DESCRIPTION

COMPOSITION FOR TREATING AND PREVENTING VIRAL INFECTIONS

BACKGROUND OF THE INVENTION

A. Cross-Reference to Related Applications [0001] This application claims the benefit of U.S. Provisional Application No.

62/381,293, filed on August 30, 2016, the content of which is incorporated into the present application by reference.

B. Field of the Invention

[0002] The present invention relates to formulations containing a compound and mixtures of compounds capable of preventing and treating viral infections such as mosquito borne viruses Zika, dengue, and chikungunya.

C. Description of Related Art

[0003] Viruses are relatively simple particles mainly made up of only a few proteins and nucleic acid(s) that contains only a few genes; however, the proteins and nucleic acids can vary greatly between virus species. The variation in virus components leads to a great variation in diseases, complications, and symptoms of virus infections. Viral infections can cause conditions that vary from benign to life threatening. Some viruses cause benign skin growths, while others cause bleeding, cancer, or failure of an infected person's immune system. [0004] Development of anti-viral drugs is challenging. The great variation between viruses makes development of a general anti-viral treatment difficult. Further, viruses require a host cell for replication, hijacking the host cell's own machinery to create viral particles; thus, treatments targeting the host dependent portion of a viral life cycle can often be harmful to the host organism. Moreover, many viral life cycles are very short in duration and many viruses have a high mutation rate, rendering treatments short lived in efficacy because of development of resistance. Currently, most anti-viral drugs target a specific subset of viruses: HIV, herpes virus, hepatitis B and C viruses, and influenza A and B viruses. There is also a great interest in developing drugs against Zika virus to battle the growing spread of Zika and the threat to unborn children. Unfortunately, these viruses only represent a very small fraction of life-threatening viruses and some of the treatments are already becoming obsolete as resistance develops. More anti-viral treatments and effective preventative measures are needed. [0005] Interest in treating and preventing Zika virus, an enveloped virus of the flavivirus genus, has increased recently because of the rapid spread of the virus. Zika virus is mainly spread through mosquito vectors, specifically the Aedes mosquito species. Infection by Zika virus has been shown to cause Zika fever, which is rarely fatal to an adult, but when Zika infection is passed from a pregnant woman to her fetus, the fetus can develop birth defects that include microcephaly, defects of the eye, hearing deficits, and impaired growth. (Center for Disease Control and Prevention, Zika, 2016). Currently there is no vaccination for Zika and the best way to prevent Zika is to avoid mosquito bites. (Id.). Further, some antibodies against Zika may actually facilitate Zika virus infection of some cell types through antibody-dependent enhancement. [0006] Similar to Zika virus, another flavivirus, dengue virus, is also spread into humans by Aedes mosquito bites. Infection by dengue virus is a leading cause of illness and death in the tropics and subtropics, where more than a third of the world's population resides. (Center for Disease Control and Prevention, Dengue, 2016). It is estimated that as many as 400 million people are infected yearly. Dengue virus infection can cause joint pain, rash, headache, and has been shown to cause dengue fever and dengue hemorrhagic fever (DHF). DHF may cause failure of the circulatory system, bleeding, and shock, and possibly death without prompt, appropriate treatment of the symptoms. (Id.). There is currently no proven and effective treatment for a dengue virus infection, but fluid replacement therapy may be useful in alleviating the symptoms. (Id.). Further, some antibodies against dengue virus actually facilitate dengue virus infection of some cell types through antibody-dependent enhancement.

[0007] Another enveloped virus transmitted to humans by Aedes mosquitos, chikungunya virus, can also cause joint pain, rash, headache, and fever. (Center for Disease Control and Prevention, Chikungunya, 2015). Though chikungunya virus infection is rarely fatal, the symptoms can be severe and disabling. (Id.). Similar to Zika virus and dengue virus infection, there is currently no treatment for a chikungunya virus infection. (Id.). [0008] Proven, effective, specific anti-viral treatments and vaccines are currently unavailable for Zika, dengue, and chikungunya viruses. Nor has any broad anti-viral treatment been shown to be effective to treat these viruses.

SUMMARY OF THE INVENTION [0009] The present invention provides a solution to the current problems facing treatment and prevention of viral infections. Specifically, it is shown herein that a compound found in elderberries is capable of preventing and treating infections by the mosquito borne viruses Zika, dengue, and chikungunya. In addition, the inventors believe that using the compound of the present invention with additional anti-viral drugs, such those that target viral budding and release and/or anti-influenza compounds, enhance the ability of the combination to prevent and treat viral infection.

[0010] In one aspect, disclosed is a composition containing a compound of Formula I.

Formula I

[0011] In some instances, the composition contains a pharmaceutically acceptable carrier. In some instances, the pharmaceutically acceptable carrier can be a naturally or a non-naturally occurring compound or structure. In some instances, the composition further contains a preservative, stabilizer, bulking agent, and/or excipient. In some instances, the preservative, stabilizer, bulking agent, and/or excipient can be a naturally or a non-naturally occurring compound or composition. In some instances, the composition is a pharmaceutical composition. In some instances, the composition is coated with a polymer. In some instances, the polymer is a naturally or a non-naturally occurring polymer. In another aspect, the composition may further comprise one or more ingredients described herein. For example, the composition may comprise one or more additional ingredients selected from one or more pH adjusters, structuring agents, and inorganic salts.

[0012] In some instances, the composition is formulated for oral administration. In some instances, the composition is one or more of a lozenge, a powder, a tablet, a delayed release capsule, a quick release capsule, a gel-cap, a gelatin, a liquid solution, and/or a dissolvable film. In some instances, the composition is formulated for topical application, intravenous administration, and/or intranasal delivery. In some instances, the composition comprises 12 to 100% by weight of the compound of Formula I. In some instances, the composition comprises 15 to 99.9% by weight of the compound of Formula I. In some instances, the composition comprises 30 to 99.9%, 40 to 99.9%, 50 to 99.9%, 60 to 99.9%, 70 to 99.9%, 80 to 99.9%, 90 to 99.9%, 95 to 99.9%, or 97 to 99.9%, or any range therein by weight of the compound of Formula I. In some instances, the composition contains a dose of the compound of Formula I. In some instances, a dose is 1 μg to 10 gram of the compound of Formula I. In some instances, the composition contains a sub-dose of the compound of Formula I. In some instances, the composition contains 0.1 mg to 3 g, 1 mg to 1 g, 1 mg to 500 mg, 1 to 300 mg, 1 to 100 mg, 1 to 10 mg, 1 mg, 2 mg, 3 mg, or 4 mg of the compound of Formula I, or any range therein.

[0013] In one aspect, the composition contains a second anti-viral drug. In some instances, the composition does not comprise a second anti-viral drug. In some instances, the second anti -viral drug inhibits viral budding or release from the host cell. In some instances, the second anti-viral drug is an anti-influenza drug. In some instances, the anti-influenza drug is oseltamivir, zanamivir, rimantadine, amantadine, peramivir, or salts thereof, or any combination thereof. In some instances, the anti-influenza drug is oseltamivir, a salt thereof, or any combination thereof. [0014] In another aspect, the composition of Formula I is synthetically obtained. In yet another aspect, the composition of Formula I is obtained from an organism. In some instances, the compound of Formula I is obtained from Sambucus nigra fruit.

[0015] Also disclosed is a method of treating or preventing a viral infection in a subject, the method comprises administering any one of the compositions of the present invention to the subject. Further, herein is disclosed a method of administering any one of the compositions of the present invention to a subject by administering any one of the compositions of the present invention to the subject. In a particular instance, the subject has been diagnosed with a viral infection. In a particular instance, any one of the compositions of the present invention is administered to a subject to prevent a viral infection in the subject.

[0016] In some aspects, disclosed is a method of treating or preventing a mosquito borne virus infection by administering any of the compositions disclosed herein. In some instances, the virus infection is a Zika virus infection. In some instances, the virus infection is a dengue virus infection. In some instances, the virus infection is a chikungunya virus infection. In some instances the virus infection is a flavivirus infection. In some instances, the infection is an alphavirus virus infection. [0017] In some aspects, disclosed herein is a method of treating or preventing an infection by an influenzavirus A and/or influenzavirus B virus by administering the compound of Formula I and a second anti -viral drug. In some instances, the second anti -viral drug inhibits viral budding or release from the host cell. In some instances, the second antiviral drug is an anti-influenza drug. In some instances, the anti-influenza drug is oseltamivir, zanamivir, rimantadine, amantadine, peramivir, or salts thereof, or any combination thereof. In some instances, the anti-influenza drug is oseltamivir, a salt thereof, or any combination thereof.

[0018] In some aspects of any of the methods described herein, the subject is administered a total sum of between 0.001 mg and 10 g of the compound of Formula I during a 24 hour period. In some instances, the subject is administered a total sum of between 0.1 and 1,500 mg, 1 and 1,500 mg, 5 and 1,000 mg, 10 and 500 mg, or any range therein, of the compound of Formula I during a 24 hour period.

