| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | June 30, 1998 |
| PATENT TITLE |
Biflavanoids and derivatives thereof as antiviral agents |
| PATENT ABSTRACT |
Substantially purified antiviral biflavanoids robustaflavone, hinokiflavone, amentoflavone, agathisflavone, volkensiflavone, morelloflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a are provided. Antiviral biflavanoid derivatives and salt forms thereof, e.g., robustaflavone tetrasulfate potassium salt, and methods for preparing the same are also disclosed. Pharmaceutical compositions which include the antiviral biflavanoids, derivatives or salts thereof are also provided. Also disclosed is an improved method for obtaining substantially pure robustaflavone from plant material. The biflavanoid compounds, derivatives or salts thereof of the invention may be used in a method for treating and/or preventing viral infections caused by viral agents such as influenza, e.g., influenza A and B; hepatitis, e.g., hepatitis B; human immunodeficiency virus, e.g., HIV-1; Herpes viruses (HSV-1 and HSV-2); Varicella Zoster virus (VZV); and measles. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 21, 1996 |
| PATENT REFERENCES CITED |
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Robustaflavone from the seed-kernels of Rhus succedanea. Phytochemistry, 1974, 13, 1617-1619. Lin, Y.M.; Chen, F.C. Agathisflavone from the drupes of Rhus succedanea. Phytochemistry, 1974B, 13, 657-658 (1974). Lin, Y,M,; Chen, F.C.; Lee, K.H. Hinokiflavone, a cytotoxic principle from Rhus succedanea and the cytoxicity of the related biflavonoids. Planta Medica, 1989, 55, 166-168. Lopez-Saez, J.A.; Perez-Alonso, M.; Negueruela, A.V. Biflavanoids of Selaginella denticulata growing in Spain. Naturforsch., 1994, 49c, 267-270. Magri, N.J.; Kinston, D.G. I. Modified Toxols, 4. Synthesis and biological activity of toxols modified in the side chain. J. Nat. Prod., 1987, 51, 298-306. Markham, K.R.; Sheppard, C.; Geiger, H. .sup.13 C NMR studies of some naturally occurring amentoflavone and hinokiflavone biflavanoids. Phytochemistry, 1987, 26, 3335-3337. Martin, P. and Friedman, L. S. In Innovations in Antiviral Development and the Detection of Virus Infections; T. M. Block; D. Junkind; R. L. 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Production of hepatitis B virus by stably transfected monocytic cell line U-937: a model for extrahepatic hepatitis B virus replication. J. Infect. Dis., 1992, 165, 929-933. Murakami, A.; Ohigashi, H.; Jisaka, M.; Irie, R.; Koshimizu, K. Inhibitory effects of new types of biflavonoid-related polyphenols; lophirone A and lophiraic acid, on some tumor promoter-induced biological responses in vitro and in vivo. Cancer Lett. (Shannon, Irel.), 1991, 58, 101-106. Nagai, T.; Miyaichi, Y.; Tomimori, T.; Suzuki, Y.; Yamada, N. Inhibition of influenza virus sialidase and anti-influenza virus activity by plant flavonoids. Chem. Pharm. Bull., 1990, 38, 1329-1332. Nagai, T.; Miyaichi, Y.; Tomimore, T.; Suzuki, Y.; Yamada, H. In vivo anti-influenza virus activity of plant flavonoids possessing inhibitory activity for influenza virus sialidase. Antiviral Res., 1990, 19: 207-217. Nagai, T.; Suzuki, Y.; Tomimore, T.; Yamada, H. Antiviral activity of plant flavonoid, 5,7,4'-trihydroxy-8-methoxyflavone, from roots of Scutellaria baicalenais against influenza A (H3N2) and B viruses. Biol. Pharm. Bull., 1995, 18, 295-299. Nagai, T.; Moriguchi, R.; Suzuki, Y.; Tomimori, T.; Yamada, H. Mode of action of the anti-influenza virus activity of plant flavonoid, 5,7,4'-trihydroxy-8-methoxyflavone, form the roots of Scutellaria baicalensis. Antiviral Res., 1995, 26, 11-25. Nakazawa, K. Chemical structure of ginkgetin. Gifu Yakka Diagaku. Kiyo, 1941, 12, 1, Chem. Abst. 59, 2759d. Ono, K.; Nakane, H.; Jukushima, M.; Chermann, J.K.; Barre-Sinlussi, F. Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases. Euro. J. Biochem., 1990, 190, 469-476. Qasim, M.A.; Roy, S.K.; Ilyas, M. Phenolic Constituents of Selaginellaceae. Indian Journal of Chemistry, 1985, 24B, 220. Ray, C. G.; Icenogle, T. B.; Minnich, L. L; Copeland, J. G.; Grogan, T. M. 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| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What we claim: 1. A method for treating an influenza infection in a mammal which comprises administering to said mammal an effective therapeutic amount of a biflavanoid selected from the group consisting of robustaflavone, amentoflavone, and derivative or salt thereof. 2. The method according to claim 1, wherein said derivative or salt comprises a robustaflavone or amentoflavone alkyl ether, ester, acid adduct, amine or sulfate. 3. The method according to claim 2, wherein said biflavonoid derivative or salt is robustaflavone tetrasulfate potassium salt. 4. A method for treating a hepatitis B viral infection in a mammal which comprises administering to said mammal an effective therapeutic amount of robustaflavone, or derivative or salt thereof. 