl Zeiss Microscopy) on _{Zeiss LSM800 instrument equipped with Airyscan detector and load bench CO 2} incubator to observe phospholipid binding protein V- FITC/PI stained samples to quantify apoptosis and cytotoxicity. The formation and formation of viral synapses between mitomycin C-treated HTLV-1+SLB1/pLenti-GFP lymphoblasts and cultured huPBMC were observed by immunofluorescence conjugate focus microscope using Plan-Apochromat 20x/0.8 objective lens Viral transmission (ie, determined by quantifying the relative percentage of ^{anti-HTLV-1 gp21 Env positive huPBMC).} Use Zen 2.5D analysis tool (Carl Zeiss Microscopy) to graphically quantify ^{the relative fluorescence intensity of DAPI, anti-HTLV-1 gp21 Env} specific (rhodamine red positive) and GFP signal. The Nikon Eclipse TE2000-U inverted microscope equipped with 633nm and 543nm He/Ne and 488nm Ar lasers and the conjugate focus fluorescent microscope on the D-Eclipse conjugate focus imaging system use Plan Fluor 10x/0.30 objective lens and DIC phase difference filter Light sheet (Nikon Instruments) to screen the colonization of HTLV-1+SLB1/pLenti-GFP T cells expressing GFP.

Example 24

Statistical Analysis

The unpaired two-tailed Student’s t test (α=0.05) was used to determine the statistical significance of the experimental data set, and the Shapiro-Wilk normality test and Graphpad Prism 7.03 software were used to calculate the P value. The P value is defined as: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). Unless otherwise stated, error bars represent SEMs from at least three independent experiments.

Example 25

Treatment of delta retroviral infection in subjects

Exemplary delta retrovirus infection containing HTLV-1

Method A. Antiviral composition therapy

An antiviral composition is administered to a subject exhibiting HTLV-1 infection, and in addition, a therapeutically relevant dose is administered to the subject for a period of time as specified in the dosing schedule. Periodically determine the subject's treatment response level. The level of therapeutic response can be determined by determining the titer of the HTLV-1 virus in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose will be escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is reached. Subjects continue to be treated with the antiviral composition as needed, and in addition, the dosage or dosing regimen can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination therapy: antiviral composition and other agents

In addition to instructing and administering to the subject one or more other therapeutic agents for the treatment of HTLV-1 infection or its symptoms, the above method A is followed. Thus, one or more other therapeutic agents can be administered before, after, or together with the antiviral composition. It is also possible to increase (or decrease) the dose of one or more other therapeutic agents. Exemplary other therapeutic agents are as described in this specification.

Example 26

Treatment of CoV infection in subjects

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

Method A. Antiviral composition therapy

An antiviral composition is administered to a subject exhibiting CoV infection, and in addition, a therapeutically relevant dose is administered to the subject for a period of time as specified in the dosing schedule. Regularly determine the subject's treatment response water flat. The treatment response level can be determined by determining the CoV virus titer in the blood or plasma of the subject. If the level of therapeutic response at a dose is too low, the dose will be escalated according to a predetermined dose escalation plan until the desired level of therapeutic response in the subject is reached. Subjects continue to be treated with the antiviral composition as needed, and in addition, the dosage or dosing regimen can be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B. Combination therapy: antiviral composition and other agents

In addition to instructing and administering to the subject one or more other therapeutic agents for the treatment of CoV infection or its symptoms, the above method A is followed. Thus, one or more other therapeutic agents can be administered before, after, or together with the antiviral composition. It is also possible to increase (or decrease) the dose of one or more other therapeutic agents. Exemplary other therapeutic agents are as described in this specification.

Example 27

Use ANVIRZEL ^TM to treat COVID-19 infection in subjects

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

Example 28

In vitro evaluation of oleandrin against COVID-19 infection

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

Prepare a stock solution of oleandrin in methanol (10mg oleandrin/mL). The aforementioned stock solution is used to prepare DMSO (in aqueous culture medium RPMI 1640 and oleandrin (20μg/mL, 10μg/mL, 1.0μg/mL or 0.1 μg/mL) 0.1% or 0.01% v/v) of the culture medium, the culture medium such as Shown below.