[0019] In some aspects of any of the methods described herein, the subject is administered any one of the compositions disclosed herein at least once a day. In some instances, the subject is administered any one of the compositions disclosed more than once a day. In some instances, the subject is administered any one of the compositions disclosed herein twice a day. In some aspects, the subject is administered any one of the compositions disclosed herein daily for at least five days. In some aspects, the subject is administered any one of the compositions disclosed herein daily for at least ten days. In some aspects, the subject is administered any one of the compositions disclosed herein daily for at least fourteen days. In some aspects, the subject is administered any one of the compositions disclosed herein every other day for at least six days. In some aspects, the subject is administered any one of the compositions disclosed herein every other day for at least ten days. In some aspects, the subject is administered any one of the compositions disclosed herein every other day for at least fourteen days. [0020] In some aspects of the invention, the composition may further comprise one or more nutraceutical and/or pharmaceutically acceptable carriers or diluents. These carriers/diluents can be adjuvants, excipients, or vehicles such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, fragrance, antibacterial agents, antifungal agents, lubricating agents, vitamins, polymers, siloxane containing compounds, essential oils, structuring agents, and dispensing agents. Each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. In some aspects of the invention, the carrier can include at least one hydrophilic polymeric compound selected from the group consisting of a gum, a cellulose ether, an acrylic resin, a carbohydrate carrier, talc, lactose, mannitol, glucose, water, gelatin, a protein-derived compound, polyvinyl pyrrolidone, magnesium stearate, and any combination thereof. Non-limiting examples of diluents/carriers are identified throughout this specification and are incorporated into this section by reference. The amounts of such ingredients can range from 0.0001% to 99.9% by weight or volume of the composition, or any integer or range in between as disclosed in other sections of this specification, which are incorporated into this paragraph by reference.

[0021] The composition can be stored for one month, 6 months, 12 months, 18 months, or 24 months at room temperature. In some aspects of the invention, the composition is formulated as a powder, a tablet, a gel-cap, a bead, an edible tablet, a dissolvable film, a liquid capable of being dispersed through the air, a gelatin, a lotion, a transdermal patch, or a liquid solution for oral administration. In some aspects of the invention, the formulated composition can be comprised in a solid nanoparticle, a lipid-containing nanoparticle, a lipid- based carrier, a sealed conduit, a straw, sealed bag, or any combination thereof. In other aspects of the invention, the composition can be formulated for administration by injection.

[0022] Kits that include the compositions of the present invention are also contemplated. In certain embodiments, the composition is comprised in a container. The container can be a bottle, dispenser, package, or a straw. The container can dispense a predetermined amount of the composition. In certain aspects, the compositions are dispensed as a pill, a tablet, a capsule, a transdermal patch, an edible chew, a cream, a lotion, a gel, spray, mist, dollop, a powder, or a liquid. The container can include indicia on its surface. The indicia can be a word, an abbreviation, a picture, or a symbol.

[0023] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0024] Also contemplated is a product that includes the composition of the present invention. In non-limiting aspects, the product can be a nutraceutical product. The nutraceutical product can be those described in other sections of this specification or those known to a person of skill in the art. In other non-limiting aspects, the product can be a pharmaceutical product. The pharmaceutical and/or nutraceutical product can be those described in other sections of this specification or those known to a person of skill in the art. Non-limiting examples of products include a pill, a tablet, an edible chew, a capsule, a cream, a lotion, a gel, a spray, a mist, a dissolving film, a transdermal patch, or a liquid, etc. [0025] "Therapeutic agent" encompasses the compounds specifically claimed herein.

It also encompasses such compounds together with nutraceutical and/or pharmaceutically acceptable salts thereof. Useful salts are known to those skilled in the art and include salts with inorganic acids, organic acids, inorganic bases, or organic bases. Therapeutic agents useful in the present invention are those compounds that affect a desired, beneficial, and often pharmacological, effect upon administration to a human or an animal, whether alone or in combination with other nutraceutical and/or pharmaceutical excipients or inert ingredients.

[0026] The term "biomarker" refers to the compound defined as the biomarker, analogues thereof, derivatives thereof, or salt forms of any analogue or derivative thereof.

[0027] The term "accurate mass" refers to a measured mass of a molecule experimentally determined for an ion of known charge. The units for accurate mass include atomic mass units (amu) and milli unified atomic mass units (mmu). The term "molecular weight" refers to the average weight of the molecule with all of the different isotopic compositions present in a compound but weighted for their natural abundance.

[0028] The term "relative abundance" refers to the abundance of a compound of interest relative to the abundance of a reference compound. In particular aspects, relative abundance is the raw intensity of a mass spectrometry peak for the compound of interest over the raw intensity of a mass spectrometry peak for a reference compound. In one non-limiting instance, the mass spectrometry peaks can be obtained by the use of DART-TOF mass spectrometry. In another particular aspect, the reference compound is a compound that is spiked, or doped, into a sample containing the compound of interest. In yet another particular aspect, the reference compound is a compound that does not exist in the sample previous to its addition to the sample for determining relative abundance. In another particular aspect, the reference compound can be curcumin.

[0029] Accurate mass and relative abundances described herein are based on experiments using particular instruments and particular settings and can change from instrument to instrument. There is variability in each measurement. Thus, the accurate mass and relative abundances are defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 20%, preferably 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. In one non-limiting embodiment, the accurate mass has an error of within +/- 20 mmu, preferably 10 mmu, more preferably within 5 mmu, and most preferably within 1 mmu. In one non-limiting embodiment, the relative abundance has an error of +/- 20%, preferably 10%), preferably within 5%, and more preferably within 1%, and most preferably within 0.5%. [0030] The term "substantially" and its variations are defined as being largely but not necessarily wholly what is specified as understood by one of ordinary skill in the art, and in one non-limiting embodiment substantially refers to ranges within 10%, within 5%, within 1%, or within 0.5%.

[0031] "Patient," "subject," or "individual" refers to a mammal (e.g., human, primate, dog, cat, bovine, ovine, porcine, equine, mouse, rat, hamster, rabbit, or guinea pig). In particular aspects, the patient, subject, or individual is a human.

[0032] "Inhibiting" or "reducing" or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result.

[0033] "Effective" or "effect" or any variation of these terms means adequate to accomplish a desired, expected, or intended result. [0034] "Treating" or "treat" or any variation of these terms includes any measurable improvement in a disease, condition, or symptom that is being treated or is associated with the disease, condition, or symptom being treated, such as an extension of life span, reduced viral load, and/or reduction of weight loss. [0035] "Preventing" or "prevent" or any variation of these terms means to slow, stop, or reverse progression toward a result. The prevention may be any slowing of the progression toward the result.

[0036] "Analogue" and "analog," when referring to a compound, refers to a modified compound wherein one or more atoms have been substituted by other atoms, or wherein one or more atoms have been deleted from the compound, or wherein one or more atoms have been added to the compound, or any combination of such modifications. Such addition, deletion or substitution of atoms can take place at any point, or multiple points, along the primary structure comprising the compound.

[0037] "Derivative," in relation to a parent compound, refers to a chemically modified parent compound or an analogue thereof, wherein at least one substituent is not present in the parent compound or an analogue thereof. One such non-limiting example is a parent compound which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, pegylations and the like.

[0038] A "therapeutically equivalent" compound is one that has essentially the same effect in the treatment of a disease or condition as one or more other compounds. A compound that is therapeutically equivalent may or may not be chemically equivalent, bioequivalent, or generically equivalent.

[0039] "Parenteral injection" refers to the administration of small molecule drugs via injection under or through one or more layers of skin or mucus membranes of an animal, such as a human.

[0040] "Bioavailability" refers to the extent to which the therapeutic agent is absorbed from the formulation.

[0041] "Systemic," with respect to delivery or administration of a therapeutic agent to a subject, indicates that the therapeutic agent is detectable at a biologically significant level in the blood plasma of the subject.

[0042] "Controlled release" refers to the release of the therapeutic agent at such a rate that blood (e.g., plasma) concentrations are maintained within the therapeutic range, but below toxic concentrations over a period of time of about one hour or longer, preferably 12 hours or longer.

[0043] "Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering a drug compound of the present invention to a mammal such as an animal or human.

[0044] "Nutraceutical acceptable carrier" refers to a nutraceutical acceptable solvent, suspending agent or vehicle for delivering a compound of the present invention to a mammal such as an animal or human.

[0045] "Pharmaceutically acceptable" ingredient, excipient or component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic response) commensurate with a reasonable benefit/risk ratio.

[0046] "Nutraceutical acceptable" ingredient, excipient or component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic response) commensurate with a reasonable benefit/risk ratio.

[0047] The term "about" or "approximately" or "substantially unchanged" are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%), and most preferably within 0.5%. Further, "substantially non-aqueous" refers to less than 5%, 4%, 3%, 2%, 1%, or less by weight or volume of water.

[0048] The use of the word "a" or "an" when used in conjunction with the term

"comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0049] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0050] The compositions and methods for their use can "comprise," "consist essentially of," or "consist of any of the ingredients or steps disclosed throughout the specification. With respect to the transitional phase "consisting essentially of," in one non- limiting aspect, a basic and novel characteristic of the compositions and methods disclosed in this specification includes the compositions' abilities to reduce or prevent viral infections from viruses such as Zika virus, dengue virus, or chikungunya virus.

[0051] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the examples, while indicating specific embodiments of the invention, are given by way of illustration only. Additionally, it is contemplated that changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0053] FIG. 1 The dose dependent inhibition of Zika virus infection of Vero cells incubated with the compound of Formula I.

[0054] FIG. 2 Survival of AG129 mice challenged with ZIKV PRVABC59.

