WO2008034832A1

WO2008034832A1 - Compositions comprising a statin and caffeine for the treatment of viral infection

Info

Publication number: WO2008034832A1
Application number: PCT/EP2007/059862
Authority: WO
Inventors: Patrick T. Prendergast
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-09-18
Filing date: 2007-09-18
Publication date: 2008-03-27
Anticipated expiration: 2009-03-18
Also published as: KR20100005063A

Abstract

The present invention relates to methods for the treatment and/or prophylaxis of a viral infection comprising the steps of (i) providing a therapeutically effective amount of a composition comprising at least one statin and caffeine and (ii) administering the same to a subject in need of treatment. The invention further extends to a pharmaceutical composition for the treatment of a viral infection wherein the composition comprises at least one statin and caffeine along with a pharmaceutically acceptable excipient, carrier or diluent. In a preferred embodiment, the viral infection is Influenza A and the statin is lovastatin hydroxy acid salt.

Description

COMPOSITIONS COMPRISING A STATIN AND CAFFEINE FOR THE TREATMENT OF VIRAL INFECTION

Field of the Invention The present invention relates to compositions comprising combinations of statins and caffeine. The invention further extends to methods for the treatment and prophylaxis of viral infections, and in particular the treatment and/or prophylaxis of type A and type B influenza. The invention further extends to kits containing the compositions of the invention. The compositions and methods of the invention have further utility in the treatment and prevention of Rhabdomyolysis and further in the prevention of damage to lung tissue following viral infection.

Background to the Invention Influenza is one of the most widely spread infections worldwide. In the United States alone, it is estimated that there are between 20,000 and 40,000 deaths each year attributable to infection with Influenza A virus. Further, there is also a risk that the Influenza virus may cause an epidemic or pandemic due to the fast spread of new strains of the virus through the human population.

Influenza viruses are orthomyxoviruses which fall into three types, A, B and C. Influenza A and B virus particles contain a genome of negative sense, single-stranded RNA divided into 8 linear segments. Co-infection of a single host with two different influenza viruses may result in the generation of "reassortant" progeny viruses having a new combination of genome segments derived from each of the parental viruses.

Influenza A viruses can be divided into subtypes according to their surface proteins, haemagglutinin (HA or H) and neuraminidase (NA or N). There are 14 known H subtypes and 9 known N subtypes. Only three H subtypes (H1 , H2 and H3) and two N subtypes (N1 and N2) have been reported as commonly circulating in humans.

Seasonal influenza epidemics in humans are associated with amino acid changes in antigenic sites in the haemagglutinin (H) and neuraminidase (N) proteins, in a process termed "antigenic drift". Major pandemics are associated with the introduction of new haemagglutinin and neuraminidase genes from animal-derived influenza viruses by reassortment into the genetic background of a currently circulating human virus, with this process being termed "antigenic shift".

At present, there are two classes of drugs commercially available for prevention and treatment of influenza virus infections in humans, M2 ion channel blockers and Neuraminidase inhibitors.

Amantadine and Rimantadine function by blocking the ion channel activity of the viral M2 protein, which is mainly required during virus entry in the early phase of the replication life cycle. Both treatments are highly effective in treating influenza A but cause significant side effects on the central nervous system, liver and kidneys. Sensitive influenza strains rapidly develop resistance in vitro and in vivo.

Oseltamivir and Zanamivir block the action of neuraminidase to prevent release of newly formed viruses from infected cells and their spread within the host. Both drugs efficiently inhibited influenza viruses in clinical studies, however escape from the selective pressures of neuraminidase inhibitors has been observed in cell culture and in patients. Many of the current anti-viral therapies are directed towards targeting viral components and are therefore prone to compensatory viral escape mechanisms.

Treatments aimed at manipulating the host to interfere with viral replication, either by enhancing antiviral responses or by inhibiting proviral activities within the host cell, have greater potential to control influenza without selective pressure on the virus itself to mutate in a compensatory manner and are therefore desirable. The possibility for a combination therapy targeting the virus and the host at the same time to minimise the opportunity for the virus to acquire resistance is also particularly appealing.

