WO2015051281A1

WO2015051281A1 - Antiviral therapies

Info

Publication number: WO2015051281A1
Application number: PCT/US2014/059109
Authority: WO
Inventors: Thomas E. MORRISON; Matthew FRIEMAN
Original assignee: Morrison Thomas E; Frieman Matthew
Priority date: 2013-10-06
Filing date: 2014-10-03
Publication date: 2015-04-09

Abstract

The disclosure provides methods of preventing, treating or ameliorating a viral infection in a mammal by administering a quinolone to the mammal. The disclosure also provides methods of preventing the transmission of a viral infection in a mammal to another mammal by administering a quinolone to the infected mammal.

Description

ANTIVIRAL THERAPIES

GOVERNMENT INTEREST

This invention was made with Government support under grant number NIH- NIAID U54 AI065357 awarded by the National Institutes of Health (NTH). The U.S. Government has certain rights in the invention.

TECHNICAL FIELD

The disclosure relates to the use of quinolone compounds, pharmaceutical compositions containing the same, and their use in the prevention or treatment of viral infections.

BACKGROUND OF DISCLOSURE

For most healthy individuals, viral infections are irritating, but not generally life- threatening. Many viral infections are successfully overcome by the immune system and treatment of the infection is provided an adjunct to address specific symptoms of the infection. However, viral infectious diseases are a serious concern in developing countries and in immunocompromised individuals.

In developing countries, the lack of adequate sanitation and consequent poor hygiene provide an environment that fosters viral infections. Poor hygiene and nutritional deficiencies may diminish the effectiveness of natural barriers, such as skin and mucous membranes or the ability of the immune system to combat the viral agent.

Additionally, the breakdown of host defenses can also occur due to conditions such as circulatory disturbances, mechanical obstruction, fatigue, smoking, genetic defects, AIDS, bone marrow transplant, cancer, and diabetes. For example, an increasingly prevalent problem in the world is opportunistic infections in individuals who are HIV positive.

Although vaccines may be available to protect against some of viral infections, these vaccinations are not always feasible (due to factors such as inadequate delivery mechanisms and economic poverty) or effective (due to factors such as delivery too late in the infection, inability of the patient to mount an immune response to the vaccine, or evolution of the pathogen). For most viral infectious agents, no vaccines are available. When protection against infection is not possible, treatment of infection is generally pursued, but very few effective antiviral therapies are available in view of the many known viral infectious agents. Chikungunya fever is an emerging, epidemic disease, caused by an arbovirus and transmitted by mosquitoes. The disease has been reported from countries of South and East Africa, South Asia and South-East Asia. Massive outbreaks of Chikungunya fever have occurred in India and in the island countries of the Indian Ocean. Although generally not a lethal disease, high rates of prolonged polyarthritis, leading to considerable disability in a proportion of the affected population, can cause substantial socio-economic impact in infected individuals. Patients with this persistent or chronic phase of arthritis are typically treated with nonsteroidal anti-inflammatory drugs (NSAIDs).

The chikungunya virus (CHIKV) is a member of the genus alphavirus and family Togaviridae. The alphaviruses are small enveloped single-stranded positive-sense R A viruses exhibiting a broad cellular tropism. The viral surfaces are covered in membrane- anchored spikes composed of triplets of heterodimers of the envelope El and E2 glycoproteins. During the course of the alphavirus life cycle, the E2 glycoprotein is responsible for receptor binding. Neutralizing antibodies that recognize epitopes in E2 have been raised against alphaviruses. As with many viral infections, no specific treatment for chikungunya virus infection is available. Currently, vector control in the form of mosquito suppression by insecticide spraying is the only way to prevent and control the outbreaks, and these methods are often ineffective. Accordingly, there is a need for providing therapies that are useful in the prevention and/or treatment of viral infections, like chikungunya.

SUMMARY OF DISCLOSURE

The present disclosure provides molecules that can prevent and treat viral infections, as well as therapeutic uses of these molecules to prevent or slow the growth and/or transmission of viral infection in a mammal.