[0055] FIGS. 3 A-C Body weight for AG129 mice challenged with ZIKV

PRVABC59. (A) AG129 mice treated with 10 mg/kg Formula I. (B) AG129 mice treated with 30 mg/kg Formula I. (C) AG129 mice treated with vehicle.

[0056] FIG. 4 Percent change in body weight for AG129 mice challenged with

ZIKV PRVABC59. [0057] FIG. 5 Mean survival of mice after infection with dengue virus- 1 (DENV-1).

Mean survival increased with treatment using 5 mg/kg and 10 mg/kg of the compound of Formula I. The mice were treated with the compound of Formula I twice daily at -2, -1, 0, +1, and +2 days relative to infection. Treatment with the compound of Formula I increased the mean survival by up to 6 days.

[0058] FIG. 6 Viral titer of DENV-1 in mice at 19 days post infection with DENV-1.

Viral titer was decreased at day 19 in mice treated twice a day with 5 mg/kg and 10 mg/kg of the compound of Formula I at -2, -1, 0, +1, and +2 days relative to infection.

[0059] FIG. 7 Mean survival of mice after infection with chikungunya virus

(CHIKV). Mean survival increased with treatment using 5 mg/kg, 15 mg/kg, and 30 mg/kg of the compound of Formula I. The mice were treated with the compound of Formula I twice daily at -2, 0, +2, , +4, +6, +8, and +10 days relative to infection. Treatment with the compound of Formula I increased the mean survival in a dose dependent manner up to 12 days.

[0060] FIG. 8 Viral titer of CHIKV in mice at 21 days past infection with CHIKV.

Viral titer was decreased at day 21 in mice treated twice a day with 5 mg/kg, 15 mg/kg, and 30 mg/kg of the compound of Formula I at -2, 0, +2, , +4, +6, +8, and +10 days relative to infection.

DETAILED DESCRIPTION

[0061] The inventors have surprisingly found that a compound that can be found in elderberries, can prevent and treat Zika, dengue, and chikungunya virus infection. In addition, the inventors believe that using the compound of the present invention with additional antiviral drugs, such as anti-influenza compounds enhance the ability of the combined compounds to treat and prevent viral infection. Without wishing to be bound by theory, it is believed that the compound and compositions disclosed herein are capable of blocking entry of viruses such as influenza, Zika, dengue, and/or chikungunya virus into a cell. Further, it is expected that the compound will have synergistic effect with other anti-viral drugs for prevention and treatment of virus infection. A. Active Compound

[0062] A folk medicine remedy for cold and flu, Sambucus nigra L. (elderberry), has been shown in clinical trials to be effective in treating influenza viruses infections. Furthermore, elderberry extract can be effective to treat Influenzavirus A and B infections when taken as a syrup. (Roxas and Jurenka 2007; Zakay-Rones et al. 1995; 2004). These studies demonstrated that relatively large dosage amounts of the extract when compared with Tamiflu® (e.g., over 1000 times larger wt./vol.) could potentially reduce the duration of flu- like symptoms. The larger dosage amounts were achieved by increasing the frequency of treatments, which can lead to a decrease in patient compliance. Anti-influenza activities of some elderberry products are attributed to the presence of three flavonoids: averionol; tristenonol; and istrocyanidin. (Roschek and Alberte 2008). Extracts containing averionol, tristenonol, and istrocyanidin have been used to show that these three compounds may specifically bind some viruses, including some influenza strains, and may inhibit HIV. (US 2009/0149530). Further, US 2009/0149530 discloses that an unidentified active ingredient from an extract that may contain averionol, tristenonol, and istrocyanidin may inhibit infection of several viruses in cell culture. {Id).

[0063] The compound disclosed herein is effective in inhibiting the mosquito borne viruses Zika, dengue, and chikungunya and is expected to inhibit viruses synergistically with other anti-viral drugs. The compound disclosed herein can be found in Sambucus nigra L. (elderberry) defined by accurate mass of 358.1121 amu and has the structure shown in Formula I.

Formula I [0064] The composition of the present invention can include the compound of

Formula I. Without wishing to be bound by theory, it is believed that the compound of Formula I blocks viral entry into a cell.

[0065] In some aspects of the invention, the compound and derivatives and analogues can be made through known synthetic methods. In some aspects of the invention, the compound can be synthetically obtained by producing the compound according to methods known to one of skill in the art in chemical synthesis. In one instance, the compound is synthesized through organic chemistry methods. In one aspect, the compound can be synthesized according to Reaction Scheme I:

Reaction Scheme I

[0066] Referring to reaction scheme I, (1) is rutin, (3) is the compound of Formula I,

(a) is DMS, K2CO3, acetone, and reflux for 70 hours, and (b) is 20% HC1 and reflux for 2 hours. [0067] In some aspects of the invention, the compound can be isolated from extracts of an organism such as fruits, plants, animals, fungi, bacteria, and/or archaea. Non-limiting examples of suitable fruits include elderberry fruit. The compound can be extracted from the organism using known extraction methods, such as contacting the extract with CO2, contacting the extract with H2O, or any combination of EtOFFFhO, and/or with any method utilizing polymer separating the extract. A non-limiting example of a polymer used for polymer separation includes ADS 5 polymer (Nankai University, China).

B. Additional Anti- Viral Agents

[0068] In some aspects, a second anti-viral drug can be combined with the compound of Formula I. In some instances, anti -viral drugs can inhibit binding of a virus to a host cell, inhibit viral entry into a host cell, prevent budding of virus from a host cell, prevent release of a virus from a host cell, prevent replication in a host cell, or destroy or inhibit the virus particle. Anti-viral drugs include those that are specific to one or a few viruses or are broad spectrum drugs against several types of viruses. Anti-viral drugs include those that are combination drugs and single drugs. Anti-influenza drugs are a non-limiting example of antiviral drugs. In one embodiment, the compositions disclosed herein further includes at least one additional anti-viral agent.

[0069] Anti-influenza agents are compounds or compositions that are used to decrease the influenza viral load or prevent viral infection. Non-limiting examples of anti- influenza agents include oseltamivir (also known as TAMIFLU®), zanamivir (RELENZA®), peramivir (RAPIVAB®) rimantadine (also known as FLUMADINE®), and amantadine (also known as SYMMETREL®). Some anti-influenza agents inhibit neuraminidase, which prevents the release of viral progeny from infected cells. Non-limiting examples of anti- influenza agents that prevent the release of viral progeny from infected cells include neuraminidase inhibitors such as oseltamivir, zanamivir, and peramivir. Some anti-influenza agents block the viral encoded M2 ion-channel. Non-limiting examples of anti-influenza agents that block the M2 ion-channel are rimantadine and amantadine. Non-limiting examples of influenza viruses include viruses of the Influenzavirus A and Influenzavirus B genus. In one instance the viruses include, but are not limited to, H1N1, H3N2, H3N5, H5N1, and Influenza B. In one embodiment, the compositions disclosed herein further includes at least one additional anti-influenza agent, which may be, but is not limited to, oseltamivir, zanamivir, peramivir, rimantadine, and amantadine.

C. Amounts of Ingredients

[0070] It is contemplated that the compositions of the present invention can include any amount of the ingredients discussed in this specification. The compositions can also include any number of combinations of additional ingredients described throughout this specification (e.g., stabilizers, fillers, pharmaceutically and/or nutraceutical acceptable salts, and/or additional pharmaceutical and/or nutraceutical ingredients). The concentrations of the any ingredient within the compositions can vary. In non-limiting embodiments, for example, the compositions can comprise, consisting essentially of, or consist of, in their final form, for example, at least about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.0010%, 0.001 1%, 0.0012%, 0.0013%, 0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019%, 0.0020%, 0.0021%, 0.0022%, 0.0023%, 0.0024%, 0.0025%, 0.0026%, 0.0027%, 0.0028%, 0.0029%, 0.0030%, 0.0031%, 0.0032%, 0.0033%, 0.0034% 0.0035%, 0.0036% 0.0037% 0.0038%, 0.0039% 0.0040%, 0.0041% 0.0042%, 0.0043% 0.0044% 0.0045% 0.0046% 0.0047%, 0.0048% 0.0049%, 0.0050% 0.0051%, 0.0052% 0.0053% 0.0054% 0.0055% 0.0056%, 0.0057% 0.0058%, 0.0059% 0.0060%, 0.0061% 0.0062% 0.0063% 0.0064% 0.0065%, 0.0066% 0.0067%, 0.0068% 0.0069%, 0.0070% 0.0071% 0.0072% 0.0073% 0.0074%, 0.0075% 0.0076%, 0.0077% 0.0078%, 0.0079% 0.0080% 0.0081% 0.0082% 0.0083%, 0.0084% 0.0085%, 0.0086% 0.0087%, 0.0088% 0.0089% 0.0090% 0.0091% 0.0092%, 0.0093% 0.0094%, 0.0095% 0.0096%, 0.0097% 0.0098% 0.0099% 0.0100% 0.0200%, 0.0250% 0.0275%, 0.0300% 0.0325%, 0.0350% 0.0375% 0.0400% 0.0425% 0.0450%, 0.0475% 0.0500%, 0.0525% 0.0550%, 0.0575% 0.0600% 0.0625% 0.0650% 0.0675%, 0.0700% 0.0725%, 0.0750% 0.0775%, 0.0800% 0.0825% 0.0850% 0.0875% 0.0900%, 0.0925% 0.0950%, 0.0975% 0.1000%, 0.1250% 0.1500% 0.1750% 0.2000% 0.2250%, 0.2500% 0.2750%, 0.3000% 0.3250%, 0.3500% 0.3750% 0.4000% 0.4250% 0.4500%, 0.4750% 0.5000%, 0.5250% 0.0550%, 0.5750% 0.6000% 0.6250% 0.6500% 0.6750%, 0.7000%, 0.7250%, 0.7500% 0.7750%, 0.8000% 0.8250%, 0.8500%, 0.8750%, 0.9000%, 0.9250%, 0.9500%, 0.9750%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or any range derivable therein, of at least one of the ingredients that are mentioned throughout the specification and claims. In non-limiting aspects, the percentage can be calculated by weight or volume of the total composition or relative abundance. A person of ordinary skill in the art would understand that the concentrations can vary depending on the addition, substitution, and/or subtraction of ingredients in a given composition.

