WO2011088431A1 - Prophylactic and treatment for virus-induced disease - Google Patents

Abstract

Many viruses gain entry to host cells via a clathrin-dependent endocytic pathway. Administering pharmaceutical compositions containing the tyrosine kinase inhibitor genistein to a mammal treats or prevents diseases and disorders caused by clathrin-dependent viruses. The compositions are effective when administered after exposure and even after development of symptoms in the mammal.

Description

PROPHYLACTIC AND TREATMENT FOR VIRUS-INDUCED DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] This application claims priority to U.S. Provisional Application Serial Nos.

61/295,787, filed January 18, 2010, and 61/425,686, filed December 21, 2010. These applications are incorporated herein in their entireties.

BACKGROUND

[02] Viruses are obligate intracellular infectious agents. Their replication and pathogenicity is critically dependent on the ability to infect cells. Viruses have evolved many strategies to gain entry into host cells, including direct fusion with the plasma membrane, macropinocytosis, a fluid uptake pathway found particularly in epithelial cells and fibroblasts, caveolar endocytosis, and clathrin-dependent endocytosis. In some cases, viruses can use more than one pathway. See Marsh et al., "Virus entry: open sesame," Cell. 2006 Feb 24;124(4):729-40; Vela et al., "Arenavirus entry occurs through a cholesterol- dependent, non-caveolar, clathrin-mediated endocytic mechanism," Virology. 2007 Dec 5;369(1):1-11 ; Pelkmans et al., "Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae," Science. 2002 Apr 19;296(5567):535-9); Martinez et al., "Characterization of Junin arenavirus cell entry," J Gen Virol. 2007 Jun;88(Pt 6): 1776-84; Pelkmans et al., "Insider information: what viruses tell us about endocytosis," Curr Opin Cell Biol. 2003 Aug; 15(4) :414-22.

[03] Clathrin-dependent endocytosis is a normal cellular process used to internalize ligand/receptor complexes and membrane from the cell surface. Several clathrin proteins combine to form a triskelion complex. Several triskelion complexes interact to produce a polyhedral lattice, which coats membrane pinched from the plasma membrane and produces a coated vesicle. The coated vesicle detaches from the plasma membrane and undergoes further processing, including disassembly of the coat followed by eventual return of the internalized membrane and protein back to the plasma membrane or for further processing into the cell. To infect cells, viruses must first bind to the cell surface. Often the viruses bind to specific receptors, activate signaling pathways, and trigger internalization of the receptor/virus complex via the clathrin-dependent pathway. See Marsh et ai, supra.

Clathrin-dependent viruses include arenaviruses, bunyaviruses, flaviviruses, and togaviruses. The Arenaviridae are enveloped, single-stranded bipartite RNA viruses, which use a clathrin-dependent endocytic route to gain entry to cells. In some areas of the world, Arenavirus infections are relatively common and can cause severe illnesses in humans that usually occurs as a result of rodent-transmitted disease.

Arenaviridae members are divided into two groups: the New World or Tacaribe complex and the Old World or LCM/Lassa complex. Viruses in these groups that cause illness in humans include Flexal virus (the disease has not been characterized); Lassa virus, which causes Lassa fever; Junin virus which causes Argentine hemorrhagic fever; Machupo virus which causes Bolivian hemorrhagic fever; Guanarito virus which causes Venezuelan hemorrhagic fever, and Sabia virus which causes Brazilian hemorrhagic fever.

Arenavirus-induced hemorrhagic fever symptoms include fever, headache, diarrhea, leucopenia, loss of strength, thrombocytopenia, edema, and abdominal pain. Hemorrhagic events include bleeding under the skin, in internal organs, or from body orifices like the mouth, eyes, or ears. Liver necrosis and vascular collapse due to coagulopathy may also occur.

Bunyaviridae is a family of negative-stranded RNA viruses that are usually found in rodents and insects, such as mosquitoes, but can infect humans. Several Bunyavirus species are known to infect humans including Hantaan virus, Crimean-Congo virus, and Rift Valley Fever virus. Human infection can be associated with disease ranging from mild asymptomatic infection to hemorrhagic fever and fatal encephalitis.

Flaviviridae are positive, single-stranded RNA viruses. Human infection is often via mosquito or tick bite. Flavivirus species known to infect humans include West Nile virus, dengue virus, tick-borne encephalitis virus, and Yellow Fever virus.

[09] Togaviruses are positive, single-stranded RNA viruses and include the genera Alphaviruses and Rubivirus. Alphaviruses, such as Eastern Equine Encephalitis (EEE) virus, Western Equine Encephalitis (WEE) virus, Venezuelan Equine Encephalitis virus (VEE), Ross River virus, and O'nyong'nyong virus, can infect humans, causing rashes, arthritis, and encephalitis. The best known member of the Rubivirus is Rubella virus, which causes Rubella.

[10] Filoviruses are negative-stranded RNA viruses that can cause hemorrhagic fever in humans. Ebola virus and Marburg virus are members of this viral family. Disease is usually associated with abrupt onset of fever, headache, myalgia, chest pain, and delirium. The case fatality rate for Ebola viral infection is 50-88% and the case fatality rate for Marburg viral infection is 23- 75%.