The coding sequences for individual CHIKV proteins (nsPl, nsP2, nsP3, nsP4, and capsid) were cloned into a yeast expression vector and galactose-inducible expression of each protein was achieved in yeast. The inventors demonstrated that expression of CHIKV nsP2 confers a slow-growth phenotype in yeast on both solid media as well as in liquid media, which was significant in identifying small molecule inhibitors that reverse growth phenotypes in yeast imparted by expression of CHIKV proteins. The inventors next optimized the assay in liquid culture for high-throughput screening, including defining plating conditions, temperature and shaking conditions, and testing the DMSO tolerance of the assay, and showed that the assay could tolerate DMSO concentrations up to 5%, which is important given that the compound libraries are in DMSO. This assay was utilized to screen the Spectrum Collection of approximately 2,000 compounds for the ability to reverse the slow growth phenotype. This screen identified a single compound, ofloxacin, which reproducibly and significantly reversed the nsP2-mediated slow-growth phenotype in yeast. The inventors generated variants of nsP2 with mutations that disrupt either nsP2 helicase activity or nsP2 protease activity, expressed these mutants in yeast, and showed that nsP2-mediated growth inhibition is dependent upon helicase activity. The inventors next developed a screening assay to test the ability of ofloxacin and related fluoroquinolones for the ability to inhibit CHIKV replication in cells. For this assay, the inventors utilized a recombinant CHIKV engineered to express green fluorescent protein (CHIKV-GFP). Vera cells were infected with CHIKV-GFP, treated with increasing doses of fluoroquinolones, and the percent GFP positive cells was quantified by flow cytometry, demonstrating that ofloxacin, levofloxacin, ciprofloxacin, and danofloxacin significantly inhibited CHIKV-GFP replication in a dose-dependent manner. The inventors also demonstrated that treatment of CHIKV-infected cells with levofloxacin dramatically reduced yields of infectious progeny virus. In preliminary experiments, these compounds inhibited replication of Ross River virus, a related alphavirus, but not the replication of respiratory syncytial virus (RSV), a negative-stranded RNA virus that does not encode a helicase.

As there are no specific therapies to treat acute or chronic CHIKV arthritis or any other alphavirus-induced disease, the inventors' identification of new antivirals and their targets will contribute to the strategies used to prevent or control CHIKV disease as well as diseases caused by other emerging alpha viruses. The inventors have thus developed a novel yeast-based HTS screening assay that can be used to identify compounds that target the helicase activity of CHIKV nsP2, which is required for efficient CHIKV replication. Furthermore, the inventors have used this assay to screen a compound library and shown that compounds identified by this screening assay inhibit CHIKV replication in cells. These findings suggest that existing quinolone antibiotics may be useful in the treatment of CHIKV infection in humans.

Thus, the present disclosure provides methods of using quinolone compounds and pharmaceutical compositions containing these compounds to treat or prevent a viral infection.

One embodiment of the disclosure is a method of treating a viral infection by administering to a mammal in need of such treatment, a therapeutically effective amount of a quinolone compound. In a preferred aspect of this embodiment, the compound is at least one of cinoxacin, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, levofloxacin, pazufloxacin, sparfloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, delafloxacin, nemonoxacin, danofloxacin, difloxacin, enrofloxacin, ibafloxacin, marbofloxacin, orbifloxacin, and sarafloxacin.

In a certain preferred embodiments, the quinolone compound is one of ofloxacin, levofloxacin, danofloxacin, and ciprofloxacin. These compounds are available

commercially, in some instances from generic pharmaceutical sources.

In preferred embodiments, the compounds are administered to the mammal as a pharmaceutical composition. Thus, the disclosure also encompasses the administration of these quinolone compounds within pharmaceutical compositions containing one or more of these compounds in admixture with at least one pharmaceutically acceptable carrier.

Another embodiment of the disclosure is a method of preventing or treating a viral infection, by administering a therapeutically effective amount of one or more quinolone compounds, or a pharmaceutically acceptable salt thereof, to a mammal in need of such treatment. Mammals in need of such treatment include those diagnosed with a viral infection and those suspected of having a viral infection. In specific embodiments of the disclosure, the mammal is infected with, or suspected of being infected with, an alphavirus. In specific embodiments of the disclosure, the mammal is infected with, or suspected of being infected with, chikungunya virus (CHIKV). In other specific embodiments of the disclosure the mammal is infected with, or suspected of being infected with, a flavi virus.

Another embodiment of this disclosure is a method of treating a viral infection by administering a therapeutically effective combination of at least one of the quinolone compounds and one or more other known anti- viral or anti-inflammatory treatments. For example, other anti-viral treatments may include administration of one or more of abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balavir, boceprevirertet, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitor, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferons including peg-interferons, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfmavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

Also provided herein are methods for the prevention, treatment or prophylaxis of a viral infection in a mammal comprising administering to the mammal in need thereof, therapeutically-effective amounts of a quinolone compound formulated as a

pharmaceutical composition.