D. Additional Components

[0071] The compound of the present invention can be formulated into any suitable composition form for administration to a human or non-human animal patient. [0072] The composition may consist of the claimed compound or compounds alone or may include the compound or compounds and any suitable additional component, such as one or more pharmaceutically and/or nutraceutical acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. 1. Excipients

[0073] Excipients employed in the compositions of the present invention can be solids, semi-solids, liquids or combinations thereof. Preferably, the excipients are solids. Compositions of the invention containing excipients can be prepared by any known technique that comprises, for example, admixing an excipient with the claimed compounds. A pharmaceutical composition of the invention contains a desired amount of the claimed compounds per dose unit and, if intended for oral administration, can be in the form, for example, of a tablet, a caplet, a pill, a hard or soft capsule, a lozenge, a cachet, a dispensable powder, granules, a suspension, an elixir, a dispersion, or any other form reasonably adapted for such administration. If intended for parenteral administration, it can be in the form, for example, of a suspension or transdermal patch. If intended for rectal administration, it can be in the form, for example, of a suppository. Presently preferred are oral dosage forms that are discrete dose units each containing a predetermined amount of the claimed compounds such as tablets or capsules.

2. Carriers / Diluents [0074] Suitable carriers or diluents illustratively include, but are not limited to, either individually or in combination, lactose, including anhydrous lactose and lactose monohydrate; starches, including directly compressible starch and hydrolyzed starches (e.g., Celutab™and Emdex™), mannitol, sorbitol, xylitol, dextrose (e.g., Cerelose™ 2000) and dextrose monohydrate, dibasic calcium phosphate dihydrate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, granular calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, celluloses including microcrystalline cellulose, food grade sources of alpha- and amorphous cellulose (e.g., RexcelJ), powdered cellulose, hydroxypropylcellulose (HPC) and hydroxypropylmethylcellulose (HPMC), calcium carbonate, glycine, clay, bentonite, block co-polymers, polyvinylpyrrolidone, and the like. Such carriers or diluents, if present, constitute in total about 5% to about 99.999%, about 10% to about 85%, and 20% to about 80%), of the total weight of the composition. The carrier, carriers, diluent, or diluents selected preferably exhibit suitable flow properties and, where tablets are desired, compressibility.

3. Disintegrant

[0075] Compositions of the invention optionally can include one or more pharmaceutically and/or nutraceutical acceptable disintegrants as excipients, particularly for tablet formulations. Suitable disintegrants include, but are not limited to, either individually or in combination, starches, including sodium starch glycolate and pregelatinized corn starches, clays, celluloses such as purified cellulose, microcrystalline cellulose, methylcellulose, carboxymethylcellulose and sodium carboxymethylcellulose, croscarmellose sodium, alginates, crospovidone, and gums such as agar, guar, locust bean, karaya, pectin and tragacanth gums. Disintegrants may be added at any suitable step during the preparation of the composition, particularly prior to granulation or during a lubrication step prior to compression. Such disintegrants, if present, constitute in total about 0.2% to about 30%, preferably about 0.2% to about 10%, and more preferably about 0.2% to about 5%, of the total weight of the composition.

4. Binders

[0076] The compositions of the present invention can include binding agents or adhesives particularly for tablet formulations. Such binding agents and adhesives preferably impart sufficient cohesion to the powder being tableted to allow for normal processing operations such as sizing, lubrication, compression and packaging, but still allow the tablet to disintegrate and the composition to be absorbed upon ingestion. Such binding agents may also prevent or inhibit crystallization or recrystallization of a co-crystal of the present invention once the salt has been dissolved in a solution. Suitable binding agents and adhesives include, but are not limited to, either individually or in combination, acacia; tragacanth, sucrose, gelatin, glucose, starches such as, but not limited to, pregelatinized starches, celluloses such as, but not limited to, methylcellulose and carmellose sodium, alginic acid and salts of alginic acid; magnesium aluminum silicate, PEG, guar gum, polysaccharide acids, bentonites, povidone, polymethacrylates, HPMC, hydroxypropylcellulose, and ethylcellulose. Such binding agents and/or adhesives, if present, constitute in total about 0.5% to about 25%, preferably about 0.75% to about 15%, and more preferably about 1% to about 10%, of the total weight of the pharmaceutical composition. Many of the binding agents are polymers comprising amide, ester, ether, alcohol or ketone groups and, as such, can be included in pharmaceutical compositions of the present invention. Polyvinylpyrrolidones is an non-limiting example of a binder used for slow release tablets. Polymeric binding agents can have varying molecular weight, degrees of crosslinking, and grades of polymer. Polymeric binding agents can also be copolymers, such as block copolymers that contain mixtures of ethylene oxide and propylene oxide units. Variation in these units' ratios in a given polymer affects properties and performance.

5. Wetting Agents

[0077] Wetting agents can be used in the compositions of the present invention. Wetting agent can be selected to maintain the crystal in close association with water, a condition that may improve bioavailability of the composition. Such wetting agents can also be useful in solubilizing or increasing the solubility of crystals. Surfactants can be used as wetting agents. Non-limiting examples of surfactants that can be used as wetting agents in compositions of the invention include quaternary ammonium compounds, for example benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride, dioctyl sodium sulfosuccinate, polyoxyethylene alkylphenyl ethers, poloxamers (polyoxyethylene and polyoxypropylene block copolymers), polyoxyethylene fatty acid glycerides and oils, for example polyoxyethylene (8) caprylic/capric mono- and di glycerides, polyoxyethylene (35) castor oil and polyoxyethylene (40) hydrogenated castor oil, polyoxyethylene alkyl ethers, for example polyoxyethylene (20) cetostearyl ether, polyoxyethylene fatty acid esters, for example polyoxyethylene (40) stearate, polyoxyethylene sorbitan esters, for example polysorbate 20 and polysorbate 80, propylene glycol fatty acid esters, for example propylene glycol laurate, sodium lauryl sulfate, fatty acids and salts thereof, for example oleic acid, sodium oleate and triethanolamine oleate, glyceryl fatty acid esters, for example glyceryl monostearate, sorbitan esters, for example sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate and sorbitan monostearate, tyloxapol, and mixtures thereof. Such wetting agents, if present, constitute in total about 0.25% to about 15%, preferably about 0.4% to about 10%), and more preferably about 0.5% to about 5%, of the total weight of the pharmaceutical composition.

6. Lubricants

[0078] Lubricants can be included in the compositions of the present invention. Suitable lubricants include, but are not limited to, either individually or in combination, glyceryl behapate, stearic acid and salts thereof, including magnesium, calcium and sodium stearates; hydrogenated vegetable oils, colloidal silica, talc, waxes, boric acid, sodium benzoate, sodium acetate, sodium fumarate, sodium chloride, DL-leucine, PEG (e.g., Carbowax™ 4000 and Carbowax™ 6000 of the Dow Chemical Company), sodium oleate, sodium lauryl sulfate, and magnesium lauryl sulfate. Such lubricants, if present, constitute in total about 0.1%> to about 10%>, preferably about 0.2% to about 8%, and more preferably about 0.25%) to about 5%, of the total weight of the composition.