RIBAVIRIN is a broad-spectrum anti-viral agent based on a purine nucleoside analogue and is the standard treatment regimen for hepatitis C. RIBAVIRIN is known to be active against various RNA viruses by inducing lethal mutagenesis of the viral RNA genome and is known to show anti-viral activity against animal coronaviruses. Although RIBAVIRIN has a marked anti-viral activity against a number of viruses, it is not acknowledged as a medicament for influenza infections. In addition, the considerable toxicity associated with RIBAVIRIN limits its utility as a medicament.

Statins are compounds that are known to have a lowering effect on levels of LDL-cholesterol in the human blood. Statins inhibit the hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, the rate- determining enzymatic step in cholesterol biosynthesis.

Currently available statins include lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin and atorvastatin. Lovastatin (disclosed in US Patent No. 4,231 ,938) and simvastatin (disclosed in US Patent No. 4,444,784 and WO 00/53566) are administered in the lactone form. After absorption, the lactone ring is opened in the liver by enzymatic hydrolysis and the active hydroxy acid is generated.

Pravastatin (disclosed in US Patent No. 4,346,227) is administered as the sodium salt. Fluvastatin (disclosed in US Patent No. 4,739,073) and cerivastatin (disclosed in US Patent No. 5,006,530 and 5,177,080) are also administered as sodium salts and are entirely synthetic compounds that are in part structurally distinct from the fungal derivatives of this class, which contain a hexahydronaphthalene ring. Atorvastatin and two new "superstatins", called rosuvastatin and pitavastatin, are administered as calcium salts.

International patent application No. WO 00/47196 relates to a method for treating a mammal suffering from hepatitis C, HIV, hepatitis B, hepatitis G, or hepatitis H by administering to the patient at least one statin or statin- like compound. However, WO 00/47196 does not consider the utility of said statin compounds in the prophylaxis of viral infections, nor in the prophylaxis or treatment of viral infections other than those listed therein.

Rhabdomyolysis is a rare but potentially life-threatening side effect of treatment with statins wherein the statin compounds can cause skeletal muscle tissue to breakdown. The principal result is a large release of CPK (creatine kinase) enzymes and other cell byproducts into the blood system, this leading to acute renal failure due to accumulation of muscle breakdown products, several of which can cause damage to the kidney. The most common symptoms of Rhabdomyolysis include severe musclular aching and muscle weakness. Treatment is with intravenous fluids, and dialysis if necessary. The present inventor has surprisingly identified a combined medicament comprising at least one statin, or at least one statin precursors, an active metabolite of a statin, or a statin-like compound along with caffeine or a salt, analogue or metabolite of caffeine, this combined medicament having substantial utility in the treatment of viral infections, and in particular influenza. The combination of the statin compound along with the caffeine compound results in a medicament which has a much higher antiviral potency than compositions comprising only a statin compound.

Accordingly, as the components of the combined medicament work in a synergistic manner, the combined medicament provides the further henceforth unforeseen advantage that a lower statin dose can be administered to a subject in order to achieve a therapeutic effect, such as the treatment and/or prevention of a subject with a viral infection. Further, the associated advantage of administering a lower dose of a statin compound has the associated advantage that the risk of side effects which may result from the statin administration, such as Rhabdomyolisis are reduced.

Summary of the Invention

According to a first aspect of the present invention there is provided a method for the treatment and/or prophylaxis of a viral infection, the method comprising the steps of: - providing a therapeutically effective amount of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof, - further providing a therapeutically effective amount of a composition comprising caffeine or a salt, analogue, derivative or metabolite thereof, and

- administering said compositions to a subject in need of treatment.