Also provided herein are methods for preventing the transmission of a viral infection comprising administering to a mammal having a viral infection a quinolone compound or a pharmaceutical composition comprising such compounds in an amount effective to prevent transmission of the infecting virus to another mammal.

Also provided herein are pharmaceutical packages comprising a quinolone compound that may optionally be formulated in admixture with at least one

pharmaceutically acceptable carrier. These packages may also include prescribing information describing the administration, and/or use of these quinolone compounds alone, or in combination with other therapies, in the prevention, treatment or amelioration of viral infections. The compounds may be administered separately, simultaneously or sequentially, with other compounds or therapies used in the prevention, treatment or amelioration of a viral infection.

Other aspects of the disclosure will be set forth in the accompanying description of embodiments, which follows and will be apparent from the description or may be learnt by the practice of the disclosure. However, it should be understood that the following description of embodiments is given by way of illustration only since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art and are encompassed within the scope of this disclosure.

BRIEF DESCRIPTION OF DRAWINGS

Figures 1 A and IB show the expression of CHIKV nsP2 in S. cerevisiae results in a slow growth phenotype. Figure 1 A shows the growth of yeast transformed with CHIKV nsP2 is less efficient compared to empty vector control cells. Figure IB shows that, as determined by anti-HA western blot, yeast transformed with CHIKV nsP2 encoding vector express the CHIKV nsP2 protein.

Figures 2A and 2B show the results of high throughput screening to identify ofloxacin as a compound that reverses the CHIKV nsP2-mediated slow growth phenotype in yeast. Figure 2A shows the results of a high-throughput screen (HTS) of a small molecule compound library (Spectrum Collection 2000). Figure 2B shows an anti-HA western blot analysis of yeast treated with DMSO or ofloxacion.

Figure 3 shows that ofloxacin and Levofloxacin inhibit CHIKV replication in vera cells. Figure 4 shows that danofloxacin inhibits CHIKV replication in vera cells.

Figure 5 shows that ciprofloxacin inhibits CHIKV replication in vera cells.

DESCRIPTION OF EMBODIMENTS

The present inventors used an antiviral screening platform (Basu D, et al. Novel influenza virus NS 1 antagonists block replication and restore innate immune function. J Virol. 2009, 83(4): 1881-91; Frieman M, et al. Yeast based small molecule screen for inhibitors of SARS-CoV. PLoS One. 2011; 6(12):e28479) to test 2000 compounds for antiviral activity. To perform this screen, coding sequences for the CHIKV nonstructural protein (nsP) 1, nsP2, nsP3, and nsP4, as well as the capsid protein, were cloned as HA- tagged fusion proteins into a galactose-inducible yeast expression vector. When S.

cerevisiae carrying these constructs were grown in the presence of 2% galactose, each CHIKV protein was expressed. Expression of nsP2 slowed the growth of yeast in liquid culture by 3 -fold at 24 hours post-induction in a highly reproducible fashion and in the presence of the compound solvent DMSO, permitting high-throughput screens for small compounds that reverse the nsP2-mediated slow-growth phenotype in yeast.

This screen identified ofloxacin as reproducibly reversing the slow-growth phenotype in the yeast-based assay. When ofloxacin and related quinolones were tested for their ability to inhibit CHIKV replication in cells, the results demonstrated that quinolone compounds inhibit CHIKV replication in a dose-dependent manner. These data suggest that quinolones, and especially fluoroquinolones, may be useful as antivirals against alphaviruses, which include CHIKV and a variety of other human pathogens.

Thus, the present disclosure is drawn to methods of treating viral infections in a mammal by administering to the mammal in need of such treatment a quinolone compound in an amount effective to treat the viral infection.

A related embodiment provides methods of preventing viral infections in a mammal by administering to the mammal a quinolone compound in an amount effective to prevent contracting of the viral infection.

A related embodiment provides methods of preventing transmission of a viral infection in a mammal by administering to a mammal having a viral infection a quinolone compound in an amount effective to prevent the transmission of the virus to another mammal.

In certain of these embodiments, the viral infection is caused by an R A virus. In certain of these embodiments, the RNA virus is an alphavirus. In certain of these embodiments, the RNA virus is a flavivirus. In certain of these embodiments, the RNA virus is a chikungunya virus. In certain of these embodiments, the viral infection is caused by a virus that encodes a helicase that is essential for viral genome replication, transcription, and/or translation.