Uninfected Vero cells in the culture (target initial cell count is 1×10 ⁶ ) were cultured in each vial of the indicated culture medium at 37° C. for 30 minutes. The virus inoculum of SARS-CoV-2 was then added to each vial to reach the target initial virus titer (approximately PFU/mL 1×10 ⁴ ). The target MOI (viral infection dose) is about 0.1. The solution was incubated at 37°C for another 2 hours to achieve Vero cell infection. Then the infected Vero cells were washed with the control vector to remove extracellular viruses and oleandrin. A new aliquot of each culture medium was added to each corresponding vial of infected Vero cells. Those that received oleandrin in the second aliquot were denoted as "+post-infection treatment", while those that did not receive oleandrin in the second aliquot were denoted as "-post-infection treatment" (Figure 23A ~23D). The virus titer of each vial was determined about 24 hours and about 48 hours after infection.

As a means to determine the potential toxicity of oleandrin against Vero cells, parallel cultures were prepared against uninfected Vero cells based on the above-mentioned culture medium.

The data obtained includes the number of viruses produced, the infectivity of progeny viruses, and the relative safety (non-toxic) of oleandrin in infected and uninfected cells.

Example 29

In vitro evaluation of oleandrin against COVID-19 virus

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

6-well plate was removed from confluent monolayers of about 10 ⁶ Vero CCL81 cells in growth media. Oleandrin was serially diluted in the culture medium, and then added to Vero-E6 cells seeded in 96-well plates. Replace the growth medium with 200μl maintenance medium, which contains 1.0μg/mL, 0.5μg/mL, 100ng/mL, 50ng/mL, 10ng/mL or 5ng/mL oleandrin, or a matched control group containing only DMSO . Before adding virus, the plate was incubated at 37°C for about 30 minutes.

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

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

Add a separate medium (without oleandrin) back to the oleandrin-treated wells on the data slide designated as "pretreatment". The medium with the specified concentration of oleandrin is added back to the wells on the data slide designated as "Duration".

The plates were fixed 24 hours (entry test) or 48 hours (replication test) after infection, and immunostained with virus-specific antibodies and fluorescently labeled secondary antibodies.

Use Operetta to image the cells and analyze the data using custom algorithms in the Harmonia software to determine the percentage of infected cells in each well.

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

Example 30

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

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

The oleandrin was serially diluted in the culture solution and added to Vero-E6 cells seeded in 96-well plates, and cultured at 37°C for 24 hours. The cell count was obtained using the CellTiter Glo assay.

The results are shown in Figure 25.

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 oleandrin treatment.

Except for using the following lower concentrations of oleandrin, the steps of Example 28 were repeated: 1 μg/mL, 0.5 μg/mL, 0.1 μg/mL, 0.05 μg/mL, 0.01 μg/mL, and 0.005 μg/mL. In addition, VERO CCL-81 cells are used instead of VERO E6 cells.

The virus titer was determined according to Example 28, and the reduction factor of the virus titer was calculated by comparing with the control sample. The results are shown in Figures 26A to 26D, 27A to 27D, and 28A and 28B.

Example 32

Sublingual liquid dosage form

The sublingual liquid dosage form consists of combining oleandrin or a composition containing oleandrin (for example, an extract containing oleandrin; 2% by weight), medium chain triglycerides (95% by weight) and flavoring agents (3% by weight) Oleandrin made by mixing. The content of oleandrin in the dosage form is about 25 μg/mL.

Example 33

Preparation of subcritical fluid extract of Oleander

By using subcritical fluid extraction instead of supercritical liquid extraction of oleander biomass, an improved method for the preparation of oleandrin-containing extracts was developed.