In certain embodiments, the statin composition may be administered to the subject simultaneously with the caffeine composition. In certain embodiments, the statin composition may be administered to the subject sequentially with the caffeine composition. Where the compounds are administered sequentially, in certain embodiments, the statin composition is administered prior to the caffeine composition. In certain further embodiments, the caffeine composition is administered prior to the statin composition.

In certain embodiments the statin composition is provided separately to the caffeine composition.

In certain embodiments, the statin composition and caffeine composition are co-administered. Co-administration of the statin composition and the caffeine composition means that these components may be administered together as a composition, or as part of the same unitary dose. As used herein, the term "co-administration" can also mean administering the statin composition and caffeine composition separately, but as part of the same therapeutic regimen or treatment program. In certain embodiments, the compositions are administered to a subject at the same time, however the compositions may also be administered separately as separate dosages or dosage forms. Where the compositions are administered separately, the "co-administration" of the compositions does not impose a restriction on the timing, frequency, dosage or order of when the compounds can be administered. In certain embodiments where the statin and caffeine compositions are provided sequentially, simultaneously or separately by different routes of administration. Further, said caffeine and statin compositions may be in the same or different forms, for example a solid and a liquid.

In certain embodiments, the statin composition and the caffeine composition are provided in a combined medicament, for example as a single composition.

In certain embodiments, the statin provided in the statin composition is at least one statin compound or a stain-like compound. The terms "statin compound" and "statin-like compound" embrace analogues, metabolites, derivatives, pharmaceutically active salts, such as hydroxy acid salts, precursors, pro-drugs, enantiomers, and stereoisomers of statins or statin- like compounds.

In certain embodiments, the statin salt is a hydroxy acid salt. In certain embodiments, said hydroxy acid salt is a metabolite of a statin pro-drug which is administered to, and metabolised by, the subject in need of treatment.

In certain embodiments, the statin metabolite results from the metabolism of a compound following administration to a subject.

In certain embodiments, the statin pro-drug is a composition which, following administration to a subject, is metabolised into a molecular or structural form which is active in terms of providing the effects desired by a statin compound. In certain embodiments, the composition is a combined medicament comprising at least one statin compound, pro-drug or metabolite thereof along with caffeine or a derivative, analogue or metabolite thereof.

In certain embodiments, the active metabolite form of the statin is selected from the group comprising, but not limited to, mevastatin, lovastatin, pravastatin, simvastatin, cehvastatin, fluindostatin, velbstatin, fluvastatin, dalvastatin, dihydrocompactin, compactin, atorvastatin, bervastatin, NK- 104, ZD-4522.

In certain further embodiments, the statin compound or statin-like compound may be an optical or geometric isomers, a nontoxic pharmacologically acceptable acid addition salt, an N-oxide, an ester, a quaternary ammonium salt, or a prodrug of any of the above-listed statin compounds.

In certain embodiments, the viral infection results from the infection of a subject by a lipid layered virus. In certain embodiments, the virus is an influenza virus, for example, Influenza type A or Influenza type B.

In certain embodiments where the viral infection infects the lungs, administration of the composition prevents damage to lung tissue caused by an immune response mounted against the viral pathogen.

Although the composition of the present invention may be administered by any specific route, in certain embodiments, the composition or compositions may be administered by the nasal route, for example, using a nebuliser so that the composition is inhaled by the subject. In certain further embodiments, the route of administration is intragastric. Further examples of suitable routes of administration are defined hereinafter. According to a second aspect of the invention there is provided a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof for use in the treatment and/or prophylaxis of viral infection.

In certain embodiments, the composition is a combined preparation or a pharmaceutical composition.

In certain embodiments, said viral infection is Influenza type A or Influenza type B.

In certain embodiments, the composition is a combined medicament. Typically, the statin compound and caffeine compound are administered simultaneously.

In certain embodiments, the composition results from the sequential administration of the statin compound and the caffeine compound to a subject.

A further aspect of the present invention provides the use of a combined preparation comprising (a) at least one statin compound, and (b) caffeine or a salt, analogue or a derivative thereof for the treatment of a viral infection in a subject.