7. Other Agents

[0079] Surfactant, emulsifier, or effervescent agents can be used in the compositions. Emulsifying agents can be used to help solubilize the ingredients within a soft gelatin capsule. Non-limiting examples of the surfactant, emulsifier, or effervescent agent include D- sorbitol, ethanol, carrageenan, carboxyvinyl polymer, carmellose sodium, guar gum, glycerol, glycerol fatty acid ester, cholesterol, white beeswax, dioctyl sodium sulfosuccinate, sucrose fatty acid ester, stearyl alcohol, stearic acid, polyoxyl 40 stearate, sorbitan sesquioleate, cetanol, gelatin, sorbitan fatty acid ester, talc, sorbitan trioleate, paraffin, potato starch, hydroxypropyl cellulose, propylene glycol, propylene glycol fatty acid ester, pectin, poly oxy ethylene (105) polyoxypropylene (5) glycol, poly oxy ethylene (160) polyoxypropylene (30) glycol, poly oxy ethylene hydrogenated castor oil, poly oxy ethylene hydrogenated castor oil 40, poly oxy ethylene hydrogenated castor oil 60, polyoxyl 35 castor oil, polysorbate 20, polysorbate 60, polysorbate 80, macrogol 400, octyldodecyl myristate, methyl cellulose, sorbitan monooleate, glycerol monostearate, sorbitan monopalmitate, sorbitan monolaurate, lauryl dimethylamine oxide solution, sodium lauryl sulfate, lauromacrogol, dry sodium carbonate, tartaric acid, sodium hydroxide, purified soybean lecithin, soybean lecithin, potassium carbonate, sodium hydrogen carbonate, medium-chain triglyceride, citric anhydride, cotton seed oil-soybean oil mixture, and liquid paraffin. E. Vehicles

[0080] Various delivery systems are known in the art and can be used to administer a therapeutic agent or composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis and the like. Methods of administration include, but are not limited to, parenteral, intra-arterial, intramuscular, intravenous, intranasal, and oral routes. The compositions can be provided in the form of tablets, lozenges, granules, capsules, pills, ampoule, suppositories or aerosol form. The compositions can also be provided in the form of suspensions, solutions, and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granulates or powders. F. Formulation and Administration

[0081] The composition may, for example, be a pharmaceutical composition

(medicament), and over the counter composition (OTC), a nutraceutical, etc. Compositions according to the present invention include formulations suitable for oral or parenteral routes. Non-limiting examples of specific routes include intradermal, subcutaneous, intramuscular, intravenous, local injection, rectal, intranasal inhalation, insufflation, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art. Such methods include the step of bringing into association the active ingredient (or ingredients) with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with a suitable carrier, such as liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Formulations of the subject invention suitable for oral administration can be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient, or as an oil-in-water liquid emulsion, water-in-oil liquid emulsion, or as a supplement within an aqueous solution, for example, a tea. The active ingredient can also be presented as bolus, electuary, or paste. Useful injectable preparations include sterile suspensions, solutions or emulsions of the compound compositions in aqueous or oily vehicles. The compositions can also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers, and can contain added preservatives. Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the compound compositions can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

[0082] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth, pastilles that include the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia, mouthwashes that include the active ingredient in a suitable liquid carrier, and chocolate comprising the active ingredients.

[0083] Formulations suitable for topical administration according to the subject invention can be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. Alternatively, a formulation can comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients, and optionally one or more excipients or diluents. Topical formulations preferably comprise compounds that facilitate absorption of the active ingredients through the skin and into the bloodstream.

[0084] Formulations suitable for intranasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns, which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for intranasal administration, such as by the non-limiting examples of a nebulizer, include aqueous or oily solutions of the agent. Formulations preferably can include compounds that facilitate absorption of the active ingredients through the skin and into the bloodstream.

[0085] Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which can contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations can be presented in unit-dose or multi-dose or multi-dose sealed containers, such as for example, ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

[0086] Liquid preparations for oral administration can take the form of, for example, elixirs, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically and/or nutraceutical acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate. [0087] For buccal administration, the compositions can take the form of the non- limiting examples of tablets or lozenges formulated in a conventional manner.

[0088] For rectal and vaginal routes of administration, the compound compositions can be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides. [0089] For nasal administration or administration by inhalation or insufflation, the compound compositions can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0090] For prolonged delivery, the compound compositions can be formulated as a depot preparation for administration by implantation or intramuscular injection. The compound compositions can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch, which slowly releases the compound compositions for percutaneous absorption, can be used. To this end, permeation enhancers can be used to facilitate transdermal penetration of the compound compositions. Suitable transdermal patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5, 164,189; U.S. Pat. No. 5, 163,899; U.S. Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475. [0091] Alternatively, other delivery systems can be employed. Liposomes and emulsions are well-known examples of delivery vehicles that can be used to deliver the compound compositions. Certain organic solvents such as dimethylsulfoxide (DMSO) can also be employed, although usually at the cost of greater toxicity.

[0092] It should be understood that in addition to the ingredients particularly mentioned above, the formulations useful in the present invention can include other agents conventional in the art regarding the type of formulation in question. For example, formulations suitable for oral administration can include such further agents as sweeteners, thickeners, and flavoring agents. It also is intended that the agents, compositions, and methods of this invention be combined with other suitable compositions and therapies. [0093] In one embodiment, the pharmaceutical and/or nutraceutical compositions of the invention can be administered locally to the area in need of treatment; such local administration can be achieved, for example, by local infusion, by injection, or by means of a catheter. In another embodiment, a compound or composition of the invention is administered in a manner so as to achieve peak concentrations of the active compound at sites of the disease. Peak concentrations at disease sites can be achieved, for example, by intravenously injecting of the agent, optionally in saline, or orally administering, for example, a tablet, capsule or syrup containing the active ingredient.

G. Other Pharmaceutical and/or Nutraceutical Agents

[0094] Pharmaceutical, OTC, and/or nutraceutical formulations of the invention can be administered simultaneously or sequentially with other drugs or biologically active agents. Examples include, but are not limited to, anti-influenza agents, antioxidants, free radical scavenging agents, analgesics, anesthetics, anorectals, antihistamines, anti-inflammatory agents including non-steroidal anti-inflammatory drugs, antibiotics, antifungals, antivirals, antimicrobials, anti-cancer actives, antineoplastics, biologically active proteins and peptides, enzymes, hemostatics, steroids including hormones and corticosteroids, etc.

H. Therapeutic Methods And Dosage

[0095] Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, or an appropriate fraction thereof, of an agent. Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated, and the efficacy and toxicity of the agent. Similarly, suitable dosage formulations and methods of administering the agents can be readily determined by those of ordinary skill in the art.

[0096] In some embodiments, a therapeutic method of the present invention can include treating a disease, condition, or disorder by administering to a subject having such disease or condition a stable formulation as described herein in an amount effective to treat the disease, condition, or disorder. In some embodiments, the subject is administered a stable formulation comprising the compounds claimed herein. The disease, condition, or disorder can be caused by an influenza virus. Further, the disease, condition, or disorder can be influenza, the flu, and/or a disease with flu like symptoms and related diseases, conditions, and disorders. For prophylactic administration, the composition can be administered to a patient at risk of developing one of the previously described conditions.

[0097] The amount of composition administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, etc. Determination of an effective dosage is well within the capabilities of those skilled in the art. In some aspects of the invention, total dosage amounts of a compound composition will typically be in the range of from about 0.0001 or 0.001 or 0.01 mg/kg of patient/day to about 100 mg/kg patient/day, but may be higher or lower, depending upon, among other factors, the activity of the components, its bioavailability, the mode of administration and various factors discussed above. Dosage amount and interval can be adjusted individually to provide plasma levels of the compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds can be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. Skilled artisans will be able to optimize effective local dosages without undue experimentation.

I. Secondary and Combination Treatments

[0098] Certain embodiments provide for the administration or application of one or more secondary or additional forms of therapies or preventative interventions. The type of therapy is dependent upon the type of disease that is being treated or prevented. The secondary form of therapy may be administration of one or more secondary pharmacological agents that can be applied in the treatment or prevention of a viral infection.

[0099] If the secondary or additional therapy is a pharmacological agent, it may be administered prior to, concurrently, or following administration of the compound of Formula I. The interval between administration of the compound of Formula I and the secondary or additional therapy may be any interval as determined by those of ordinary skill in the art. For example, the compound of Formula I and the secondary or additional therapy may be administered simultaneously, or the interval between treatments may be minutes to weeks. In embodiments where the agents are separately administered, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that each therapeutic agent would still be able to exert an advantageously combined effect on the subject. For example, the interval between therapeutic agents may be about 12 h to about 24 h of each other and, more preferably, within about 6 hours to about 12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. In some embodiments, the timing of administration of a secondary therapeutic agent is determined based on the response of the subject to the compound of Formula I.

[00100] Examples of secondary treatments useful with methods disclosed herein are: anti -viral drugs, inhibitors of viral binding to a host cell, inhibitors of viral entry into a host cell, inhibitors of budding of virus from a host cell, inhibitors of release of a virus from a host cell, inhibitors of replication in a host cell, virucides, anti-influenza drugs such as, but not limited to, oseltamivir, zanamivir, rimantadine, amantadine, peramivir, or salts thereof. Further, secondary treatments can include treatments for the symptoms of a viral infection or the complications caused by a virus infection, such as fluid replacement therapy, fever reducers, pain reducers, etc. Secondary treatments can also include dietary supplements such as vitamins C, E and D, calcium, zinc, selenium, curcumin, folate, bioflavonoids, resveratrol, and plant extracts.

J. DART-TOF Mass Spectrometry

[00101] In a non-limiting example, the compounds of the present invention can be identified using Direct Analysis in Real Time (DART) Time of Flight/Mass Spectrometry (TOF/MS). Specifically, a JEOL DART™ AccuTOF-mass spectrometer from Jeol USA of Peabody, MA (JMS-T100LC) can be used. Accurate mass can be determined by subtracting the mass of a proton (1.007825 amu) from the measured mass of the ions produced from the sample. The mass of compounds may be determined in a sample by directly introducing the sample to the ion stream by means of a Dip-IT sampler and a Dip-IT sampler holder (ionSense™). While no sample preparation is required for a simple analysis with the DART, a chemical doped/spiked solution can be used for quantitation relative to a known quantity. As a non-limiting example, curcumin is not present in elderberry extract and can therefore be used to create a quantitative chemical profile of the bioactive molecules. The settings for the DART ion source can be the following:

Gas: He

Flow: 2.52 LPM @ 50 PSI

Temperature: 250 C

Needle Voltage: 3000V

Grid Electrode Voltage: 250V

Discharge Electrode Voltage: 400V

The settings for the JEOL AccuTOF MS can be the following:

Peaks Voltage: 1000V

Orifice 1 Temperature: 120 C

Detector Voltage: 2600V

Reflectron Voltage: 990.0V

[00102] Samples can be analyzed in six replicates by DART-TOF MS. These six replicates can be analyzed to create a single, averaged, filtered, and statistically significant DART fingerprint of the sample. This processed fingerprint can then be used to determine the presence of the markers by comparison of masses. A simple mass comparison can be sufficient to determine the presence of a target compound in any extract or mixture of chemicals. [00103] All MS have a mass tolerance - a range of acceptable reported masses surrounding the predicted [M+H] or [M-H] value. For the AccuTOF, that mass tolerance is less than 20 millimass units (mmu) (predicted mass +/-10 mmu). Given the same sample and ion source, other TOF-MS may have a higher or lower mass tolerance.