As used herein, the term "compound" means a chemical or biological molecule such as a simple or complex organic molecule, a peptide, a protein or an oligonucleotide.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

"Pharmaceutically-acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, or alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically-acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Such conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. Pharmaceutically acceptable salts are those forms of compounds, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically-acceptable salt forms of quinolone compounds are synthesized from quinolone compounds which contain a basic or acidic moiety by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in at page 1418 of Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985.

The term "therapeutically-effective amount" of a compound of this disclosure means an amount effective to inhibit the progression or transmission of viral infection following administration to a mammal having a viral infection.

The quinolone compounds used in making the pharmaceutical compositions of the present disclosure may be purchased commercially. The quinolone compounds, including the salts of these compounds, may also be prepared in ways well known to those skilled in the art of organic synthesis. The compounds may be prepared using the reactions performed in solvents appropriate to the reagents and materials employed and suitable for the transformation being effected. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule must be compatible with the reagents and reactions proposed. Such restrictions to the substituents, which are compatible with the reaction conditions, will be readily apparent to one skilled in the art and alternate methods must then be used.

Also provided herein are pharmaceutical compositions containing quinolone compounds and a pharmaceutically-acceptable carrier, which are media generally accepted in the art for the delivery of biologically active agents to animals, in particular, mammals. Pharmaceutically-acceptable carriers are formulated according to a number of factors well within the purview of those of ordinary skill in the art to determine and accommodate. These include, without limitation: the type and nature of the active agent being

formulated; the subject to which the agent-containing composition is to be administered; the intended route of administration of the composition; and, the therapeutic indication being targeted. Pharmaceutically-acceptable carriers include both aqueous and nonaqueous liquid media, as well as a variety of solid and semi-solid dosage forms. Such carriers can include a number of different ingredients and additives in addition to the active agent, such additional ingredients being included in the formulation for a variety of reasons, e.g., stabilization of the active agent, well known to those of ordinary skill in the art. Descriptions of suitable pharmaceutically-acceptable carriers, and factors involved in their selection, are found in a variety of readily available sources, such as Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985. This disclosure further provides methods of treating a mammal afflicted with a viral infection, or preventing the transmission of such virus in another mammal, which includes administering to the mammal a pharmaceutical composition comprising a quinolone compound. Such compositions generally comprise a therapeutically effective amount of a quinolone compound in an amount effective to prevent, ameliorate, lessen or inhibit the transmission of the viral infection. Such amounts typically comprise from about 0.1 to about 100 mg of the compound per kilogram of body weight of the mammal to which the composition is administered. Therapeutically effective amounts can be administered according to any dosing regimen satisfactory to those of ordinary skill in the art.

Administration may be, for example, by various parenteral means. Pharmaceutical compositions suitable for parenteral administration include various aqueous media such as aqueous dextrose and saline solutions; glycol solutions are also useful carriers, and preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffering agents. Antioxidizing agents, such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or in combination, are suitable stabilizing agents; also used are citric acid and its salts, and EDTA. In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben, and

chlorobutanol.

Alternatively, compositions can be administered orally in solid dosage forms, such as capsules, tablets and powders; or in liquid forms such as elixirs, syrups, and/or suspensions. Gelatin capsules can be used to contain the active ingredient and a suitable carrier such as, but not limited to, lactose, starch, magnesium stearate, stearic acid, or cellulose derivatives. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of time. Compressed tablets can be sugar- coated or film-coated to mask any unpleasant taste, or used to protect the active ingredients from the atmosphere, or to allow selective disintegration of the tablet in the gastrointestinal tract.

A preferred formulation of the disclosure is a mono-phasic pharmaceutical composition suitable for parenteral or oral administration for the prevention, treatment or prophylaxis of a viral infection, consisting essentially of a therapeutically-effective amount of a quinolone compound, and a pharmaceutically acceptable carrier. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn may depend upon crystal size and crystalline form.

Alternatively, delayed absorption of a parenterally-administered drug is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include

poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the therapeutic compounds of the present disclosure.

Formulations of quinolones suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or an oil-in- water or water-in-oil liquid emulsions, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a compound or compounds of the present disclosure as an active ingredient. A compound or compounds of the present disclosure may also be administered as bolus, electuary or paste.

Liquid dosage forms for oral administration of the quinolone compounds include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Formulations of the quinolone compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more quinolone compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Formulations of the present disclosure which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

The ointments, pastes, creams and gels may contain, in addition to an active ingredient, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

For intranasal administration, quinolone compounds may be administered by means of nose drops or a liquid spray, such as by means of a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and Medihaler (Riker).