5. The method according to claim 4, wherein said derivative or salt comprises a robustaflavone alkyl ether, ester, acid adduct, amine or sulfate. 6. The method according to claim 5, wherein said derivative or salt is robustaflavone tetrasulfate potassium salt. 7. A pharmaceutical composition comprising a therapeutically effective amount of purified robustaflavone or derivative or salt thereof and a pharmaceutically acceptable carrier therefor. 8. The composition according to claim 7, wherein said derivative or salt comprises robustaflavone alkyl ether, ester, acid adduct, amine or sulfate. 9. The composition according to claim 7, wherein said derivative or salt is robustaflavone tetrasulfate potassium salt. 10. Robustaflavone tetrasulfate potassium salt. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to substantially pure antiviral biflavanoids, e.g., robustaflavone, biflavanoid derivatives and salts thereof such as esters, ethers, amines, sulfates, ethylene oxide adducts, and acid salts, and pharmaceutical compositions containing the same. The present invention also relates to method for extracting substantially pure robustaflavanone from plant material. The present invention also relates to a method for preventing and/or treating viral infections such as hepatitis B, influenza A and B, and HIV. BACKGROUND OF THE INVENTION Viruses, an important etiologic agent in infectious disease in humans and other mammals, are a diverse group of infectious agents that differ greatly in size, shape, chemical composition, host range, and effects on hosts. After several decades of study, only a limited number of antiviral agents are available for the treatment and/or prevention of diseases caused by viruses such as hepatitis B, influenza A and B and HIV. Because of their toxic effects on a host, many antiviral agents are limited to topical applications. Accordingly, there is a need for safe and effective antiviral agents with a wide-spectrum of anti-viral activity with reduced toxicity to the host. Since the identification of the human immunodeficiency virus (HIV) as the causative agent of AIDS,.sup.36,46 the search for safe and effective treatments for HIV infection has become a major focus for drug discovery groups around the world. Investigations into the molecular processes of HIV have identified a number of macromolecular targets for drug design, such as HIV-1 reverse transcriptase (HIV-RT), protease and integrase enzymes, and regulatory proteins (e.g., TAT and REV). Other targets are enzymes which aid in virus attachment and fusion. HIV-RT is an essential enzyme in the life cycle of HIV, which catalyzes the transcription of HIV-encoded single-stranded RNA into double-stranded DNA. Furthermore, the RNA-dependent DNA polymerase function of HIV-RT does not have an analogous process in mammalian metabolism, and thus is a suitable target for a chemotherapeutic agent. The hepatitis B virus (HBV) infects people of all ages. It is one of the fastest-spreading sexually transmitted diseases, and also can be transmitted by sharing needles or by behavior in which a person's mucus membranes are exposed to an infected person's blood, semen, vaginal secretions, or saliva. While the initial sickness is rarely fatal, ten percent of the people who contract hepatitis are infected for life and run a high risk of developing serious, long-term liver diseases, such as cirrhosis of the liver and liver cancer, which can cause serious complications or death..sup.1 The World Health Organization lists HBV as the ninth leading cause of death. It is estimated that about 300 million persons are chronically infected with HBV worldwide, with over 1 million of those in the United States. The Center for Disease Control estimates that over 300,000 new cases of acute HBV infection occurs in the United States each year, resulting in 4,000 deaths due to cirrhosis and 1,000 due to hepatocellular carcinoma..sup.2 The highest rates of HBV infections occur in Southeast Asia, South Pacific Islands, Sub-Saharan Africa, Alaska, Amazon, Bahai, Haiti, and the Dominican Republic, where approximately 20% of the population is chronically infected..sup.3 Hepatitis B virus (HBV) infection is currently the most important chronic virus infection, but no safe and effective therapy is available at present. The major therapeutic option for carriers of HBV is alpha interferon, which can control active virus replication. However, even in the most successful studies, the response rate in carefully selected patient groups has rarely exceeded 40%..sup.5.6 One of the reasons cited for interferon failure is the persistence of viral supercoiled DNA in the liver..sup.7 Clinical exploration of many promising antiviral agents such as nucleoside analogues is hampered because their aspecific