The dry and powdered biomass is placed in the extraction chamber, and then it is sealed. Carbon dioxide (about 95% by weight) and alcohol (about 5% by weight; methanol or ethanol) are injected into the chamber. The internal temperature and pressure of the chamber keep the extraction medium in the subcritical fluid phase rather than the supercritical fluid phase during most or substantially all of the extraction time period: the temperature is between about 2°C and about 16°C (about 7°C to about 8°C). ℃), the pressure is in the range of about 115 to about 135 bar (about 124 bar). The extraction time is about 4 hours to about 12 hours (about 6 hours to about 10 hours). Then the extraction environment is filtered and the supernatant is collected. from up Carbon dioxide is discharged from the clear liquid, and the resulting crude extract is diluted into ethanol (about 9 parts of ethanol: about 1 part of the extract), and frozen at about -50°C for at least 12 hours. The solution was thawed and filtered (100 micron pore size filter). The filtrate was concentrated to about 10% of its original volume, followed by sterile filtration (filter with 0.2 micron pore size). Next, the concentrated extract was diluted with a 50% aqueous ethanol solution to a concentration of about 1.5 mg of extract per mL of the solution.

The resulting subcritical fluid (SbCL) extract contains oleandrin extractable from oleander and one or more other compounds, and the aforementioned one or more other compounds are as defined in this specification.

Example 34

In vitro evaluation of oleandrin against COVID-19 virus

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

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

Example 35

In vivo evaluation of oleandrin against COVID-19 virus

The purpose of this study is to determine the efficacy of oleandrin-containing extracts (OCE) in the treatment of subjects who have been infected with the COVID-19 virus.

Prior to sublingual, buccal, or oral administration of the OCE prepared according to the dosage form of Example 32, subjects who were sufficiently representative of a large population distribution and exhibiting COVID-19 infection were evaluated to determine their clinical status. By dripping the liquid into the oral cavity of the subject, the composition is safely administered to the subject. The dosage regimen is about 0.5 mL per dose, four doses per day (approximately one dose every six hours), approximately every About 50 micrograms of oleandrin per day. Optionally, only half of the total daily dose has to be administered. All subjects recovered completely.

Example 36

Preparation of Ethanol Extract of Oleander

The purpose of this study is to prepare ethanol extracts by extracting oleander biomass with ethanol aqueous solution.

The ground dry leaves were repeatedly treated with an aqueous ethanol solution (90% v/v ethanol; 10% v/v water). The combined ethanol supernatant was combined and filtered, and then concentrated by vacuum evaporation to reduce the amount of ethanol and water, to provide a crude ethanol extract containing about 25 mg of oleandrin/mL extract (with about 50% v/ v Ethanol content).

Example 37

Preparation of a dosage form containing a combination of oleander extracts

The purpose of this study is to prepare a dosage form as in Example 32. The difference is that the part (1% by weight) of the ethanol extract of Example 36 and the part (1% by weight) of the SbCL extract of Example 33 are different from each other. Chain triglycerides (95% by weight) and flavoring agents (3% by weight) are combined.

Example 38

In vivo evaluation of digitonin against COVID-19 virus

The purpose of this study is to determine the efficacy of a composition containing digitonin (DCC) in the treatment of subjects who have been infected by the COVID-19 virus. Use a commercially available dosage form containing digitonin.

Evaluate subjects exhibiting COVID-19 infection before oral or systemic administration of DCC to determine clinical status. The commercially available composition is as described in this specification. Each safe dosing regimen is described in the respective NDA and package insert. The composition is safely administered to each subject as a predetermined mode of administration. Perform clinical monitoring to determine treatment response and titrate the dose accordingly.