The term "a combined preparation" or "combined medicament" or "combined composition", as used herein defines a "kit of parts" in the sense that the combination partners, i.e. (a) the statin composition, and (b) the caffeine composition, as defined above, can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i.e., simultaneously or at different time points. The parts of the kit of parts can then be administered simultaneously or administered sequentially or separately, for example chronologically staggered at different time points, and with equal or different time intervals for any part of the kit of parts.

Typically the time intervals of the administration of the dosage forms are chosen such that the effect on the treated disease resulting from the combined use of the parts is larger than the effect which would be obtained by use of only any one of the combination partners (a) and (b).

The ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can be varied, for example, in order to cope with the needs of a subject to be treated.

Typically the amount of the statin composition and the caffeine composition is selected such that a synergism results from the administration of the compositions, synergism being an effect which is more than an additive effect resulting from the combination of the statin composition and the caffeine composition. Further advantages may be the reduction in side effects or reduction in effective dosage of one or both compositions.

A further aspect of the present invention provides for use of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof in the preparation of a medicament for the treatment and/or prevention of viral infection.

According to a yet further aspect of the invention, there is provided a pharmaceutical composition comprising at least one statin or statin-like compound, or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof along with at least one diluent, carrier or excipient.

In certain embodiments, the pharmaceutical composition will comprise a statin composition as a primary active ingredient and caffeine as a secondary active ingredient in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, borate-buffered saline containing trace metals or the like. In certain embodiments, the pharmaceutical composition may further comprise one or more excipients, preservatives, solubilizers, buffering agents, lubricants, fillers, or stabilizers.

In certain embodiments, said pharmaceutical composition comprises the statin or the caffeine compound in a pharmaceutically acceptable salt form.

According to a yet further aspect of the present invention, there is provided a process for preparing a pharmaceutical composition comprising at least one statin or statin-like compound along with caffeine or a salt, analogue, derivative or metabolite thereof, said process comprising admixing at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof along with at least one pharmaceutically acceptable diluent, excipient or carrier.

In certain embodiments, the pharmaceutically acceptable diluent, excipient or carrier may be chosen based on the intended route of administration of the resulting pharmaceutical composition. In certain embodiments the composition is formulated in beta-hydroxycyclodextrin.

In various further aspects, the present invention extends to a pharmaceutical pack comprising one or more compartments wherein at least one compartment comprises one or more of the compounds as defined herein, or a composition or combined medicament as defined herein or a medicament as defined herein.

In various further aspects, the present invention extends to a kit of parts for use by a consumer for use in the prevention or treatment of at least one condition selected from the group comprising; a viral infection, for example, influenza type A or influenza type B, lung tissue damage resulting from an immune response mounted against a viral pathogen, and Rhabdomyolysis. Said kits may typically comprise a pharmaceutical composition comprising at least one statin along with a pharmaceutically acceptable carrier, vehicle or diluent and caffeine along with a pharmaceutically acceptable carrier, vehicle or diluent. In certain embodiments, said kit may further comprise instructions providing direction to a subject as to how to administer said pharmaceutical compositions. In certain embodiments, the statin compound and the caffeine compound may be combined in the same pharmaceutical composition.

The inventor of the present invention has further identified that the compositions of the present invention have utility in preventing or ameliorating damage to lung tissue caused by the immune response mounted by a subject in response to a viral pathogen.

Accordingly, a yet further aspect of the invention provides for use of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof in the preparation of a medicament for the prevention of damage to the lungs or to lung tissue caused by an immune response mounted against infection of a subject by a viral pathogen.

In certain embodiments, the composition is a pharmaceutical composition.

A yet further aspect of the invention provides a method for preventing damage to lung tissue of a subject caused by an immune response following the infection of the subject by a viral pathogen, the method comprising administering a therapeutically effective amount of a combined preparation comprising (a) at least one statin or statin-like compound, or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof, and (b) caffeine or a salt, analogue or a derivative thereof to the subject.