[11] Options for virus therapy, particularly with arenaviruses, are limited. While vaccines have proven effective they are only useful prophylactically. Moreover, vaccines are not available for all viruses. Treatment options for hemorrhagic fever viruses, such as the arenaviruses, are particularly inadequate. Supportive care and ribavirin remain the only options for arenaviral infections in humans; however, treatment can be ineffective because of the time frame before the disease is recognized.

[12] Development of new treatment options is hampered because of the potential for aerosol production, person-to-person spread and the ability to cause lethal or debilitating disease in humans. Indeed, many viruses, including arenavirus species, Marburg, and Ebola, are listed as CDC biothreat agents and NIAID category A priority pathogens. Therefore, surrogate models must be used to research and develop new treatments.

[13] There is a need in the art to identify novel therapeutic modalities capable of preventing and treating virus-induced disease, particularly disease induced by hemorrhagic fever viruses. SUMMARY

[14] In one aspect of the invention, a method is provided to administer a tyrosine kinase inhibitor to a mammal to prevent disease or disorder caused by infection with a clathrin-dependent virus.

[15] In another aspect of the invention, a method is provided to administer a tyrosine kinase inhibitor to a mammal to treat disease or disorder caused by infection with a clathrin-dependent virus.

[16] In another aspect of the invention, a method is provided to administer a tyrosine kinase inhibitor to a mammal to reduce the incidence of successful infections caused with a clathrin-dependent virus.

[17] In another aspect of the invention, a method is provided to administer a tyrosine kinase inhibitor to a mammal to inhibit the onset of symptoms caused by infection with a clathrin-dependent virus.

BRIEF DESCRIPTION OF THE FIGURES

[18] Figure 1 illustrates the study design of the Syrian golden hamster treatment protocol.

[19] Figure 2 illustrates the study activities.

[20] Figure 3 shows statistical comparison of survival and time-to-death of the animals.

[21] Figure 4 shows the Kaplan-Meier curves associated with time to death for each group. Group 1 (PIRV-infected mock-treated control); Group 2 (Hamsters treated once with genistein 24 hours prior to infection); Group 3 (PIRV-infected hamsters treated with genistein 24 hours prior to infection and every day post-challenge until the end of study); Group 4 (post-exposure prophylactic regimen); Group 5 (therapeutic regimen); Group 6 (mock- infected and treated with genistein 24 hours before mock-infection and every day post-challenge until the end of study). [22] Figure 5 shows the animal temperature data for the animals in Group 1.

[23] Figure 6 shows the animal temperature data for the animals in Group 2.

[24] Figure 7 shows the animal temperature data for the animals in Group 3.

[25] Figure 8 shows the animal temperature data for the animals in Group 4.

[26] Figure 9 shows the animal temperature data for the animals in Group 5.

[27] Figure 10 shows the animal temperature data for the animals in Group 6.

[28] Figure 1 1 shows that genistein inhibition of infection by Pirital virus, Pichende virus, West Nile Virus, and Dengue virus.

[29] Figure 12 shows genistein inhibition of infection by Lassa virus, Marburg virus, and Ebola virus pseudotypes.

[30] Figure 13 shows tyrphostin AG 1478 inhibition of infection by Lassa virus, Marburg virus, and Ebola virus pseudotypes.

[31] Figure 14 shows inhibition of Lassa Virus- VSV (LASV-VSV) pseudotype infection by genistein and tyrphostin.

[32] Figure 15 shows inhibition of Ebola Virus- VSV pseudotype (EBOV-VSV) infection by genistein and tyrphostin.

[33] Figure 16 shows inhibition of replication-competent Ebola Virus-GFP (EBOV-GFP) infection by genistein and tyrphostin.

[34] Figure 17 shows synergistic inhibition of replication-competent Ebola Virus- GFP (EBOV-GFP) infection by genistein and tyrphostin

[35] DETAILED DESCRIPTION

[36] We have found that administering genistein prevents virus-associated disease (e.g., reduces the incidence of successful infection in certain virus-associated diseases) if provided prophylactically. We have also found that genistein can inhibit the onset and/or ameliorate the disease or disease symptoms if provided subsequent to virus exposure, and even if provided subsequent to the appearance of symptoms. We have also found that tyrphostin can be used in place of, or in combination with, genistein. Moreover, we found that sub- optimal concentrations of genistein and tyrphostin can be combined to achieve synergistic inhibition of viral infection.

Genistein is an isoflavone commonly found in plants such as lupin, fava beans, soybeans, kudzu, and psoralea. Genistein is primarily known as a tyrosine kinase inhibitor. However, Genistein is known to affect other enzymes, including DNA Topoisomerase (Markovits et al., "Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II," Cancer Res. 1989 Sep 15;49(18):5111-7). Like other isoflavones, genistein may also exert effects via estrogen-like properties.

Genistein appears to inhibit infection of some viruses like Simian virus 40 (SV40) (Akiyama et al., "Genistein, a specific inhibitor of tyrosine-specific protein kinases," J Biol Chem. 1987 Apr 25;262(12):5592-5., 1987; Damm et al., "Clathrin- and caveolin-1 -independent endocytosis: entry of simian virus 40 into cells devoid of caveolae," J Cell Biol. 2005 Jan 31 ;168(3):477-88.; Pelkmans et al., "Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae," Science. 2002 Apr 19;296(5567):535-9). Similar results have been obtained in other in vitro systems, including Arenaviruses (Vela et al., "Genistein treatment of cells inhibits arenavirus infection," Antiviral Res. 2008 Feb;77(2): 153-6) and HIV- 1 (Stantchev et al., "The tyrosine kinase inhibitor genistein blocks HIV-1 infection in primary human macrophages," Virus Res. 2007 Feb;123(2):178- 89).