body distribution leads to significant toxic side effects. Recently, however, a new nucleoside analogue, 2',3'-dideoxy-3'-thiacytidine (3TC), was discovered and found to be extremely potent against HBV replication with only minimal side effects. .sup.8-10 Influenza is a viral infection marked by fever, chills, and a generalized feeling of weakness and pain in the muscle, together with varying signs of soreness in the respiratory tract, head, and abdomen. Influenza is caused by several types of myxoviruses, categorized as groups A, B, and C.sub.4. These influenza viruses generally lead to similar symptoms but are completely unrelated antigenically, so that infection with one type confers no immunity against the other. Influenza tends to occur in wavelike epidemics throughout the world; influenza A tends to appear in cycles of two to three years and influenza B in cycles of four to five years. Influenza is one of the few common infectious diseases that are poorly controlled by modern medicine. Its annual epidemics are occasionally punctuated by devastating pandemics. For example, the influenza pandemic of 1918, which killed over 20 million people and affected perhaps 100 times that number, was the most lethal plague ever recorded. Since that time, there have been two other pandemics of lesser severity, the so-called Asian flu of 1957 and the Hong Kong flu of 1968. All of these pandemics were characterized by the appearance of a new strain of influenza virus to which the human population had little resistance and against which previously existing influenza virus vaccines were ineffective. Moreover, between pandemics, influenza virus undergoes a gradual antigenic variation that degrades the level of immunological resistance against renewed infection..sup.4 Anti-influenza vaccines, containing killed strains of types A and B virus currently in circulation, are available, but have only a 60 to 70% success rate in preventing infection. The standard influenza vaccine has to be redesigned each year to counter new variants of the virus. In addition, any immunity provided is short-lived. The only drugs currently effective in the prevention and treatment of influenza are amantadine hydrochloride and rimantadine hydrochloride..sup.11-13 While the clinical use of amantadine has been limited by the excess rate of CNS side effects, rimantadine is more active against influenza A both in animals and human beings, with fewer side effects..sup.14,15 It is the drug of choice for the chemoprophylaxis of influenza A..sup.13,16,17 However, the clinical usefulness of both drugs is limited by their effectiveness against only influenza A viruses, by the uncertain therapeutic efficacy in severe influenza, and by the recent findings of recovery of drug-resistant strains in some treated patients..sup.18-22 Ribavirin has been reported to be therapeutically active, but it remains in the investigational stage of development..sup.23,24 While the search for viable therapeutics for treatment of both HBV and influenza infections has been moderately successful, therapeutic agents for HIV are severely limited. Furthermore, there are no known safe and therapeutic treatments for HBV, influenza and HIV. In HBV, with the possible exception of the drug 3TC, the use of nucleoside-based antiviral agents leads to toxicity, probably due to cross-inhibition of cellular mitchondrial DNA. Clearly, there is a need for a new class of antiviral agents which could minimize the toxicity associated with cross-inhibition. In influenza, amantadine and rimantadine have been shown to be moderately effective against only influenza A viruses; with amantadine having excessive side effects. Recently, strains of influenza A resistant to amantadine and rimantadine have been isolated. Accordingly, there is a need for new types of therapeutic antiviral agents against both influenza A and influenza B, as well as against HBV and HIV. SUMMARY OF THE INVENTION The present invention relates to substantially purified antiviral biflavanoids, derivatives and salts thereof and pharmaceutical compositions containing the same; an improved method for extracting substantially pure robustaflavonone from plant material; methods for preparing derivatives and salts from antiviral biflavanoids; and methods for treating and/or preventing viral infections using the antiviral biflavanoids, derivatives and salts thereof. The present invention provides substantially purified biflavanoids comprising robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a and pharmaceutical compositions containing the same are disclosed. Scheme I illustrates the chemical structures of these biflavanoids. The biflavanoids of the invention, extractable from from plant materials derived from a variety of natural sources such as Rhus succedanea and Garcinia multiflora, were found to be effective in inhibiting viral activity