A further aspect of the invention provides a composition for use in the prevention of lung tissue damage caused by an immune response mediated by a subject against a viral pathogen, said composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof. Without wishing to be bound by theory, the present inventor predicts that caffeine or a salt, analogue, derivative or metabolite thereof as defined herein has a role in lipolysis, a process which involves calcium receptors. This caffeine molecule functions to synergistically enhance the antiviral mode of action of the statin in the composition.

Furthermore, the inventor has surprisingly found that the risk of developing Rhabdomyolysis, a condition commonly associated with statin administration, is significantly reduced by administering the statin along with caffeine or a salt, analogue, derivative or metabolite thereof.

Accordingly, the inventor has recognised that the synergistic combination of a statin compound and caffeine will also have utility in relation to improving treatment or therapy of a subject who is receiving or being administered a statin compound for the purpose of lowering cholesterol levels. As such, the administration of both a statin compound and caffeine to a patient has the surprising advantage of requiring a lower dosage of statin compound for it to cause a cholesterol lowering effect comparable to a higher daily dosage of statin alone.

Accordingly, a further aspect of the present invention provides for a method for lowering blood cholesterol levels in a subject, the method comprising the steps of:

- providing a therapeutically effective amount of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof,

- further providing a therapeutically effective amount of a composition comprising caffeine or a salt, analogue, derivative or metabolite thereof, and - administering said compositions to a subject in need of treatment.

In certain embodiments, the at least one statin or statin like compound or the analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof is, or forms part of a medicament that is, commercially available for lowering blood cholesterol levels in a subject.

A further aspect of the present invention provides for use of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof in the preparation of a medicament for lowering blood cholesterol levels.

A yet further aspect of the present invention provides a composition comprising a statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof along with caffeine or a salt, analogue, derivative or metabolite thereof for use in the lowering blood cholesterol levels in a subject.

A yet further aspect of the present invention provides for a pharmaceutical composition for use in the reduction of cholesterol levels in a subject, said composition comprising at least one statin compound or a pharmaceutically active salt or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof along with a pharmaceutically acceptable excipient, carrier or diluent.

A further aspect of the present invention provides for a method for the treatment and/or prophylaxis of Rhabdomyolysis, the method comprising the steps of: - providing a therapeutically effective amount of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof, - further providing a therapeutically effective amount of a composition comprising caffeine or a salt, analogue, derivative or metabolite thereof, and

- administering said compositions to a subject in need of treatment.

In one embodiment, the at least one statin or statin-like compound or the analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof is, or forms part of a medicament that is, commercially available for lowering blood cholesterol level.

A further aspect of the present invention provides for use of a combined medicament comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof for the treatment and/or prophylaxis of Rhabdomyolysis.

A further aspect of the present invention provides for use of a therapeutic composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof in the preparation of a medicament for the treatment and/or prophylaxis of Rhabdomyolysis.

A further aspect of the invention provides a combined medicament comprising a statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof for use in the treatment or prevention or Rhabdomyolysis.

A yet further aspect of the present invention provides for a pharmaceutical composition for the treatment and/or prophylaxis of Rhabdomyolysis, wherein said composition comprises at least one statin compound or a pharmaceutically active salt or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof along with a pharmaceutically acceptable excipient, carrier or diluent.

The present inventor has also identified that the therapeutic composition of the present invention comprising a statin or statin pro-drug composition along with caffeine or a salt, analogue, derivative or metabolite thereof has utility when administered to a subject along with a further antiviral compound. Such a composition would provide a combination therapy which may have utility in relation to a viral infection which has a particularly high pathogenicity and prove effective at reducing the development of viral drug resistance.

Accordingly, a further aspect of the present invention provides a method for preventing and/or treating an influenza viral infection, the method comprising the steps of:

- providing a therapeutically effective amount of a composition comprising at least one statin or statin pro-drug composition along with caffeine or a salt, analogue, derivative or metabolite thereof,

- administering same to a subject in need of treatment, and

- further administering a therapeutically effective amount of a secondary antiviral compound. In certain embodiments, the antiviral compound is administered along with the combined medicament composition of the present invention. However, in further embodiments, the antiviral compound may be administered before or after the composition has been administered.