Despite promising in vitro observations, however, moving from in vitro to in vivo efficacy has not been demonstrated. See e.g., Andres et al, "Soy isoflavones and virus infections," J Nutr Biochem. 2009 Aug;20(8):563-9. Indeed, while genistein has been investigated in breast cancer, it remains unclear whether it is beneficial or hazardous. Bouker et al., "Genistein: does it prevent or promote breast cancer?" Environ Health Perspect. 2000 Aug;108(8):701-8. Additionally, because genistein is a broad and non- specific kinase inhibitor, it may have affects on various general kinases necessary for cellular activity.

[40] We have discovered that genistein may be used in a pharmaceutical composition to therapeutically or prophylactically treat mammals potentially exposed to, actually exposed to, or infected with, a clathrin-dependent virus. Tyrphostins may also be used to therapeutically or prophylactically treat mammals potentially exposed to, actually exposed to, or infected with, a clathrin-dependent virus. Tyrpohstins are, like genistein, tyrosine kinase inhibitors. See. Levitzki and Mishani, "Tyrphostins and other tyrosine kinase inhibitors," Annu Rev Biochem. 2006;75:93-109. We also discovered that combining both genistein and tyrphostin gave a synergistic inhibitory effect. Thus, when used in combination the concentrations of each tyrosine kinase inhibitor may be used at sub-optimal levels yet retain good antiviral effect.

[41] A clathrin-dependent virus uses, at least in part, the clathrin endocytic pathway to enter a host cell. For example, the clathrin-dependent virus may be an Arenavirus, such as a Lassa virus, a Junin virus, a Machupo virus, a Guanarito virus, or a Sabia virus. The compositions may be used to reduce the incidence of, prevent, treat, inhibit the onset of, or ameliorate disease or symptoms arising from infection by other viruses that use a clathrin-dependent entry mechanism to gain access to host cells. For example, the virus may be a Bunyavirus such as a Hantaan virus, a Crimean-Congo hemorrhagic fever virus, or a Rift Valley Fever virus. The virus may be a Flavivirus such as a West Nile virus, a dengue virus, a tick-borne encephalitis virus, or a Yellow Fever virus. The virus may be a Togavirus, such as an Alphavirus or a Rubivirus. For example, the Alphavirus may be an Eastern Equine Encephalitis (EEE) virus, a Western Equine Encephalitis (WEE) virus, a Venezuelan Equine Encephalitis (VEE) virus, a Ross River virus, or an O'nyong'nyong virus. The virus may be a SARS-coronavirus (SARS-CoV) or highly pathogenic avian influenza. The virus may also be a filovirus such as an Ebola virus, (e.g., Ebola-Reston) or Marburg virus.

[42] The compositions will be administered in a therapeutically effective dose range. For example, the tyrosine kinase inhibitor dose range may have a lower limit of: about 5 mg/kg of body weight, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 75 mg/kg, about 100 mg/kg, about 150 mg/kg, about 200 mg/kg. The dose may have an upper limit of about 10 mg/kg of body weight, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 75 mg/kg, about 100 mg/kg, about 150 mg/kg, about 200 mg/kg, about 300 mg/kg, about 500 mg/kg, about 1000 mg/kg. "About" the recited amount means a variation of plus or minus 10% of the recited amount. Usually, the dose range is expected to be about 5 mg/kg to about 50 mg/kg. More often, the dose range is expected to be about 10 mg/kg to about 20 mg/kg.

[43] The compositions may be administered to maximize the effective dose. For example, the compositions may be administered once a day, twice a day, three times a day or four times a day.

[44] The compositions may be administered prophylactically. For example, the compositions can be administered to someone about to enter a situation with a known arenavirus infection or threat of infection, such as a region of the world known to have endemic arenavirus problems a contaminated hospital site, or in the event of bioterrorism. The compositions may be administered therapeutically. For example, they may be administered immediately postexposure to the virus, or several days subsequent to an apparent infection event. Preferably, administration is started prior to exposure. If administration is started shortly after exposure, administration is preferably initiated as soon after exposure as possible; i.e., within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, or within 6 days. The compositions may also be administered shortly after a disease symptom is observed; i.e., within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days of detecting a symptom. Preferably, the inhibitor is administered immediately following symptom detection.

[45] The compositions may contain any source of genistein or tyrphostin. For example, suitable sources include genistein purified from natural plants sources. Genistein may also be prepared synthetically; for example, as reported in Dixon and Ferreira, "Genistein," Phytochemistry. 2002 Jun;60(3):205-1 1. Tyrphostins are available commercially; for example, Tyrphostin AG1478 is available from Cell Signaling Technology, Inc., Danvers, MA 01923.

[46] The compositions comprise a therapeutically effective amount of genistein and/or tyrphostin formulated together with one or more pharmaceutically acceptable carriers or excipients.