Example 39

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

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

In order to quantify the genomic copy of the sample, 200 μl of the sample was extracted with TRIzol LS (Ambion, Carlsbad, CA) at a volume ratio of 5:1 using a standard manufacturer's protocol and resuspended in 11 μl of water. According to the previously disclosed test (26), SARS-CoV-2 was detected by qRT-PCR on the extracted RNA. In short, the following primers and probes were used to amplify the N gene: forward primer [5'-TAATCAGACAAGGAACTGATTA-3'] (SEQ ID NO. 1); reverse primer [5'-CGAAGGTGTGACTTCCATG-3'] ( SEQ ID NO. 2); Probe [[5'-FAM-GCAAATTGTGCAATTTGCGG-TAMRA-3'; (SEQ ID NO. 3)]. Using iTaq Universal Probe One-Step kit (BioRad, Hercules, CA), prepare 20μl reaction mixture according to the manufacturer's instructions: reaction mixture (2x: 10μL), iScript reverse transcriptase (0.5μL), primer ( 10μM: 1.0μL), probe (10μM: 0.5μL), extracted RNA (4μL) and water (3μL). A thermal cycler StepOnePlus ^TM real-time PCR system (Applied Biosystems) was used for qRT-PCR reaction. The reaction was carried out at 50°C for 5 minutes and 95°C for 20 seconds, followed by reaction at 95°C for 5 seconds and 60°C for 30 seconds in 40 cycles. The positive control RNA sequence (nucleotides 26,044-29,883 of the COVID-2019 genome) is used to estimate the number of RNA copies of the N gene in the sample being evaluated.

As used in this specification, the term "about" or "approximately" refers to ±10%, ±5%, ±2.5%, or ±1% of a specific value. As used in this manual, the term "substantially" means "to a large extent" or "at least most" or "more than 50%".

The foregoing is a detailed description of specific embodiments of the present invention. It should be understood that although this specification has described specific embodiments of the present invention for illustrative purposes, various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited except for the scope of the attached patent application. According to the content of the present invention, all the embodiments disclosed and claimed in this specification can be manufactured and implemented without undue experimentation.

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

<110> Phoenix Biotechnology, Inc. (Phoenix Biotechnology, Inc.)

<120> Methods and compositions for the treatment of coronavirus infections

<130> PBI-22-PCT9

<140> TW 110101732

<141> 2021-01-15

<150> US 63/002735

<151> 2020-03-31

<150> US 63/010246

<151> 2020-04-15

<150> US 63/014294

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<151> 2020-04-29

<150> US 63/021512

<151> 2020-05-07

<150> US 63/029530

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<150> US 63/034800

<151> 2020-06-04

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<151> 2020-06-08

<150> US 63/042656

<151> 2020-06-23

<150> US 63/051576

<151> 2020-07-14

<160> 3

<170> PatentIn version 3.5.1

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<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Forward (5’ end to 3’ end) primer sequence

<400> 1

<210> 2

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse (5’ end to 3’ end) primer sequence

<400> 2

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe sequence (5’-FAM-sequence-TAMRA-3’); FAM is 6-fluorescein amidite; TAMRA is Carboxytetramethylrhodamine

<400> 3

Claims (27)