[00104] In another non-limiting example, the compounds of the present invention can be determined by DART TOF/MS by using a JEOL DART™ AccuTOF-mass spectrometer from Jeol USA of Peabody, MA (JMS-T100LC) executed in the positive ion mode ([M+H] ⁺ ) using the following settings for the DART ion source:

Gas: He

Flow: 3.98 L/min

Needle voltage: 3500 V

Temperature: 300 °C

Electrode 1 Voltage: 150 V

Electrode 2 Voltage: 250 V,

The settings for the JEOL AccuTOF MS can be the following: Peaks Voltage: 1000 V

Orifice 1 Voltage: 20 V

Ring Lens Voltage: 5 V

Orifice 2 Voltage: 5 V

Detector Voltage: 2550V

[00105] Calibrations can be performed internally with each sample using a 10%

(weight/volume) solution of PEG 600 from Ultra Chemical of North Kingston, RI that provided mass markers throughout the required mass range of 100-1000 amu. Calibration tolerances can be held to 5 mmu. Samples can be introduced into the DART He plasma using the closed end of a borosilicate glass melting point capillary tube until a signal is achieved in the total-ion chromatogram (TIC). The next sample can then be introduced when the TIC returned baseline levels. K. Kits

[00106] In another aspect of the present invention, kits for treating or preventing a disease, condition or disorder as described herein. For instance, compositions of the present invention can be included in a kit. A kit can include a container. Containers can include a bottle, a metal tube, a laminate tube, a plastic tube, a dispenser, a straw, a pressurized container, a barrier container, a package, a compartment, or other types of containers such as injection or blow-molded plastic containers into which the dispersions or compositions or desired bottles, dispensers, or packages are retained. The kit and/or container can include indicia on its surface. The indicia, for example, can be a word, a phrase, an abbreviation, a picture, or a symbol.

[00107] The containers can dispense a predetermined amount of the composition. In other embodiments, the container can be squeezed (e.g., metal, laminate, or plastic tube) to dispense a desired amount of the composition. The composition can be dispensed as a spray, an aerosol, a liquid, a fluid, a semi-solid, or a solid. In a preferred embodiment, the composition is dispensed as a tablet or lozenge. The containers can have spray, pump, or squeeze mechanisms. A kit can also include instructions for employing the kit components as well the use of any other compositions included in the container. Instructions can include an explanation of how to apply, use, and maintain the compositions. The compositions can, if desired, be presented in a pack or dispenser device, which can contain one or more unit dosage forms containing the compound compositions. The pack can, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

EXAMPLES

[00108] The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. EXAMPLE 1

(SAFETY)

[00109] The compound of Formula I was tested to determine genotoxicity and safety for oral administration in vivo. It was found that the compound did not increase levels of genetic mutations in a bacterial reverse mutation test (Ames). Further, it was found that the compound showed no effect when orally administered to rats (Acute Oral Toxicity Test) at doses up to 2.0 g/kg body weight.

[00110] Mutagenicity: Briefly, the Ames Test used multiple histidine auxotrophic Salmonella typhimurium bacterial strains and multiple tryptophan auxotrophic Escherichia strains that contain frameshift or point mutations causing the histidine or tryptophan auxotroph characteristic. The bacterial strains are cultured in the presence of a small amount of histidine or tryptophan, respectively, with/without the compound of Formula I. The mutation rate of the background (no compound of Formula I) and the test concentrations of the compound are determined by the number of bacteria that continue to proliferate after the histidine or tryptophan in the culture is depleted. The ability to proliferate after histidine or tryptophan is depleted indicates that mutations were induced in the bacteria that corrected (reverse) the frameshift or point mutations causing the auxotrophic characteristics in the parent strains. The rate at which the bacteria reverse in the presence of test compound is compared to the background reversion rate. An increase in reversion over background indicates that a compound may be a potential mutagen.

[00111] Toxicity in vivo: Briefly, five female rats were administered 2000 mg/kg body weight of the compound of Formula I by intragastric intubation. The rats were observed for 14 days and signs of death or other forms of toxicity were recorded.

[00112] Results: The compound of Formula I showed not mutagenicity at concentrations up to 1 mg/100 μΐ. Also, the compound of Formula I showed no signs of toxicity at 2000 mg/kg body weight in the Acute Oral Toxicity Test. EXAMPLE 2

(TREATMENT AND PREVENTION OF ZIKA INFECTION IN VITRO)

[00113] Inhibition and prevention of Zika virus infection by the compound of Formula

I in vitro was determined. It was determined that the compound of Formula I inhibits Zika virus infection in cell culture.

[00114] Zika Infection Inhibition: A viral plaque reduction neutralization test (PRNT) was used to determine prevention of viral infection. Briefly, the compound of Formula I was dissolved in 200 μΐ DMSO and then diluted in Dulbecco' s Modified Eagle Medium (DMEM), pH 7.2. Plaque forming units (PFU) of Zika virus strain were incubated with the compound of Formula I or control dilutions for 1 hour at room temperature. The PFUs were then allowed to infect confluent African green monkey fibroblast-like kidney cells (Vero E6 cells) for 1 hour at room temperature. The plaques were visualized by staining with neutral red.

[00115] Results: Pre-incubation of virus with the compound of Formula I decreased the PFUs bound to and/or found in Vero cells over virus not pre-exposed to the test compound. See Fig. 1. In vitro the compound of Formula I had an ICso value for Zika virus of approximately 3.0 μg/ml (8.37 μΜ). Further, 100% inhibition of Zika infection was achieved at 8 μg/ml.

EXAMPLE 3

(TREATMENT AND PREVENTION OF ZIKA INFECTION IN Vrvo)

[00116] Inhibition and prevention of Zika virus infection by the compound of Formula

I in vivo was determined. It was found that the compound of Formula I inhibits Zika virus infection in vivo. Specifically, for mice infected with Zika virus, treatment with the compound of Formula I delayed mortality in a dose dependent manner, delayed the onset of symptoms of the Zika infection, delayed weight loss due to the Zika infection, and decreased the Zika viral load.

[00117] Briefly, two groups of AG129 mice, each containing six mice received two daily oral doses of the compound of Formula I ("Formula I") for 9 days at a concentration of 10 and 30 mg/kg, respectively. On Day 0, these animals and six vehicle treated mice were challenged with a single subcutaneous dose of lxlO ⁵ PFU of ZIKV strain PRVABC59. The viral loads in the Formula I treated mice were compared with that in the vehicle treated mice. The mice were also observed daily for signs of morbidity including weight loss.

[00118] Materials: Inoculum - ZIKV, PRVABC59 Strain, 1.Ox 10 ⁷ PFU/mL viral titer, manufactured by Southern Research. Viral Diluent - VP-SFM (GIBCO) with 10v/v% FBS (SIGMA) (formulated at Southern Research). Test solution of Formula I - prepared fresh daily and diluted to the appropriate concentration with pure cottonseed oil (Acros Organics). Mice - AG129 (IFN-a y/pR-/-) from Marshall BioResources, 8-9 weeks of age at the start of the study and weighing 17.4-26.2 g. 9 male mice and 9 female mice were used. The mice were quarantined for 5 days prior to the study to determine general health and acceptability for use in the study.

[00119] Methods: Study Design Overview - Prior to Study Day -1, eighteen (18) AG129 mice were randomized into respective groups of six animals each (3 female/3 male) according to sex/weight using Provantis Software as outlined in Table 1. On Study Day -1, Groups 1 and 2 received an oral administration of Formula I twice daily at doses of 10 mg/kg and 30 mg/kg, respectively. Group 3 received a mock administration (vehicle treatment). On Study Day 0, all animals were subcutaneously inoculated with lxlO ⁵ PFU of ZIKV strain PRVABC59 as specified in Table 1. All animals were monitored twice daily for mortality for 21 days and underwent clinical observations from Study Days 9-17. Weights were recorded daily and animals with a >30% weight loss (compared to Day 0) were euthanized. SST blood was collected according to the time points in Table 2, or upon moribund euthanasia, and viral load was measured using ZIKV specific RT-qPCR. Upon euthanasia, brain, kidney, spleen, and liver were harvested and stored at -70°C or below until shipment.

[00120] Table 1. Study Design

*Animals were subcutaneously inoculated with 0.2mL of 5x10 PFU/mL ZIKV [00121] Table 2. Key Study Procedures

¹ Tissue Harvest was performed on unscheduled/scheduled euthanized animals.