In preferred embodiments, the antiviral compound may be selected from the group comprising ribavirin, amantadine, rimantadine, oseltamivir (TAMIFLU ™) and zanamivir.

According to a yet further aspect of the present invention, there is provided use of a therapeutic composition comprising at least one statin or statin- like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof and an antiviral compound in the preparation of a combined medicament for the treatment or prevention of infection with an Influenza viral infection.

In certain embodiments, the anti-viral compound may be selected from, but is not limited to, the group comprising ribavirin, amantadine, rimantadine, oseltamivir (TAMIFLU ™) and zanamivir.

A yet further aspect of the present invention provides for a pharmaceutical composition for the treatment of an Influenza viral infection in a subject, wherein said composition comprises a composition comprising at least one statin or statin pro-drug composition and caffeine or a salt, analogue, derivative or metabolite thereof and an antiviral compound along with a pharmaceutically acceptable excipient, carrier or diluent.

In certain embodiments, the composition is formulated in beta- hydroxycyclodextrin. In certain embodiments, the anti-viral compound may be selected from the group comprising ribavirin, amantadine, rimantadine, oseltamivir (TAMIFLU ™) and zanamivir.

Description of the Figures

The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention, and further with reference to the figures as described briefly below.

Figure 1 shows a safety profile of lovastatin alone, and in combination with caffeine, in the treatment of H3N2 infection in the cotton rat;

Figure 2 shows a comparison between treatment of H3N2 with lovastatin alone and treatment with other antiviral compounds, namely ZANAMIVIR and OSELTAMIVIR;

Figure 3 shows the percentage of mice infected with H5N1 testing positive for viral detection following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric) or TAMIFLU™ (PCR analysis);

Figure 4 shows the percentage of mice infected with H5N1 showing pathological damage to the lungs following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric) or TAMIFLU™; Figure 5 shows the percentage of mice infected with H5N1 testing positive for viral detection following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric) or TAMIFLU™ (PCR and histopathology analysis);

Figure 6 shows the percentage of mice infected with H3N2 testing positive for viral detection following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), RIBAVIRIN or TAMIFLU ™ (PCR analysis);

Figure 7 shows the percentage of mice infected with H3N2 showing pathological damage to the lungs following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), RIBAVIRIN or TAMIFLU ™;

Figure 8 shows the percentage of mice infected with H3N2 testing positive for viral detection following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), RIBAVIRIN or TAMIFLU ™ (PCR and histopathology analysis);

Figure 9 shows the percentage of mice infected with H1 N1 testing positive for viral detection following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), RIBAVIRIN or TAMIFLU ™ (PCR analysis);

Figure 10 shows the percentage of mice infected with H1 N1 showing pathological damage to the lungs following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), RIBAVIRIN or TAMIFLU ™; Figure 11 shows the percentage of mice infected with H1 N1 testing positive for viral detection following administration of lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), RIBAVIRIN or TAMIFLU ™ (PCR and histopathology analysis);

Figure 12 shows the preventative effect of lovastatin and caffeine compared to that of TAMIFLU ™ following infection with influenza of strain H3N2 (results for controls are also shown - no drug or no virus);

Figure 13 shows the preventative effect of lovastatin and caffeine compared to that of TAMIFLU ™ following infection with H1 N1 (results for controls are also shown - no drug or no virus);

Figure 14 shows the therapeutic effect of lovastatin and caffeine compared to that of TAMIFLU ™ following infection with H3N2 (results for controls are also shown - no drug or no virus); and

Figure 15 shows the therapeutic effect of lovastatin and caffeine compared to that of TAMIFLU ™ following infection with H1 N1

(results for controls are also shown - no drug or no virus).