A method for treating COVID-19 (SARS-CoV-2) coronavirus infection, which is characterized by administering one or more therapeutically effective doses of oleandrin to subjects in need every day, and the treatment period is at least about 5 days to About one month or multiple months. The method of claim 1, wherein the total daily dose of oleandrin is independently selected from the following at each occurrence: about 1 μg to about 180 μg, about 1 μg to about 120 μg, about 5 μg to about 100 μg, about 10 μg to about 80 μg, about 10 μg to about 75 μg, about 15 μg to about 120 μg, about 15 μg to about 100 μg, about 15 μg to about 80 μg, about 30 μg to about 120 μg, about 30 μg to about 100 μg, about 30 μg to about 90 μg, about 40 μg to about 70 μg, About 1 μg, about 5 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg , About 160 μg, about 170 μg, about 180 μg, about 140 μg to about 315 μg, about 20 μg to about 750 μg, about 12 μg to about 300 μg, about 12 μg to about 120 μg, about 0.01 μg to about 100 mg, about 0.01 μg to about 100 μg, about 0.5 To about 100 μg, about 1 to about 80 μg, about 1.5 to about 60 μg, about 1.8 to about 60 μg, and about 1.8 to about 40 μg. The method according to claim 1, wherein the dose includes about 0.05~0.5mg/kg/day, about 0.05~0.35mg/kg/day, about 0.05~0.22mg/kg/day, about 0.05~0.4mg/kg /Day, about 0.05~0.3mg/kg/day, about 0.05~0.5μg/kg/day, about 0.05~0.35μg/kg/day, about 0.05~0.22μg/kg/day, about 0.05~0.4μg/kg /Day or about 0.05~0.3μg/kg/day, which is based on the unit amount of oleandrin per kg of subject's body weight per day. The method according to claim 1, wherein a) the maximum daily dose of oleandrin is: about 100 μg/day, about 80 μg/day, about 60 μg/day, about 40 μg/day, about 38.4 μg/day, or about 30 μg /Day; and/or b) The minimum daily dose of oleandrin is: about 0.5μg/day, about 1μg/day, About 1.5 μg/day, about 1.8 μg/day, about 2 μg/day, or about 5 μg/day. The method according to claim 1, wherein one dose is administered twice a day or about every 12 hours, and the amount of oleandrin in the dose is about 0.25 to about 50 μg, or about 0.9 to 15 μg. The method according to claim 1, wherein one or more doses of oleandrin are 2 to 10 doses/day, 2 to 8 doses/day, 2 to 6 doses/day, 2 to 4 doses/day, 2 Dose/day, 3 doses/day, 4 doses/day, 5 doses/day, 6 doses/day, 7 doses/day, 8 doses/day, 9 doses/day or 10 doses/day. The method according to claim 1, wherein oleandrin is present in a composition comprising at least one extract obtained from a biomass containing oleandrin. The method according to claim 5, wherein the extract is independently selected from the group consisting of hot water extract, solvent extract, and supercritical fluid extract each time it occurs. The method according to claim 1, wherein after the 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/mL, about 0.005 to In the range of about 8 ng/mL, about 0.01 to about 7 ng/mL, about 0.02 to about 7 ng/mL, about 0.03 to about 6 ng/mL, about 0.04 to about 5 ng/mL, or about 0.05 to about 2.5 ng/mL, which It is based on the amount of oleandrin per mL of plasma. A method for treating COVID-19 (SARS-CoV-2) coronavirus infection is characterized by administering a plurality of therapeutically effective doses of oleandrin to subjects suffering from the infection. The method according to claim 10, wherein the therapeutically effective dose of the plural doses is one or plural doses administered daily on two or more days a week. The method according to claim 11, wherein the administration is continued for one or more weeks every month. The method according to claim 12, wherein the administration is continued every year for one month or more Months. The method according to claim 10, wherein oleandrin is present in a composition comprising at least one extract obtained from a biomass containing oleandrin. The method according