[47] As used herein, the term "pharmaceutically acceptable carrier or excipient" means a non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. For example, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[48] The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally or by injection.

[49] The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.

[50] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylsulfoxide (DMSO) dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[51] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, phosphate buffered saline (PBS), Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[52] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. [53] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide- polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[54] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the tyrosine kinase inhibitor(s) with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[55] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) humectants such as glycerol, d) disintegrating agents such as agaragar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[56] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract or, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

[57] Optionally, the tyrosine kinase inhibitor(s) may be contained in micelles to improve oral bioavailability. See, for example, Kwon et al., "Pharmaceutical evaluation of genistein-loaded pluronic micelles for oral delivery," Arch Pharm Res. 2007 Sep;30(9):l 138-43.

[58] The tyrosine kinase inhibitor(s) may be in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. See, for example, S. M. Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977).

[59] The tyrosine kinase inhibitor(s) may be in the form of a pharmaceutically acceptable prodrug. The term "pharmaceutically acceptable prodrugs" as used herein refers to genistein prodrugs that are suitable for use in contact with mammals with undue toxicity, irritation, allergic response, and the like. "Prodrug," as used herein means a compound which is convertible in vivo by metabolic means (for example, by enzymatic hydrolysis) to genistein. Genistein prodrugs have particular application for oral administration to approve oral bioavailability. Methods of modifying drugs into prodrugs are known in the art. See, for example, U.S. Pat. No. 6,204,257 to Stella et al. and H. Bundgaard, Design of Prodrugs: Bioreversible derivatives for various functional groups and chemical entities. In: Design of Prodrugs H. Bundgaard, Editor, Elsevier, Amsterdam (1985), pp. 1-93 chapter 1.

[60] The virus-induced disease or symptoms prevented or ameliorated by administration of the tyrosine kinase inhibitor(s) include, but are not limited to, at least one of fever, headache, diarrhea, leucopenia, loss of strength, thrombocytopenia, edema, abdominal pain; bleeding under the skin, in internal organs, or from body orifices like the mouth, eyes, or ears. Neurological disease, liver necrosis and vascular collapse due to coagulopathy may also be prevented or ameliorated.

[61] Liver necrosis or liver function is measured by clinical chemistry parameters such as elevated AST (Aspartate Amino Transferase) and ALT (Alanine Amino Transferase), while vascular collapse, in this case, may be measured by coagulation parameters like Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT). Analysis is performed at Study Day 0 and at Study Day 14, or at termination.

[62] The tyrosine kinase inhibitor(s) may be administered to any mammal. For example, the mammal may be a human, a non-human primate, an artiodactyl, such as a pig, a camel, a sheep, a cow, a goat or a deer. The mammal may be a perissodactyl such as a horse, a donkey, or a zebra.

[63] The contents of all of the above cited articles are incorporated by reference as if set forth fully herein.

EXAMPLE 1

Genistein Prevents Morbidity In A Syrian Golden Hamster Arenavirus Model

[64] Pirital virus (PIRV) infection in Syrian golden hamsters leads to morbidity, fever, lethargy, hemorrhagic fever manifestations, viremia, and replication in select tissues and results in 100% mortality within 8 days after challenge. 165] Hamsters were treated with genistein then challenged with PIRV as described in Table 1. The PIRV virus used (Pirital virus-488) was administered intra- peritoneally (IP) at a dose of 10⁴ PFU/ml. Genistein was prepared in phosphate-buffered saline (PBS) with < 0.1 % DMSO and administered intra- peritoneally (IP) as a dose of 15 mg/kg. Groups 1-5 were challenged with PIRV. Group 6 was treated with genistein 24 hours before mock-infection and every day post-challenge until the end of study. Group 1 received no genistein. Group 2 was treated once with genistein 24 hours before infection. Group 3 was treated with genistein 24 hours before infection and daily post-challenge until the end of study. Group 4 (post-exposure prophylactic regimen) was treated with genistein 24 hours post-infection and every day after until the end of the study. Group 5 (therapeutic regimen) received genistein at the first sign of disease and every day after until the end of the study.

[66] Animals began losing weight 2 days after infection and most were lethargic 3 to 4 days after infection. All untreated animals began to exhibit a petechial rash on their abdomen four days after challenge. The appearance of the rash was used as a trigger to treat Group 5, the therapeutic group. One animal each in Groups 5 and 3 did not develop a petechial rush until 7 days post-infection. However, the Group 5 animal displayed other disease signs and was treated on day 5 post-infection (p.i). By day 5 after infection, some animals exhibited huddled posture with ruffled fur. Animals also displayed ecchymosis in the lower region of the abdomen. Group 1 animals began displaying epistaxis on day 4 or 5 p.i. Most animals, including the treated animals, exhibited epistaxis by day 6 p.i. The first animal deaths occurred on day 6 p.i. Animals were either classified as moribund or found dead. None of the animals exhibited signs of neurological disease. Further, epistaxis was the only form of hemorrhaging detected at day 6 pi. By day 7 p.i., some animals began hemorrhaging from their eyes. Additionally, some animals exhibited neurological symptoms such as body rocking. Notably, the body rocking was different from shivering at earlier time-points that was likely fever-induced. More deaths occurred on day 7. By day 8, animals that were rocking on day 7 exhibited hind limb paralysis and were euthanized. Several animals began hemorrhaging from their rectum. All virus-infected non-treated animals (6/6) died or were euthanized by day 8. See Figure 4.