2 Maximum volume.

[00122] Preparation of Challenge Virus - The virus stocks were thawed in a 37 ± 1°C waterbath, vortexed, and diluted with virus diluent to yield a concentration of 5xl0 ⁵ PFU/mL. The final inoculum volume was 0.2 mL per animal and was administered by subcutaneous injection. Each syringe was filled with 0.2 mL of virus inoculum and kept on ice until transferred to the animal facility for dosing. An aliquot of the virus inoculum was taken to be back-titrated by standard plaque assay to confirm the actual delivered dose.

[00123] Preparation of Formula I - Formulations and dilutions of the compound of Formula I were prepared fresh each day.

[00124] Blood Processing - Collected whole blood samples were processed to serum and stored at -70°C or below until testing. [00125] Formula I Administration - On Study Days -1 through 7, mice in Groups 1 and 2 were administered with O. lmL of a Formula I per dose via oral gavage. Dosing was performed twice daily 8 hours apart (+/- 15 minutes) at the concentrations specified in Table 1. Exact dosing information was captured using Provantis software. [00126] Virus Challenge - On Study Day 0, the injection site was wiped with alcohol and all mice were inoculated subcutaneously with 0.2 mL of wild type Zika virus at a challenge dose of l .Ox lO ⁵ PFU per animal as outlined in Table 1.

[00127] Clinical Observations - Between Study Day 9 and moribund euthanasia, all surviving animals were observed for clinical illness or changes in behavior once daily and more frequently if necessary. Expected signs included rough coat, hunched posture, lethargy, abnormal respirations, paralysis, and skin lesions. Clinical observations were recorded in Provantis.

[00128] Weight - For all animals, weight was determined at the time points indicated in

Table 2. Body weight data was recorded in Provantis. [00129] Blood Collection - On Study Day 6, all animals were bled approximately 200- 250 uL into SST tubes via submandibular vein puncture. Terminal blood was collected from all mice at moribund euthanasia via cardiac puncture and the maximum amount of blood was collected to a volume not to exceed 1.0 mL.

[00130] Euthanasia/Tissue Harvest - All animals were euthanized or found dead during the study. Euthanasia was performed by asphyxiation with CO2. A combination of weight loss (less than 30%), dehydration, lethargy, partial body paralysis, and signs of any other unforeseen distress and/or pain, constituted acceptable endpoints for early euthanasia. Any animal that was observed to have a weight loss of equal to or greater than 30% from baseline level (Study Day 0) was euthanized. Following euthanasia, tissues were collected. [00131] Tissue Collection - Tissue samples were collected from animals upon moribund euthanasia (tissues were not collected from animals found dead). Following euthanasia, brain, kidney, liver, and spleen were aseptically removed. Tissues were immediately frozen on dry ice and stored at -70°C or below until shipment. [00132] Challenge Inoculate Back Titration - An aliquot of the challenge inocula was back-titrated by standard plaque assay on Vero cells to confirm the actual delivered dose.

[00133] Viral Load Determination By RT-qPCR - Viral load was measured in serum samples using Zika specific RT-qPCR. Viral RNA was isolated from serum using the QIAamp Viral RNA mini kit (Qiagen, #52906) and stored at -20°C or below until testing. Serially diluted, quantified RNA was used to generate a standard curve that allows for the absolute quantitation of viral RNA in each sample. All RT-qPCR reactions were run on the QuantStudios 6 using the standard cycle, under the following conditions: 53°C for 5 min, 95°C 1 min. followed by 45 cycles of 95°C for 5 seconds and 60°C for 50 seconds. [00134] Results: Briefly, survival analysis revealed that all mice had 100% mortality in all experimental groups with a geometric mean survival of 8.9 days for the vehicle treated group as opposed to 12.4 days for 10 mg/kg and 13.5 days for 30 mg/kg Formula I treated groups. Delayed mortality was evident in the Formula I treated animals. There was around a 4 and 12 fold reduction in viral load in the 30 mg/kg and 10 mg/kg Formula I treated mice, respectively, at the time of euthanasia. In summary, at the tested concentrations of 10 and 30 mg/kg, Formula I treatment resulted in delayed mortality and a reduction in viral load.

[00135] Mortality - During the study period, all mice were monitored twice daily for signs of mortality and morbidity. Mortality rates and survival times for each group are shown in Table 3 and the percent surviving mice per group is shown in FIG. 2. One mouse each from the 10 mg/kg and 30 mg/kg treated groups were found dead on Day 3 without significant weight loss. These animals were excluded from the mean survival analysis. Mouse 11012 (vehicle treated group) was found dead on Day 6 but was included in the analysis as a steady decline in weight was observed prior to the animal being found dead. Animal 10996 (vehicle treated) was also included in the analysis despite being found dead on Day 8 as the animal had a significant viral load on Day 6. The in-life phase was completed by Day 16 with a geometric mean survival of 8.9 days for the vehicle treated group, 12.4 days for the 10 mg/kg and 13.5 days for the 30 mg/kg treated animals. This represents an increased lifespan of 39.3% and 51.7% in 10 mg/kg and 30 mg/kg treated animals, respectively, over vehicle control animals. In summary, there was a delay in mortality in Formula I treated animals as compared to vehicle treated animals. [00136] Table 3. Mortality Following Challenge

11013 Found dead on day Mildly rough coat; mild lethargy; Hunched 13

13 posture; Anorexic

Group 3: Vehicle Treatment; ZIKV Challenge lxlO ⁵ PFU

10996 Found dead on day 8 N/A 8

11004 Moribund euthanasia Dyspnea; Eye closed; Hunched posture; 13

on day 13 Tremors when moving; Immobility;

Anorexic; Lethargic; Rough coat; Weakness

in both hind limbs

10998 Moribund euthanasia Mildly rough coat; Mild dyspnea; Hunched 12

on day 12 posture; Immobility

11005 Moribund euthanasia Dyspnea; Hunched posture; Squinting; 8

on day 8 Mildly lethargic; Rough coat

11012 Found dead on day 6 -21.9% weight loss (Post Challenge Day 6) 6

11009 Moribund euthanasia Dyspnea; Edema; Swelling; Eye sunken; Eye 8

on day 8 closed; Hunched posture; Mild ocular

discharge; Lethargic; Rough coat

[00137] Body Weights and Clinical Observations - Prior to and following challenge, mice were examined for signs of morbidity including weight loss. The daily weight change for each mouse is illustrated in FIG. 3 A-C. For each group, mean of the percentage body weight change from Day 0 is illustrated in FIG. 4. The percent body weight change for individual mice from Day 0 is depicted in Table 4. Individual animals within groups differed on the time of weight loss onset, but most animals experienced a steady decline in weight over multiple days during the study. In general, animals in the vehicle treated group experienced an earlier onset of weight loss than those in the Formula I treated groups. Overall, the difference in weight loss patterns observed between these groups is in accordance with their mortality data.

[00138] In most cases, weight loss preceded other signs of infection. In addition to weight loss, animals experienced a range of clinical signs including rough coat, lethargy, hunched posture, dyspnea, ocular discharge, stiffness, weakness in limbs, and occasionally, limb paralysis. A few exceptions were animals 10997, 11008 and 10996, all of which were found dead but did not exhibit appreciable levels of weight loss. In contrast, animal 11012 was found dead on Day 6 after showing consistent weight loss over a period of 6 days. [00139] Table 4: Body Weight Change (in %) from Study Day 0

Table 4: Body Weight Change (in %) from Study Day 0 (continued)

[00140] Viral Load by RT-qPCR - Blood was collected on Day 6 and at the time of moribund euthanasia and processed for serum. Blood was not collected from animal 10997 (10 mg/kg treated group) and 1 1008 (30 mg/kg treated group) both of which were found dead prior to initiation of clinical observations. Blood was also not collected from 10995 (lOmg/kg treated group), 1 1013 (30 mg/kg treated group), and 10996 and 1 1012 (vehicle treated group) at the time of euthanasia, as these mice were found dead before serum could be collected. RNA extracted from the serum was subjected to quantitative RT-PCR (RT-qPCR) analysis to determine the absolute ZIKV, PRVABC59 genome copy numbers per mL of serum. The results of the RT-qPCR analysis are presented in Table 5.

[00141] RT-qPCR analysis of genome copy equivalents/mL (GEq/mL) on Day 6 revealed high viral load in all ZIKA PRVABC59 infected animals, with the individual viremia ranging from 7.70E+06 to 8.18E+07 (geometric mean of 2.20E+07) GEq/mL for the 10 mg/kg Formula I treated group, 5.95E+06 to 4.60E+07 (geometric mean of 1.82E+07) GEq/mL for the 30 mg/kg Formula I treated group and 1.54E+06 to 3.71E+07 (geometric mean of 1.28E+07) GEq/mL for the vehicle treated animals. A decline in viral load was evident in all 3 groups at the time of moribund euthanasia (compared to Day 6) but a majority of animals exhibited clinical manifestation of Zika infection. Individual viral load at this time ranging from 1.03E+03 to 8.22E+03 (geometric mean of 3.56E+03) GEq/mL for the 10 mg/kg Formula I treated group, 8.03E +03 to 1.64E+04 (geometric mean of 1.1 1E+04) GEq/mL for the 30 mg/kg Formula I treated group and 2.63E+03 to 8.52E+05 (geometric mean of 4.32E+04) GEq/mL for the vehicle control group.