to claim 14, wherein the extract is independently selected from the group consisting of hot water extract, solvent extract, and supercritical fluid extract each time it occurs. The method according to claim 14, wherein the extract comprises a combination of oleandrin and one or more compounds extracted from the biomass. The method according to claim 16, wherein the one or more compounds comprise one or more cardiac glycoside precursors, one or more glycosomal components of cardiac glycosides, or a combination thereof. The method according to claim 10, wherein the administration is systemic, parenteral, buccal, intestinal, intramuscular, subcutaneous, sublingual, oral, oral, or a combination thereof. The method according to claim 10, wherein the antiviral composition is administered immediately after infection, or at any time within 1 to 5 days after infection, or at the earliest time after the diagnosis of viral infection. The method according to claim 10, wherein the antiviral composition is administered as a primary antiviral therapy, an auxiliary antiviral therapy, or a combined antiviral therapy, or the administration comprises: the composition and at least one other antiviral composition Or co-administered with at least one other composition used to treat symptoms associated with the viral infection. The method according to claim 10, wherein the total daily dose of oleandrin is independently selected from the following at each occurrence: about 1 μg to about 180 μg, about 1 μg to about 120 μg, about 5 μg to about 100 μg, about 10 μg to about 80 μg, about 10 μg to about 75 μg, about 15 μg to about 120 μg, about 15 μg to about 100 μg, about 15 μg to about 80 μg, about 30 μg to about 120 μg, about 30 μg to about 100 μg, about 30 μg to about 90 μg, about 40 μg to about 70 μg, About 1μg, about 5μg, about 10μg, About 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg , About 140 μg to about 315 μg, about 20 μg to about 750 μg, about 12 μg to about 300 μg, about 12 μg to about 120 μg, about 0.01 μg to about 100 mg, about 0.01 μg to about 100 μg, about 0.5 to about 100 μg, about 1 to about 80 μg , About 1.5 to about 60 μg, about 1.8 to about 60 μg, and about 1.8 to about 40 μg. The method according to claim 10, wherein the dose includes about 0.05 to 0.5 mg/kg/day, about 0.05 to 0.35 mg/kg/day, about 0.05 to 0.22 mg/kg/day, and about 0.05 to 0.4 mg/kg /Day, about 0.05~0.3mg/kg/day, about 0.05~0.5μg/kg/day, about 0.05~0.35μg/kg/day, about 0.05~0.22μg/kg/day, about 0.05~0.4μg/kg /Day or about 0.05~0.3μg/kg/day, which is based on the unit amount of oleandrin per kg of subject's body weight per day. The method according to claim 10, wherein a) the maximum daily dose of oleandrin is: about 100 μg/day, about 80 μg/day, about 60 μg/day, about 40 μg/day, about 38.4 μg/day, or about 30 μg /Day; and/or b) The minimum daily dose of oleandrin is: about 0.5 μg/day, about 1 μg/day, about 1.5 μg/day, about 1.8 μg/day, about 2 μg/day, or about 5 μg/day. The method according to claim 10, wherein one dose is administered twice a day or about every 12 hours, and the amount of oleandrin in the dose is about 0.25 to about 50 μg, or about 0.9 to 15 μg. The method according to claim 10, wherein one or more doses of oleandrin are 2 to 10 doses/day, 2 to 8 doses/day, 2 to 6 doses/day, 2 to 4 doses/day, 2 Dose/day, 3 doses/day, 4 doses/day, 5 doses/day, 6 doses/day, 7 doses/day, 8 doses/day, 9 doses/day or 10 doses/day. The method according to claim 10, wherein after the 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/mL, about 0.005 to About 8ng/mL, about 0.01 to about 7ng/mL, about 0.02 to about 7ng/mL, It is in the range of about 0.03 to about 6 ng/mL, about 0.04 to about 5 ng/mL, or about 0.05 to about 2.5 ng/mL, which is based on the amount of oleandrin per mL of plasma. The method according to claim 16, wherein the extract comprises