[67] Pathology studies in Syrian Hamster model shows that PIRV-infected animals had swollen cecums and lower intestines that contained blood, foci on the liver and spleen. The spleens were extremely necrotic and necrotic foci were found on the brain, further suggesting that the virus had infected the brain.

[68] Several animals treated with genistein survived. Several animals treated with genistein only once, prior to the infection, (Group 2) survived. Notably the temperature profile of Group 3 animals; i.e., those treated prior to infection and daily, returned to normal suggesting recovery from disease (Fig. 7). Animals from Group 4 (post-exposure prophylactic) and Group 5 (therapeutic regimen) also survived. The numbers of animals surviving was as follows: Group 1 : 0/6; Group 2: 3/6; Group 3: 4/6; Group 4: 2/6; Group 5: 2/6, and Group 6: 3/3. See Figure 4.

[69] Previously, survival of hamsters infected with PIRV has not been reported.

The effectiveness of genistein both prophylactically and as a treatment represents a substantial advance.

[70] Abnormal temperatures, hematology, clinical chemistry, and coagulation parameters associated with PIRV infection in hamsters all rebounded to normal levels in the treated animals.

[71] Lower viremia and inhibition of viral replication in select tissues were also observed in treated animals when compared to mock-treated animals. These data demonstrate pre-exposure protective efficacy of genistein as an antiviral against PIRV-induced hemorrhagic fever in the Syrian golden hamster model.

EXAMPLE 2

Genistein Treats Arenavirus-Induced Fever In The Syrian Hamster Model

[72] The hamsters in Example 1 were implanted with telemetry units to monitor temperature and activity during the study. [73] Normal hamsters exhibit a diurnal temperature pattern, which was observed prior to infection in all groups and throughout the study in group 6 (Figures 5- 10). Infection of the animals with PIRV disrupted this temperature pattern. Animals exhibited febrile temperatures 1 day post infection. The febrile temperatures remained constant in PIRV-infected animals until the final hours or days of life when the temperatures declined. Hypothermia was observed in several animals.

[74] Group 2 (Fig. 6) and Group 3 (Fig. 7) surviving animals all recovered from febrile temperatures. Animals 546, 549, and 566 recovered from both febrile and hypothermic temperatures.

EXAMPLE 3

Genistein Inhibits infection by Dengue Virus, West Nile Virus, Pirital Virus, and Pichende Virus

[75] Infection of host cells with viruses from various families including Dengue Virus (DENV), West Nile Virus (WNV), Pirital Virus (PIRV), and Pichende Virus (PICV) was inhibited in cells pre-treated with various concentrations genistein (Fig. 11). Cells were treated with various concentrations of genistein and infected with different viruses. The carrier control represents the average of the percent inhibition of cells infected with all of the viruses. The inhibition was concentration-dependent and the standard error was less than 10%.

EXAMPLE 4

Genistein And Tyrphostin Each Inhibit Infection By Lassa Virus, Marburg Virus, And Ebola Virus pseudotypes

[76] The antiviral activity of genistein and tyrphostin AG 1478 was tested using VSV virus pseudotyped with the viral envelope proteins from Lassa Virus (LASV) or Ebola Virus (EBOV) and expressing the luciferase protein as a marker for infection. Pseudotyped viruses allows for determining whether the drugs inhibit the viral entry process since these viruses are replication- deficient and only capable of one round of transduction.

[77] HEK 293 cells were treated with indicated concentrations of Genistein (Figure 12) or Tyrphostin AG1478 (Fig. 13) for 1 h prior to the addition of virus. Lassa envelope protein pseudotyped VSV (LASV-VSV), Marburg envelope protein pseudotyped VSV (MARV-VSV) or Ebola pseudotyped VSV (EBOV- VSV) were then added (MOI, 0.1) to the cells and incubated for an additional 16 h in the presence of each drug. Each virus encoded Luciferase as an infection marker. Luciferase activity in the cells was counted and the means ± SDs of the results of three independent experiments are shown.

[78] Transduction of the host cells with the LASV-VSV or EBOV-VSV pseudotypes was inhibited when cells were pre-treated with genistein or tyrphostin AG 1478. Furthermore, the inhibition was found to be dose dependent. As observed with the EBOV-VSV pseudotype, MARV-VSV transduction was also inhibited in cells pre-treated with genistein or tyrphostin AG1478. Additionally, the drugs did not appear to cause cellular toxicity at the concentrations used and the inhibitors did not inhibit Sindbis virus infection (data not shown).

[79] These data demonstrate that treating cells with the kinase inhibitors genistein or tyrphostin AG1478 inhibit the transduction of cells with pseudotypes that contain envelope proteins from EBOV, MARV, and LASV. Since these pseudotypes are replication independent, these data suggest that the drugs inhibit entry of these pseudotyped viruses into host cells

EXAMPLE 5

Genistein And Tyrphostin Synergistically Inhibit Virus Infection

[80] Genistein and tyrphostin AG 1478 were used as a cocktail to determine whether a synergistic antiviral effect of the two drugs could be observed. These cocktails consisted of various amounts of each drug ranging from 100 μΜ to 0 μΜ. [81] HEK 293 cells were treated with indicated concentrations of Genistein and Tyrphostin AG 1478 in various combinations for 1 h prior to the addition of virus. Lassa envelope protein pseudotyped VSV (LASV-VSV)(Fig. 14) and Ebola pseudotyped VSV (EBOV-VSV)(Fig. 15) were then added (MOI, 0.1) to the cells and incubated for an additional 16 h in the presence of each drug combination. Each virus encoded Luciferase as an infection marker. Luciferase activity in the cells was counted and the means of the results of three independent experiments are shown.