[00142] Table 5. Serum Viral Load

10999 M Day 1 1 E 4.60E +07 8.03E +03

1 1002 M Day 15 E 2.57E +07 1.64E +04

1 1003 M Day 15 E 3.66E +07 1.29E +04

1 1007 F Day 14 E 7.65E +06 9.07E +03

1 1008 F Day 3 D N/A N/A

1 1013 F Day 13 D 5.95E +06 N/A

Geometric Me u 1 ik 1.82E+07 I .1 1 +04

Group 3 : \ ehicle ZIKV Challenge 1x10 ⁵ PFU

10996 M Day 8 D 1.76E +07 N/A

1 1004 M Day 13 E 1.41E +07 9.64E +03

10998 M Day 12 E 2.44E +07 2.63E +03

1 1005 F Day 8 E 3.71E +07 8.52E +05

1 1012 F Day 6 D N/A N/A

1 1009 F Day 8 E 1.54E +06 1.61E +05

Geometric Me u l it T 1.28E+07 4.32E+04

N/A = Not applicable, animal found dead therefore no sample.

[00143] Discussion and Conclusions - The purpose of this study was to evaluate the anti -viral potency of Formula I in AG129 mice challenged with a lethal dose of ZIKA PRVABC59. In this study, all mice had succumbed to infection within 16 days. Delayed mortality was evident in the Formula I treated animals. In accordance with the delay in mortality, there was around a 4 and 12 fold reduction in viral load in the 30 mg/kg and 10 mg/kg Formula I treated mice, respectively, at the time of euthanasia.

EXAMPLE 4

(TREATMENT AND PREVENTION OF DENGUE INFECTION)

[00144] Treatment and prevention of dengue virus infection by the compound of

Formula I was determined in vivo using a mouse model. It was determined that the compound of Formula I prevents dengue viral infection, treats dengue viral infection, and reduces dengue viral titer in vivo.

[00145] Dengue Infection Inhibition: A mouse model of dengue viral infection was used to determine if dengue virus infection can be prevented and treated by the compound of Formula I. Briefly, C57BL/7 mice were either not treated or treated twice daily with 5 mg/kg or 10 mg/kg of the compound of Formula I at -2, -1, 0, +1, and +2 days relative to infection with highly adapted mouse dengue virus-1 (DENV-1). 13 mice were used for each treatment group. Survival and weight of the mice were recorded.

[00146] Dengue viral titer: One mouse from each of the untreated and treatment groups in the dengue infection inhibition experiment described above was sacrificed at day 19 relative to infection and used to determine dengue virus titer in the mouse.

[00147] Results: Mice infected with DENV-1 that did not receive any treatment survived an average of 19 to 20 days post infection. (See FIG. 5). Infected mice that received the compound of Formula I survived up to an average of 6 days longer than the untreated mice (see FIG. 5) and did not lose weight in comparison with the untreated mice (not shown). Viral titer in the sacrifice mouse from each group was decreased at day 19 in mice treated with the compound of Formula I in comparison to the untreated mouse. (See FIG. 6). These results suggest that the compound of Formula I is well tolerated and can prevent and treat dengue virus infection.

EXAMPLE 5

(TREATMENT AND PREVENTION OF CHIKUNGUNYA INFECTION)

[00148] Treatment and prevention of chikungunya virus infection by the compound of

Formula I was determined in vivo using a mouse model. It was determined that the compound of Formula I prevents chikungunya viral infection, treats chikungunya viral infection, and reduces chikungunya viral titer in vivo.

[00149] Chikungunya Infection Inhibition: A mouse model of chikungunya viral infection was used to determine if chikungunya virus infection can be prevented and treated by the compound of Formula I. Briefly, C57BL/7 mice were either untreated or treated twice daily with 5 mg/kg, 15 mg/kg, or 30 mg/kg of the compound of Formula I at -2, 0, +2, +4, +6, +8, and +10 days relative to infection with chikungunya virus (CHIKV). 11 mice were used for each treatment group. Survival was recorded.

[00150] Chikungunya viral titer : One mouse from each of the untreated and treatment groups in the CHIKV infection inhibition experiment described above was sacrificed at day 21 relative to infection and used to determine CHIKV titer in the mouse. [00151] Results: Mice infected with CHIKV that did not receive any treatment survived an average of 22 to 23 days post infection. (See FIG. 7). Infected mice that received the compound of Formula I survived up to an average of 12 days longer than the untreated mice (see FIG. 7). Viral titer in the sacrifice mouse from each group was decreased at day 21 in mice treated with the compound of Formula I in comparison to the untreated mouse. (See FIG. 8). These results suggest that the compound of Formula I can prevent and treat chikungunya virus infection.

EXAMPLE 6

(SYNERGY) [00152] Because of the predicted method of action of the compound of Formula I, it is believed that the compound will act synergistically with other antiviral compounds that act through a separate mechanism. To determine synergism with other compounds/compositions, the compound of Formula I disclosed herein can be tested in combination with one or more anti-viral treatments and or drugs. The anti-viral drugs can include one or more anti- influenza drugs. Combination studies can show competitive, additive, or synergistic interactions for treatment and/or prevention of viral infection, cell viability, cellular toxicity, side effects, etc. of the combination in cell culture, animal studies, human studies, etc. Non- limiting examples of studies can include those described above and herein as well as those known to one of skill in the art. As a non-limiting example, the combination of the compound of Formula I and oseltamivir may be tested. As a non-limiting example, the combination of Formula I and oseltamivir is expected to show synergistic activity against at least influenza and flu like illnesses.

[00153] A non-limiting example of a combination assay that can be performed to determine the competitive, additive, or synergistic interactions of a combination can utilize an interaction matrix commonly used to look at drug interactions and synergy. In one instance, the interaction matrix is used in a prevention and/or treatment study of influenza virus infection in cell culture. Briefly, the experiment can have 25 samples: 4 with a first test compound/composition (such as the compound of Formula I) alone, 4 with a second test compound/composition (such as oseltamivir) alone, 1 with no chemistries, and the remaining 16 can be combinations of the first and second test compounds/compositions. 1 :4 dilutions of the first test compound/composition from a starting concentration (such as 1 mg/ml for the compound of Formula I) and 1 :4 dilutions of the second test compound/composition from a starting concentration (such as 1.0 μg/ml for oseltamivir) can be tested. The infection and culture of the influenza virus can occur in the constant presence of the inhibitory compounds. In this way, the experiment simulates a patient infected while on prophylactic treatment and tests prevention of infection by the first test compound/composition alone, the second test compound/composition alone, and the combination of the two at a range of concentrations. The data can be analyzed with the methodology of Berenbaum to determine competitive, additive, or synergistic interactions. (Berenbaum 1977).

[00154] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

Chikungunya: Center for Disease Control and Prevention. November 16, 2015 [cited August 23, 2016].

https://www.cdc.gov/chikungunya/

Dengue: Center for Disease Control and Prevention. January 19, 2016 [cited August 18, 2016].

https://www.cdc.gov/dengue/

Relenza: Consumer Questions and Answers: U.S. Food and Drug Administration. January 25, 2013. [cited 2015 May 22].

http://www.fda.gov/Dmgs/DrugSafety/PostmarketDrugSafetyInfor mationforPatientsa ndProviders/ucml 88870.htm

Zika Virus: Center for Disease Control and Prevention. August 16, 2016 [cited 2016 August 17].

http : //www . cdc . gov/ zika/index . html

Berenbaum M C. (1977) Synergy, additivism and antagonism in immunosuppression. A critical review. Clin Exp. Immunol. 28(1): 1-18.

Fink R, Roschek Jr B and Alberte R S. (2009) HIV type-1 entry inhibitors with a new mode-of-action. Antiviral Chemistry and Chemotherapy 19(6):243-255..

Kong F. (2009) Pilot clinical study on a proprietary Elderberry extract: efficacy in addressing influenza symptoms. Online Journal of Pharmacology and PharmacoKinetics 5:32- 43.

Nagai T, Miyaichi Y, Tomimori T, Suzuki Y and Yamada H. (1990) Inhibition of influenza virus sialidase and anti -influenza virus activity by plant flavonoids. Chemical & Pharmaceutical Bulletin 38: 1329-1332.

Roschek Jr. B, and Alberte R S. (2009) Pharmacokinetics of cyaniding and anti-influenza phytonutrients in an elder berry extract determined by LC-MS and DART TOF-MS. Online Journal of Pharmacology and Pharmacokinetics 4: 1-17. Roschek Jr. B, Fink R C, McMichael M D, Li D and Alberte R S. (2009) Elderberry flavonoids bind to and prevent H1N1 Infection in vitro. Phytochemistry 70(10): 1255- 1261.

Roxas M and Jurenka J. (2007) Colds and influenza: a review of diagnosis and conventional, botanical, and nutritional considerations. Alternative Medicine Review 12:25-48.

Zakay-Rones Z, Varsano N, Zlotnik M, Manor O, Regev L, Schlesinger M and Mumcuoglu M. (1995) Inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract {Sambucus nigra L.) during an outbreak of influenza B Panama. Journal of Alternative and Complementary Medicine 1 :361-369. Zakay-Rones Z, Thorn E, Wollan T and Wadstein J. (2004) Randomized study of the efficacy and safety of oral elderberry extract in the treatment of influenza A and B virus infections. Journal of International Medical Research 32: 132-140.

US 2009/0149530