and may be used in a method for treating and/or preventing a broad range of viral infections such as Influenza A and B, hepatitis B and HIV-1, HSV-1, HSV-2, VZV, and measles. It has been discovered that robustaflavone effectively inhibits activity of influenza A and B viruses, hepatitis B, HIV-1, HSV-1 and HSV-2. Hinokiflavone and morelloflavone exhibited similar activity against various strains of HIV-1. Anti-viral biflavanoid derivatives and salts and pharmaceutical compositions containing the same are also contemplated by the invention. Representative derivatives include ethers, e.g., methyl ethers, esters, amines, ethylene oxide adducts, and polymers such as trimers and tetramers of apeginin. Representative salts include sulfates and acid salts. Methods for preparing these derivatives and salts are also provided. It has been discovered for instance that salts of robustaflavone, e.g., robustaflavone tetrasulfate potassium salt, effectively inhibits hepatitis B activity. Scheme I illustrates several examples of biflavanoid derivatives. An improved method for extracting robustaflavone from plant material is also provided. According to this method, a substantially pure robustaflavone in greater yields can be obtained through the use of a particular solvent mixture comprising toluene/ethanol/pyridine. The improved extraction method eliminates the use of benzene and requires smaller volumes of pyridine from the prior reported methods. Finally, a method for treating and/or preventing viral infections using antiviral biflavanoids is described. Representative viral infections include influenza A and B viruses, hepatitis B and human immunodeficiency virus (HIV-1), HSV-1, HSV-2, VZV, and measles. Accordingly, it is an object of the invention to provide substantially purified antiviral biflavanoids robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a. It is another object of the invention to provide antiviral derivatives and salt forms of biflavanoids robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a as well as method of preparation thereof. A representative example of an antiviral biflavanoid derivative includes robustaflavone tetrasulfate potassium salt. It is yet another object of the invention to provide pharmaceutical compositions which include at least one antiviral biflavanoids such as robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, GB-2a, derivatives or salts thereof. It is a further object of the invention to provide an improved method for obtaining substantially pure robustaflavone and in greater yields than prior procedures. It is yet a further object of the invention to provide a method for treating and/or preventing viral infections which comprises administering an antivirally effective amount of a biflavanoid. Representative viral infections are caused by viral agents such as influenza, e.g., influenza A and B; hepatitis, e.g., hepatitis B; human immunodeficiency virus, e.g., HIV-1; HSV-1, HSV-2, VZV, and measles. These and other objects of the invention will become apparent in light of the detailed description below. ##STR1## DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the effect of treatment with robustaflavone in DMSO on mean arterial oxygen saturation (mean SaO.sub.2 (%)) in Influenza A virus-infected mice as described in Example 10. FIG. 2 illustrates the effect of treatment with robustaflavone in DMSO on mean lung scores in Influenza A virus-infected mice as described in Example 10. FIG. 3 illustrates the effect of treatment with robustaflavone in DMSO on mean lung weights in Influenza A virus-infected mice as described in Example 10. FIG. 4 illustrates the effect of treatment with robustaflavone in DMSO on mean virus titers in Influenza A virus-infected mice as described in Example 10. FIG. 5 illustrates the effect of treatment with robustaflavone in CMC on mean arterial oxygen saturation (mean SaO.sub.2 (%)) in Influenza A virus-infected mice as described in Example 10. FIG. 6 illustrates the effect of treatment with robustaflavone in CMC on mean lung scores in Influenza A virus-infected mice as described in Example 10. FIG. 7 illustrates the effect of treatment with robustaflavone in CMC on mean lung weights in Influenza A virus-infected mice as described in Example 10. FIG. 8 illustrates the effect of treatment with robustaflavone in CMC on mean virus titers in Influenza A virus-infected mice as described in Example 10. DETAILED DESCRIPTION OF THE INVENTION All references and patents cited herein are hereby incorporated by reference in their entirety. In one embodiment of the invention, substantially pure biflavanoids robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a, derivatives and salts of the biflavanoids, and pharmaceutical compositions containing the same are disclosed. Methods for extracting and isolating the biflavanoids were previously reported..sup.28,37,39,40,53-55 