[82] Either drug added to the cells at a concentration of 100 μΜ caused 90% to 99% inhibition of LASV-VSV transduction. However, adding genistein to 100 μΜ of tyrphostin AG1478 did not detectable enhance inhibition of LASV- VSV. By contrast, adding various concentrations of genistein to 50 μΜ of tyrphostin AG 1478 caused an increase in the inhibition of LASV-VSV transduction, revealing the synergistic effects obtained using lower concentrations of the inhibitors. This same pattern was generally observed the concentration of tyrphostin AG1478 was lowered in concert with the addition of increasing concentrations of genistein. Similarly, increasing tyrphostin AG1478 concentration at lower genistein concentrations resulted in improved inhibition of LASV-VSV pseudotype transduction. These data suggested an additive or synergistic antiviral effect of the two kinase inhibitors on host cells pre-treated with the drugs and transduced with the LASV-VSV pseudotype.

[83] Similar data was obtained with EBOV-VSV pseudotype. (Fig. 15). To determine whether the cocktail also had a synergistic antiviral effect on EBOV-VSV pseudotype transduction the cocktails were prepared with various amounts of each drug and the cells were pre-treated with each cocktail mix before the addition of the EBOV-VSV pseudotype. As with the LASV-VSV pseudotype, 100 μΜ of tyrphostin AG1478 resulted in 96%-99% inhibition of EBOV-VSV transduction regardless of the concentration of genistein (Fig. 15). Lowering the concentration of tyrphostin AG 1478 resulted in a concentration dependent reduction in inhibition. Both with LASV-VSV and EBOV-VSV, 100 μΜ of tyrphostin AG 1478 appeared to cause greater inhibition of viral transduction. As with the LASV-VSV data, the EBOV-VSV data suggests a synergistic or additive antiviral effect of these two kinase inhibitors.

EXAMPLE 6

Genistein And Tyrphostin Synergistically Inhibit Viral Infection by Replication-Competent Ebola virus

[84] After observing inhibition of EBOV and LASV pseudotype transduction in host cells treated with the kinase inhibitors, we next tested the affects of the kinase inhibitors on recombinant replication competent EBOV expressing GFP (EBOV-GFP), in vitro.

[85] HE 293 cells were treated with combinations of genistein and tyrphostin AG 1478 at indicated concentrations for 1 h prior to the addition of virus. Recombinant replication competent EBOV expressing GFP was then added (MOI, 0.1 ) to the cells and incubated for an additional 20 h in the presence of each drug combination. Cells infected with EBOV-GFP were then fixed in 10% Formalin for 24 hr according to BSL4 protocol and stained with DAPI. Cells were then imaged using and images were analyzed. DAPI was used to count total cells number and identify cells boundaries while GFP was used to count cells infected with EBOV-GFP.

[86] We found that incubating cells with 100 μΜ of tyrphostin AG 1748 resulted in 96-99% inhibition of both EBOV and LASV (Figure 16). EBOV Infection was higher in host cells treated with lower concentrations of tyrphostin AG 1478, demonstrating concentration-dependent inhibition of the drug. Pre- treatment with 100 μΜ of tyrphostin AG 1478 was more effective at inhibiting EBOV infection than genistein.

[87] Notably, combining genistein and tyrphostin AG 1478 at lower concentrations of each drug resulted in a treatment that provided enhanced inhibition against EBOV infection (Figure 16) compared with each drug alone. Using MacSynergy II software to analyze the effects of genistein and tyrphostin AG1478 on EBOV infection we determined that combining the drugs had a synergistic effect. The three dimensional synergy plot in Figure 17 was generated using the MacSynergy II program and shows a subtraction of the theoretical additive surface from experimentally observed surface. The theoretical additive surface represents theoretical surface if genistein and tyrphostin had an additive affect. This surface is calculated based on effects of genistein only and tyrphostin. The higher the peaks the more significant is the synergy between two drugs. Figure 17 shows synergy plot with 99.9% statistical significance or above. The three-dimensional synergy plot would equal zero if the drugs only had an additive effect and would be negative if the drugs had an antagonistic effect. The positive plot demonstrates a synergistic effect of the two drugs.

EXAMPLE 7

In Vitro Genistein Virus Inhibition By Plaque Assay

[88] In vitro plaque assay inhibition experiments are performed with the following virus species: Flexal virus (arenavirus), Hantaan virus (bunyavirus), West Nile virus (flavivirus) and Venezuelan Equine Encephalitis virus (alphavirus). Genistein inhibition is determined at a variety of concentrations ranging from 1 μg to 50 μg. Genistein is added to cells at the following time-points: prior to exposure, at time of exposure, 10 minutes after exposure, 30 minutes after exposure, 60 minutes after exposure, 8 hours after exposure, and 24 hours after exposure.