oleandrin and one or more compounds selected from the group consisting of cardiac glycosides, glycosides, aglycones, steroids, triterpenes, polysaccharides, sugars Classes, alkaloids, fats, proteins, oleandroside, ordonoside, oleanolic acid, ursolic acid, betulinic acid, oleandrin, oleandrin A, betulin (Urso-12-ene-3β) ,28-diol), 28-nor urso-12-ene-3β-ol, urso-12-ene-3β-ol, 3β,3β-hydroxy-12-oleanolic acid-28-acid, 3β,20α-dihydroxyurso-21-ene-28-acid, 3β,27-dihydroxy-12-ursoene-28-acid, 3β,13β-dihydroxyurso-11-ene-28-acid , 3β,12α-dihydroxyoleanane-28,13β-lactone, 3β,27-dihydroxy-12-oleanolic-28-acid, homogalacturonic acid, arabinogalacturonic acid, green Original acid, caffeic acid, L-quinic acid, 4-coumarol coenzyme A, 3-O-caffeic quinic acid, 5-O-caffeic quinic acid, cardiac glycoside B-1, cardiac glycoside B- 2. Oleandrin, ganglioside, nerolin, ordonoside H, 3-β-O-(D) composed of galacturonic acid, rhamnose, arabinose, xylose and galactose -Glyoside)-5-β,14β-dihydroxy-cardonosta-20(22)-nonolactone pectin polysaccharide, MW is in the range of 17000~120000D or MW is about 35000D, about 3000D, about 5500D or About 12000D of polysaccharides, cardiac glycosides, cardiac glycosides, cardiac glycosides N-1, cardiac glycosides N-2, cardiac glycosides N-3, cardiac glycosides N-4, pregnane, 4,6-diene-3,12,20- Triketone, 20R-hydroxyprogesterone-4,6-diene-3,12-dione, 16β,17β-epoxy-12β-hydroxyprogesterone-4,6-diene-3,20-dione, 12β-Hydroxypregna-4,6,16-triene-3,20-dione (Ouyidienone A), 20S,21-dihydroxypregna-4,6-diene-3,12- Dione (Ouyi Dienone B), oleander coumaric acid, iso-oleander coumaric acid, oleandrolic acid, oleandene, 8α-Methoxy Heliphae-18-acid, 12-ursoene, mannoside, neriumoside , 3β-O-(D-Glyoside)-2α-hydroxy-8,14β-epoxy-5β-cardiotoner-16:17,20:22-dienolactone, 3β-O-(D- Glycoside)-2α,14β-dihydroxy-5β-cardiotoner-16:17,20:22-dienolactone, 3β,27-dihydroxy-urso -18-ene-13,28-lactone, 3β,22α,28-trihydroxy-25-nor-lupin-1(10),20(29)-diene-2-one, cis-carinine (3β-Hydroxy-28-Z-p-coumarinoxy-Uso-12-ene-27-acid), trans-carinin (3-β-hydroxy-28-E-p-coumarinoxy -Usuo-12-ene-27-acid), 3β-hydroxy-5α-cardiotoner-14(15), 20(22)-dienolactone (β-anhydrourosaglycone), 3β-O- (D-digital glycoside)-21-hydroxy-5β-cardiotonol-8, 14,16,20(22)-tetraenolide (oleandrin-A-3β-D-digitalin), Cucurbita aglycone, Ouyi Dienone A, 3β,27-dihydroxy-12-Usole-28-acid, 3β,13β-Dihydroxyurso-11-ene-28-acid, 3β-Hydroxy Urso-12-ene-28-aldehyde, 28-methyl urso-12-ene-3β-alcohol, urso-12-ene-3β-alcohol, urso-12-ene-3β, 28-diol, 3β,27-dihydroxy-12-oleanolene-28-acid, (20S,24R)-epoxydammarane-3β,25-diol, 20β,28-epoxy-28α-methoxy Taraxacum steroid-3β-alcohol, 20β,28-epoxy taraxacum ster-21-en-3β-ol, 28-nor-urso-12-ene-3β,17β-diol, 3β-hydroxyurso- 12-ene-28-aldehyde, α-neriursate, β-neriursate, 3α-Acetylphenoxy-Uso-12-ene-28-acid, 3β-Acetylphenoxy-Uso-12-ene- 28-acid, oleanolic acid, canardenone, 3β-p-hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursoene-28-acid, 28-hydroxy-20(29)-lupin Ene-3,7-dione, kanerocin, 3α-hydroxy-urso-18,20-diene-28-acid, D-salmonose, D-dipyranose, ganglioside, neringoside, iso Sesame oleic acid, gentiobioside ganglioside, gentiobioside qingming anthocyanin, gentiobioside oleandrin, oleandrin C, 12β-hydroxy-5β-cardiotoner-8,14,16,20(22 )-Tetranolactone, 8β-hydroxy-digitoxin, △16-8β-hydroxy-digitoxin, △16-matrine, urfacol, arbutal, 2'7(right Coumadin oxygen) ursolic acid, oleandrol, 16-anhydro-deacetyl-ganglioside, 9-D-hydroxy-cis-12-octadecanoic acid, adiglycin, oleandrin B, α- Fragrant, β-sitosterol, campesterol, rubber, capric acid, caprylic acid, choline, cornerin, cortenerin, deacetylated oleandrin, diacetyl-ganglioside, oleandrin, and raft Tysamine, quercetin, quercetin-3-rhamnoside, quercetin, rosagi nin, rutin, stearic acid, stigmasterol, digoxigenin, urehitoxin and ursaglycin.

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