Moreover, methods for preparing derivatives such as the acetate.sup.37,38 and methyl ethers.sup.39,40 for several of these biflavanoids are also reported. Representative methods for preparing biflavanoid derivatives are illustrated in the examples below. Applicants have determined that these biflavanoids, especially robustaflavone, were surprisingly effective in inhibiting one or more activities of viruses such as Influenza A and B, hepatitis B and HIV-1, HSV-1, HSV-2, VZV, and measles. Approximately 100 biflavanoids have been isolated to date, since the first biflavanoid, a biflavone, was isolated in 1929 by Furukawa from ginkgo biloba L. as a yellow pigment..sup.44,45,61 Biological activities of several biflavanoids, such as ginkgetin, have been reported. For instance, peripheral vasodilatation, anti-bradykinin, and anti-spasmogenic activities have been observed..sup.48,62 Garcinikolin stimulates RNA synthesis in rat hepatocyte suspensions..sup.57 Also, agathisflavone, kolaviron, GB-1 and GB-2 have hepatoprotective activity..sup.33,49 Hinokiflavone, kayaflavone, bilobetin, lophirone A, lophiraic acid, and sotetusflavone demonstrate inhibitory action on the genome expression of the Epstein-Barr virus (EBV). 51,52,60 GB-1 exhibits molluscicidal activity,.sup.65 while daphnodorin A, daphnodorin B, and daphnodorin D possess antimicrobial activity..sup.34 Hinokiflavone exhibits cytotoxicity against tissue cultured cells of human mouth epidermoid carcinoma (KB)..sup.56 Amentoflavone and morelloflavone exhibit an inhibitory effect on lipid peroxidation,.sup.41,59,66 and kolaviron produced hypoglycemic effects..sup.50 None of these references, however, disclose or suggest that robustaflavone, hinokiflavone, morelloflavone, amentoflavone, agathisflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a and GB-2a, especially robustaflavone and its tetrasulfate potassium salt, have an inhibitory effect against at least one of influenza, e.g., influenza A and B; hepatitis, e.g., hepatitis B; human immunodeficiency virus, e.g., HIV-1; HSV-1, HSV-2, VZV, and measles. In another embodiment of the invention, an improved method for extracting substantially pure robustaflavone from natural sources is also provided. Robustaflavone, 1, a naturally occurring biflavanoid, was previously isolated, purified, and identified from the seed-kernels of Rhus succedanea..sup.25 Other sources of robustaflavone include: seed kernel of Rhus succedanea L.;.sup.25 leaves of Selaginella lepidophylla;.sup.27 leaves of Anacardium occidentale;.sup.28 leaves and branches of Podocarpus neriifolius D. Doa;.sup.29 Selaginella denticulata;.sup.30 and Selaginella willdenowii..sup.31 The drupes of wax-tree, Rhus succedanea L (Anacardiaceae), are of great economic importance in that they yield Japan wax. Earlier work on this species has shown the presence of fustin and fisetin in the wood, rhoifolin in leaves, japanic acid in the wax, and ellagic acid, fatty acids, and flavanoids in the seed kernels. Further studies of the pigment in the seed kernels of wax-tree led to the isolation of eight biflavanoids, four of which were new. Concentration of the ethanol extract of the seed kernels yielded, successively, fractions of ellagic acid, pigment A (hinokiflavone and robustaflavone) and pigment B (amentof lavone). Further concentrations gave a crude yellow pigment C which, when subjected to silica gel column chromatography, afforded fractions C.sub.I, (rhusflavanone, succedaneaf lavanone and neorhusf lavanone), C.sub.II. (rhusflavone), and C.sub.III (agathisflavone). A prior method for extracting and isolating substantially pure robustaflavone from plant material was reported..sup.55 This method, however, used large quantities of benzene and pyridine which is undesirable for use in large scale extractions and produced mediocre yields of robustaflavone. The Applicants discovered an improved extraction method which eliminates benzene and greatly reduced the amount of pyridine and produced at least double the quantities of substantially pure robustaflavone compared to the prior method. According to this embodiment of the invention, a solvent mixture comprising toluene/ethanol/formic acid at a volume ratio ranging about 10-30:2-10:1, preferably about 20:5:1, was found to be useful. This particular solvent mixture was found to be especially useful in large scale extractions. An example of an extraction via the improved extraction method of the invention is illustrated in the examples below. In yet another embodiment of the invention, a method for treating and/or preventing viral infections in mammals comprising administering an antivirally effective amount of a biflavanoid such robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a. In practicing this invention, administration of robustaflavone or derivatives thereof is preferred. Examples of mammals include