EXAMPLE 8

Drug Toxicity Treatment In The Syrian Golden Hamster

[89] Drug toxicity and pharmacokinetics is measured in the hamster at the following doses: 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 300 mg/kg, 500 mg/kg, and 1000 mg/kg.

EXAMPLE 9

In Vivo Genistein Inhibition Of Flexal Virus (FLEV) In The Syrian Golden Hamster Animal Model (901 Pre-exposure therapy is assessed by genistein treatment of the hamsters 24 hours prior to FLEV exposure followed by daily treatment. Post-exposure prophylactic treatment is assessed by administering genistein 8 hours after FLEV exposure and daily for 14 days. Therapeutic effect is assessed by administering genistein at the first sign of disease, and then daily for 14 days. Typically, the first sign of disease is fever.

EXAMPLE 10

[91] In Vivo Genistein Inhibition Of Haantan Virus In The Male Neonate Norway Rat Animal Model

[92] Pre-exposure therapy is assessed by genistein treatment of the neonate rats 24 hours prior to Haantan virus exposure followed by daily treatment. Postexposure prophylactic treatment is assessed by administering genistein 8 hours after Haantan virus exposure and daily for 14 days. Therapeutic effect is assessed by administering genistein at the first sign of disease, and then daily for 14 days. Typically, the first sign of disease is fever.

EXAMPLE 11

In Vivo Genistein Inhibition Of West Nile Virus In The Syrian Golden Hamster Animal Model

[93] Pre-exposure therapy is assessed by genistein treatment of the hamsters 24 hours prior to West Nile virus exposure followed by daily treatment. Postexposure prophylactic treatment is assessed by administering genistein 8 hours after West Nile virus exposure and daily for 14 days. Therapeutic effect is assessed by administering genistein at the first sign of disease, and then daily for 14 days. Typically, the first sign of disease is fever.

EXAMPLE 12

In Vivo Genistein Inhibition Of Venezuelan Equine Encephalitis (VEE) Virus In A Mouse Model [94] Pre-exposure therapy is assessed by genistein treatment of the mice 24 hours prior to VEE virus exposure followed by daily treatment. Post-exposure prophylactic treatment is assessed by administering genistein 8 hours after VEE virus exposure and daily for 14 days. Therapeutic effect is assessed by administering genistein at the first sign of disease, and then daily for 14 days. Typically, the first sign of disease is fever.

[95] Reference List

Akula, S., Hurley, D., Wixon, R., Wang, C, Chase, C, 2002. Effect of genistein on replication of bovine herpes virus type 1. American Journal of Veterinary Research 63, 1124-1128.

Andres, A., Donovan, S. M., Kuhlenschmidt, T. B., Kuhlenschmidt, M. S., 2007. Isoflavones at concentrations present in soy infant formula inhibit Rotavirus infection in vitro. The Journal of Nutrition 2068-2073.

Ang, F., Wong, A., Ng, M., Chu, J., 2010. Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virology Journal 7, 24.

Bhattacharyya, S., Warfield, K. L., Ruthel, G., Bavari, S., Aman, M. J., Hope, T. J., 2010. Ebola virus uses clathrin-mediated endocytosis as an entry pathway. Virology 401 , 18-28.

Han, Y., Caday, C. G., Nanda, A., Cavenee, W. K., Huang, H.-J. S., 1996. Tyrphostin AG 1478 Preferentially Inhibits Human Glioma Cells Expressing Truncated Rather than Wild-Type Epidermal Growth Factor Receptors. Cancer Research 56, 3859-3861.

Hensley, L. E., Mulangu, S., Asiedu, C, Johnson, J., Honko, A. N., Stanley, D., Fabozzi, G., Nichol, S. T., Ksiazek, T. G., Rollin, P. E., Wahl-Jensen, V., Bailey, M., Jahrling, P. B., Roederer, M., Koup, R. A., Sullivan, N. J., 2010. Demonstration of Cross-Protective Vaccine Immunity against an Emerging Pathogenic Ebolavirus Species. PLoS Pathog 6, el 000904. Jin, ML, Park, J., Lee, S., Park, B., Shin, J., Song, K. J., Ahn, T. 1., Hwang, S. Y., Ahn, B. Y., Ahn, K., 2002. Hantaan Virus Enters Cells by Clathrin- Dependent Receptor-Mediated Endocytosis. Virology 294, 60-69.

Lecot, S., Belouzard, S., Dubuisson, J., Rouille, Y., 2005. Bovine Viral Diarrhea Virus Entry Is Dependent on Clathrin-Mediated Endocytosis. The Journal of Virology 79, 10826-10829.

Martinez, M. G., Cordo, S. M., Candurra, N. A., 2007. Characterization of Junin arenavirus cell entry. Journal of General Virology 88, 1776-1784.

McCormick, J. B., King, I., Webb, P., Johnson, K., O'Sullivan, R., Smith, E., Tripple, S., Tong, T., 1986. Lassa fever: effective therapy with Ribavirin. The New England Journal of Medicine 314, 20-26.

Sbrana, E., Mateo, R. I., Xiao, S.-Y., Popov, V. L., Newman, P. C, Tesh, R. B., 2006. Clinical laboratory, virologic, and pathologic changes in hamsters experimentally infected with Pirital virus (Arenaviridae): a rodent model of Lassa fever. Am J Trop Med Hyg 74, 1096-1 102.