Drops, such as eye drops or nose drops, may be formulated with an aqueous or nonaqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered by means of a simple eye dropper-capped bottle or by means of a plastic bottle adapted to deliver liquid contents dropwise by means of a specially shaped closure.

The quinolone formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

The dosage formulations provided by this disclosure may contain the therapeutic quinolone compounds, either alone or in combination with other therapeutically active ingredients, and pharmaceutically acceptable inert excipients. The term 'pharmaceutically acceptable inert excipients' includes at least one of diluents, binders, lubricants/glidants, coloring agents and release modifying polymers.

Another embodiment relates to the use of any of the quinolone compounds or compositions in the preparation of a medicament for the inhibition of the growth or transmission of a viral infection in a mammal.

Another embodiment of the disclosure relates to the use of any of the quinolone compounds or compositions for the inhibition of the growth or transmission of a viral infection in a mammal.

The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present disclosure. The examples are not intended to limit the disclosure, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. EXAMPLES

Example 1

A screening platform that can be performed at Biosafety level 2 with large compound libraries, rather than with live virus at Biosafety level 3 which, increases costs and limits feasibility, was used. To perform this screen, coding sequences for the CHIKV nonstructural protein (nsP) 1, nsP2, nsP3, and nsP4, as well as the capsid protein, were cloned as HA-tagged fusion proteins into a galactose-inducible yeast expression vector. When S. cerevisiae carrying these constructs were grown in the presence of 2% galactose, each CHIKV protein was expressed. In addition, we found that expression of nsP2 slowed the growth of yeast in liquid culture by 3 -fold at 24 hours post-induction in a highly reproducible fashion and in the presence of the compound solvent DMSO, permitting high-throughput screens for small compounds that reverse the nsP2-mediated slow-growth phenotype in yeast.

This first example demonstrates that expression of CHIKV nsP2 in S. cerevisiae results in a slow growth phenotype. Yeast were transformed with an empty vector or a vector encoding an HA-tagged CHIKV nsP2 gene under control of a galactose-inducible promoter. Figure 1 A shows that in the presence of galactose, but not glucose, the growth of yeast transformed with CHIKV nsP2 is less efficient compared to empty vector control cells. Figure IB shows that, as determined by anti-HA western blot, in the presence of galactose, but not glucose, yeast transformed with CHIKV nsP2 encoding vector express the CHIKV nsP2 protein.

Example 2

High throughput screening (HTS) was used to identify compounds that reverse the CHIKV nsP2-mediated slow growth phenotype. Figure 2 shows that HTS identified ofloxacin as a compound effectively reversing the CHIKV nsP2-mediated slow growth phenotype.

Figure 2A shows the results of a high-throughput screen (HTS) of a small molecule compound library (Spectrum Collection 2000) was performed with yeast transformed with an empty vector or a vector encoding the CHIKV nsP2 gene under control of a galactose inducible promoter. Yeast were grown in the presence of galactose and treated with DMSO (vehicle control) or compounds (in duplicate). Yeast growth was monitored by optical density (00) over the course of 48 hours. Toxic compounds were removed. Hits were identified as compounds in which both replicates where> 2 standard deviations from the mean. These criteria identified a single compound, ofloxacin, which reproducibly rescued the CHIKV nsP2-mediated slow growth phenotype. Figure 2B shows an anti-HA western blot analysis of yeast treated with DMSO or ofloxacion demonstrates that ofloxacin does not diminish expression of CHIKV nsP2.

Example 3

This example demonstrates that ofloxacin and levofloxacin inhibit CHIKV replication in vera cells. Vera cells were mock-inoculated or inoculated with a CHIKV (moi =0.01) engineered to express green fluorescent protein (GFP) from a second 26S subgenomic promoter (CHIKVGFP). After a one hour adsoprtion, cells were washed and media containing vehicle (NaOH) or various amounts of either ofloxacin or levofloxacin was added to cells. At 24 HPI, the cells were trypinized, fixed in 2% paraformaldehyde, and Figure 3 shows the percent of cells that were GFP-positive, as determined by flow cytometry. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test.

Example 4

This example demonstrates that danofloxacin inhibits CHIKV replication in vera cells. Vera cells were mock-inoculated or inoculated with a CHIKV (moi= 0.01) engineered to express green fluorescent protein (GFP) from a second 265 subgenomic promoter (CHIKV-GFP). After a one hour adsoprtion, cells were washed and media containing vehicle (NaOH) or various amounts of either levofloxacin (Levo) or danofloxacin (Dano) was added to cells. At 24 HPI, cells were trypsinized, fixed in 2% paraformaldehyde, and Figure 4 shows the percent of cells that were GFP-positive as determined by flow cytometry. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test.