Detailed Description of the Invention

The present invention relates to the unexpected identification by the inventor that a composition comprising a statin compound and caffeine, when administered to a subject has utility in the prevention and/or treatment of an Influenza viral infection. In certain embodiments, the viral infection may be selected from the group comprising, but not limited to, Influenza virus, Hepatitis C, Hepatitis B, Hepatitis G, Hepatitis H and Respiratory Syncytical Virus (RSV).

Typically, the Influenza virus is a type A Influenza virus, for example avian influenza (or "bird flu"), which may be transmissible to humans. In certain embodiments, the type A Influenza virus has a haemagglutinin component of subtype H1 , H3 or H5. Typically, the type A Influenza virus is selected from the group consisting of H1 N1 , H3N2 and H5N1.

In an alternative embodiment, the Influenza virus is a type B Influenza virus.

In certain embodiments, the viral infection may be caused by infection by a subject with one or more flavivirus or togavirus including, but not limited to: California encephalitis virus, St. Louis encephalitis virus, western equine encephalitis virus, eastern equine encephalitis virus, Colorado tick fever virus, LaCrosse encephalitis virus, Japanese encephalitis virus, yellow fever virus, Venezuelan equine encephalitis virus, Murray valley fever virus, tick-borne encephalitis viruses, GB virus A, GB virus B, GB virus C, Dengue virus 1 , Dengue virus 2, Dengue virus 3, Dengue virus 4, Semliki Forest virus and Sindbis virus.

In certain further embodiments, the viral infection may be derived from a rubivirus, such as human rubella virus, pestiviruses such as mucosal disease viruses, for example, bovine virus diarrhoea virus, hog cholera virus and sheep border disease virus, retroviruses, such as a human immunodeficiency virus, for example HIV1 or HIV2, simian immunodeficiency viruses, a recombinant human simian immunodeficiency virus, a feline immunodeficiency virus, a feline or murine leukaemia virus, a feline or murine sarcoma virus, Rote viral infections in children and marburg viruses.

In certain embodiments, the viral infection may result in a viral skin lesion, such as a cutaneous herpes skin lesion and, as such, the invention extends to the treatment of viral conditions such as herpes simplex, herpes zoster and Molluscum contagiosum.

In certain embodiments, the statin may be at least one statin hydroxy acid salt. In certain embodiments, the statin is lovastatin hydroxy acid salt, for example, lovastatin hydroxy acid sodium salt. Lovastatin hydroxy acid sodium salt is also known by the name mevinolinic acid, the compound being characterised in that it has a molecular weight of 444.54 and a CAS number [75225-50-2].

In certain embodiments, where the statin compound is lovastatin, it is defined by the following chemical structure:

In a further embodiment, the lovastatin hydroxy acid sodium salt, also known by the name mevinolinic acid, has the structural formula:

When reference is made herein to a "statin", this term is intended to embrace the statin in free base form or pharmaceutically acceptable salts of such statins. Furthermore, said statin may extend to an enantiomer or diasterioisomers of said statin compound, wherein the desired functional activity of said statin compound is maintained in that form. Further, when reference is made to a "pro-drug" of a statin, it is further intended to embrace salts and analogues of the pro-drug.

The invention extends to pro-drugs of the compounds of the present invention. As herein defined, a "pro-drug" is a compound which is transformed in vivo to yield a compound of the present invention. The term "pro-drug" may be further expanded to include pro-drug esters, said pro-drug ester being a chemical derivative of the statin compound that is rapidly transformed in vivo to yield the compound, for example by hydrolysis in the blood. Accordingly, in such embodiments, the term "prodrug ester" refers to derivatives of the compound of the present invention formed by the addition of any of several ester forming groups that are hydrolysed under physiological conditions.