humans, primates, bovines, ovines, porcines, felines, canines, etc. Examples of viruses may include, but not be limited to, HIV-1, HIV-2, herpes simplex virus (type 1 and 2) (HSV-1 and 2), varicella zoster virus (VZV), cytomegalovirus (CMV), papilloma virus, HTLV-1, HTLV-2, feline leukemia virus (FLV), avian sarcoma viruses such as rous sarcoma virus (RSV), hepatitis types A-E, equine infections, influenza virus, arboviruses, measles, mumps and rubella viruses. More preferably the compounds of the present invention will be used to treat a human infected with hepatitis and/or influenza virus. Preferably the compounds of the present invention will also be used to treat a human exposed or infected (i.e., in need of such treatment) with the human immunodeficiency virus, either prophylactically or therapeutically. Antiviral biflavanoids and derivatives thereof may be formulated as a solution of lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or in buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium choride or sodium citrate. Alternatively, the compounds of the present invention may be encapsulated, tableted or prepared in an emulsion (oil-in-water or water-in-oil) syrup for oral administration. Pharmaceutically acceptable solids or liquid carriers, which are generally known in the pharmaceutical formulary arts, may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch (corn or potato), lactose, calcium sulfate dihydrate, terra alba, croscarmellose sodium, magnesium stearate or stearic acid, talc, pectin, acacia, agar, gelatin, maltodextrins and microcrystalline cellulose, or collodial silicon dioxide. Liquid carriers include syrup, peanut oil, olive oil, corn oil, sesame oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 10 mg to about 1 g per dosage unit. The dosage ranges for administration of biflavanoids or derivatives thereof are those which produce the desired affect whereby symptoms of infection are ameliorated. For example, as used herein, a pharmaceutically effective amount for influenza or hepatitis infection refers to the amount administered so as to maintain an amount which suppresses or inhibits circulating virus throughout the period during which infection is evidenced such as by presence of anti-viral antibodies, presence of culturable virus and presence of viral antigen in patient sera. The presence of anti-viral antibodies can be determined through use of standard ELISA or Western blot assays for example. The dosage will generally vary with age, extent of the infection, the body weight and counterindications, if any, for example, immune tolerance. The dosage will also be determined by the existence of any adverse side effects that may accompany the compounds. It is always desirable, whenever possible, to keep adverse side effects to a minimum. One skilled in the art can easily determine the appropriate dosage, schedule, and method of administration for the exact formulation of the composition being used in order to achieve the desired effective concentration in the individual patient. However, the dosage can vary from between about 0.001 mg/kg/day to about 150 mg/kg/day, but preferably between about 1 to about 50 mg/kg/day. The pharmaceutical composition may contain other pharmaceuticals in conjunction with biflavanoids and derivatives thereof to treat (therapeutically or prophylactically) antiviral infections. For example, other pharmaceuticals may include, but are not limited to, other antiviral compounds (e.g., AZT, ddC, ddI, D4T, 3TC, acyclovir, gancyclovir, fluorinated nucleosides and nonnucleoside analog compounds such as TIBO derivatives, nevirapine, saquinavir, .alpha.-interfon and recombinant CD4), immunostimulants (e.g., various interleukins and cytokines), immunomodulators and antibiotics (e.g., antibacterial, antifungal, anti-pneumocysitis agents). The following examples are illustrative and do not serve to limit the scope of the invention as claimed. In these examples, eleven biflavanoids, amentoflavone (1), agathisflavone (2), robustaflavone (3), hinokiflavone (4), volkensiflavone (5), morelloflavone (7), rhusflavanone (9), succedaneaflavanone (11), GB-1a (13), GB-1a 7"-O-.beta.-glucoside (15), and GB-2a (16), isolated from Rhus succedanea and Garcinia multiflora, and their methyl ethers, acetate and sulfate potassium salt, volkensiflavone hexamethyl ether (6), morelloflavone heptamethyl ether (8), rhusflavanone hexaacetate (10) succedaneaflavanone hexaacetate (12), GB-1a hexamethyl ether (14) and robustaflavone tetrasulfate potassium salt were evaluated for their antiviral activities. The inhibitory activities against HIV-1 RT and various viruses including herpes viruses (HSV-1, HSV-2, HCMV, and VZV), and respiratory viruses (influenza A, influenza B. RSV, parainfluenza 3, adenovirus 5, and measles) were investigated |
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