Example 5

This example demonstrates that ciprofloxacin inhibits CHIKV replication in vera cells. Vera cells were mock-inoculated or inoculated with a CHIKV (moi =0.01) engineered to express green fluorescent protein (GFP) from a second 265 subgenomic promoter (CHIKV-GFP). After a one hour adsoprtion, cells were washed and media containing vehicle (HCI) or various amounts of cirpofloxacin was added to cens. At 24 HPI, cells were trypsinized, fixed in 2% paraformaldehyde, and Figure 5 shows the percent of cells that were GFP-positive as determined by flow cytometry. Significance was determined by one way ANOVA followed by Tukey's multiple comparison test.

The foregoing examples of the present disclosure have been presented for purposes of illustration and description. Furthermore, these examples are not intended to limit the disclosure to the form disclosed herein. Consequently, variations and modifications commensurate with the teachings of the description of the invention, and the skill or knowledge of the relevant art, are within the scope of the present invention. The specific embodiments described in the examples provided herein are intended to further explain the best mode known for practicing the invention and to enable others skilled in the art to utilize the invention in such, or other, embodiments and with various modifications required by the particular applications or uses of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.

Claims

What is claimed is:

1. A method of preventing, treating or ameliorating a viral infection in a mammal comprising administering to a mammal in need of such treatment, a therapeutically effective amount of a quinolone compound.

2. The method of Claim 1, wherein the compound inhibits a viral helicase enzyme.

3. The method of Claim 1, wherein the compound is a quinolone.

4. The method of Claim 1, wherein the compound is a fluoroquinolone.

5. The method of Claim 1, wherein the compound is a at least one of Cinoxacin, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, levofloxacin, pazufloxacin, sparfloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, delafloxacin, nemonoxacin, danofloxacin, difloxacin, enrofloxacin, ibafloxacin, marbofloxacin, orbifloxacin, and sarafloxacin.

6. The method of Claim 1 , wherein the viral infection is caused by an RNA virus.

7. The method of Claim 1, wherein the viral infection is caused by a flavivirus.

8. The method of Claim 1, wherein the viral infection is caused by an alpha virus.

9. The method of Claim 8, wherein the alphavirus is a chikungunya virus.

10. The method of Claim 1, wherein the quinolone compound is administered orally.

11. The method of Claim 1 , wherein the quinolone compound is administered

parenterally.

12. The method of any one of claims 1-11, wherein the quinolone compound is

administered to the mammal in a pharmaceutical composition.

13. The method of claim 10, wherein the pharmaceutical composition is a mono-phasic pharmaceutical composition suitable for parenteral or oral administration consisting essentially of a therapeutically-effective amount of the quinolone compound, and a pharmaceutically acceptable carrier.

14. A method of preventing transmission of a viral infection in a mammal, comprising administering a therapeutically effective amount of a quinolone compound, or a pharmaceutically acceptable salt thereof, to a mammal having a viral infection.

15. The method of any one of claims 1-14, wherein the compound is administered in conjunction with another anti-viral or anti-inflammatory treatment.

16. The method of claim 15, wherein the other anti-viral treatment is the

administration of at least one of Abacavir, Aciclovir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Balavir,

Boceprevirertet, Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Entry inhibitors,

Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Fusion inhibitor, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine,

Interferons including peg-interferons, Lamivudine, Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nucleoside analogues, Oseltamivir, Penciclovir, Peramivir, Pleconaril, Raltegravir, Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine, Telaprevir, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada,

Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir, and Zidovudine.

17. A pharmaceutical package comprising a quinolone compound and prescribing information for the compound specifying the administration of the quinolone compound for the prevention, treatment or amelioration of a viral infection in a mammal.

18. Use of compound of Claim 5 in the manufacture of a medicament for the treatment of disease Y.

19. Use of a composition of Claim 13 in the preparation of a medicament for the

treatment of a viral infection in a mammal.

20. A compound of Claim 5 for use in the treatment of a viral infection in a mammal.

21. Composition of Claim 13 for use in the treatment of a viral infection in a mammal.

22. A method of treating an individual at risk of a viral infection comprising

administering an effective amount of a compound of Claim 5 to the individual in need thereof.