Simmons, G., Rennekamp, A. J., Chai, N., Vandenberghe, L. H., Riley, J. L., Bates, P., 2003. Folate Receptor Alpha and Caveolae Are Not Required for Ebola Virus Glycoprotein-Mediated Viral Infection. The Journal of Virology 77, 13433-13438.

Towner, J., Sealy, T., Ksiazek, T. G., Nichol, S. T., 2007. High-Throughput Molecular Detection of Hemorrhagic Fever Virus Threats with Applications for Outbreak Settings. The Journal of Infectious Diseases 196, S205-S212.

Vela, E., Knostman, K., Warren, R., Garver, J., Stammen, R., 2010a. The disease progression associated with Pirital virus infection in the Syrian golden hamster. Journal of Infectious Diseases and Immunity 2, 15-23.

Vela, E. M., Bowick, G. C, Herzog, N. K., Aronson, J. F., 2008. Genistein treatment of cells inhibits arenavirus infection. Antiviral Research 77, 153- 156. Vela, E. ML, Colpitis, T., Zhang, L., Davey, R., Aronson, J., 2008. Pichinde virus is trafficked through a dynamin 2 endocytic pathway that is dependent on cellular Rab5- and Rab7-mediated endosomes. Archives of Virology 153, 1391-1396.

Vela, E. M., Knostman, K. A., Mott, J. M., Warren, R. L., Garver, J. N., Vela, L. J., Stammen, R. L., 2010b. Genistein, a general kinase inhibitor, as a potential antiviral for arenaviral hemorrhagic fever as described in the Pirital virus-Syrian golden hamster model. Antiviral Research 87, 318-328.

Vela, E. M., Zhang, L., Colpitts, T. M., Davey, R. A., Aronson, J. F., 2007. Arenavirus entry occurs through a cholesterol-dependent, non-caveolar, clathrin-mediated endocytic mechanism. Virology 369, 1-1 1.

Xiao, S.-Y., Zhang, H., Yang, Y., Tesh, R. B., 2001. Pirital virus (Arenaviridae) infection in the Syrian golden hamster, Mesocricetus auratus: A new animal model for arenaviral hemorrhagic fever. Am J Trop Med Hyg 64, 11 1-1 18.

Yura, Y., Yoshida, H., Sato, M., 1993. Inhibition of herpes simplex virus replication by genistein, an inhibitor of protein-tyrosine kinase. Archives of Virology 132, 451-461.

The contents of all of the above cited articles are incorporated by reference as if set forth fully herein.

Claims

WHAT IS CLAIMED IS:

1. A method of preventing a virus-induced disease or disorder in a mammal, comprising at least one administration of a composition comprising a tyrosine kinase inhibitor to the mammal,

wherein the virus enters a host cell via a clathrin-dependent mechanism.

2. The method of claim 1 wherein the administration is oral or intra-peritoneal

3. The method of claim 1 wherein the tyrosine kinase inhibitor is selected from the group consisting of a genistein, a tyrphostin, and mixtures thereof.

4. The method of claim 1 wherein the virus is an arenavirus.

5. The method of claim 5 wherein the arenavirus is selected from the group consisting of: a Lassa virus, a Junin virus, a Machupo virus, a Guanarito virus, a Flexal virus, and a Sabia virus.

6. The method of claim 1 wherein the mammal is a human.

7. The method of claim 1 wherein the virus is a filovirus.

8. The method of claim 5 wherein the filovirus is selected from the group consisting of an Ebola virus and a Marburg virus.

9. A method of treating a virus-induced disease or disorder in a mammal, comprising at least one administration of a composition comprising a tyrosine kinase inhibitor to the mammal,

wherein the virus is a virus that enters a host cell via a clathrin- dependent mechanism.

10. The method of claim 9 wherein the administration is oral or intra-peritoneal.

1 1. The method of claim 9 wherein the clathrin-dependent virus is an arenavirus.

12. The method of claim 1 1 wherein the arenavirus is selected from the group consisting of: a lassa virus, a Junin virus, a Machupo virus, a Guanarito virus, and a Sabia virus.

13. The method of claim 9 wherein the mammal is a human.

14. The method of claim 9 wherein the clathrin-dependent virus is a filovirus.

15. The method of claim 14 wherein the filovirus is selected from the group consisting of an Ebola virus and a Marburg virus.

16. The method of claim 9 wherein the tyrosine kinase inhibitor is selected from the group consisting of a genistein, a tyrphostin, and mixtures thereof.

17. The method of claim 9 wherein the tyrosine kinase inhibitor is administered shortly after identification of a symptom associated with a clathrin-dependent virus infection.

18. The method of claim 9 wherein the tyrosine kinase inhibitor is administered shortly after exposure of the mammal to the clathrin-dependent virus.

19. A method of reducing the incidence of successful infection in a virus-induced disease or disorder in a mammal, comprising at least one administration of a composition comprising a tyrosine kinase inhibitor to the mammal, wherein the virus enters a host cell via a clathrin-dependent mechanism.

20. The method of claim 19 wherein the tyrosine kinase inhibitor is selected from the group consisting of a genistein, a tyrphostin, and mixtures thereof.