In certain embodiments, the statin pro-drug may be selected from the group consisting of, but not limited to: lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin and atorvastatin. In certain embodiments, the statin pro-drug may be a pharmaceutically active salt of lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin or atorvastatin. In certain embodiments, the statin is a hydroxy acid salt of mevastatin, lovastatin, pravastatin, simvastatin, cerivastatin, fluindostatin, velbstatin, fluvastatin, dalvastatin, dihydrocompactin, compactin, atorvastatin, bervastatin, NK-104, or ZD-4522.

In certain embodiments, the statin compound is an enantiomer or stereoisomer of lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin or atorvasta mevastatin, lovastatin, pravastatin, simvastatin, cerivastatin, fluindostatin, velbstatin, fluvastatin, dalvastatin, dihydrocompactin, compactin, atorvastatin, bervastatin, NK-104, or ZD-4522tin.

In certain embodiments, the statin is pravastatin hydroxy acid salt, simvastatin hydroxy acid salt or mevastatin hydroxy acid salt.

In certain embodiments, where the statin compound is mevastatin, it is defined by the following chemical structure:

In certain embodiments, where the statin compound is pravastatin, it is defined by the following chemical structure:

defined by the following chemical structure:

Statins combined with caffeine are not only effective at inhibiting infection by a virus, such as a type A influenza virus, but are also effective in reducing the speed with which the virus spreads to uninfected cells. As the statin / caffeine composition acts to prevent viral entry and exit from infected cells, this serves to increase synergistically the antiviral properties of the composition.

In certain embodiments of the statin compounds provided herein, said statins may comprise a free carboxylic acid or a free amine group. In certain further embodiments, statins for use in the present invention may contain lactone moieties, said lactone moieties existing in equilibrium with the free carboxylic acid form. These lactones may be maintained as carboxylates by preparing pharmaceutically acceptable salts of the lactone. As herein defined, the term "caffeine" as defined herein may be further referred to using its systematic name, 1 ,3,7-thmethyl-1 /-/-puhne- 2,6(3H,7/-/)-dione. Caffeine can also be referred to as 1 ,3,7- trimethylxanthine, trimethylxanthine, theine, mateine, guaranine and methyltheobromine caffeine, and may be defined by the following chemical structure:

The term "compound" is intended to encompass isomeric forms (such as stereoisomers and/or geometric and/or optical isomers and mixtures thereof), chemical derivatives, mimetics, solvates and salts of the compounds.

Some of the compounds/agents of the present invention may exist as stereoisomers and/or geometric isomers, e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all of the individual stereoisomers and geometric isomers of those compounds and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).

The present invention also includes the use of solvate forms of the compounds/agents of the present invention. The terms used in the claims encompass these forms.

The present invention also includes the use of pro-drug forms of the compounds/agents of the present invention. The terms used in the claims encompass these forms. Examples of pro-drugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form the compounds of the present invention which are pharmacologically active.

As defined herein, the term "metabolite" means any substance which results from, or is produced by the metabolism or digestion by a subject of a compound administered to said subject. In pharmaceutical terms, the phrase relates to the product which remains after digestion or metabolism, particularly by the liver, has occurred.

In certain embodiments of the present invention, the statin compound or caffeine compound may be a mimetic. As used herein, the term "mimetic" relates to any compound which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as the statin compound or caffeine compound of the invention.

In certain embodiments of the present invention, the statin compound or caffeine compound may be a derivative. The term "derivative" as used herein includes chemical modification of the statin compound or caffeine compound, said derivative retaining the desired chemical activity of said composition. In certain embodiments of the present invention, the statin compound or caffeine compound may be chemically modified compounds. The chemical modification of a compound or agent may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, van der Waals interaction or dipole interaction between the agent and the target.

The compounds and/or agents of the present invention may be administered as pharmaceutically acceptable salts. In certain embodiments, the invention extends to pharmaceutically acceptable salts of the statin compounds, and in particular to pharmaceutically acceptable salts of the carboxylic acids or amine groups present thereon, these being present where said statin molecule comprises a free carboxylic acid group, or a free amine group.

As herein defined, the expression "pharmaceutically acceptable salts" extends to both pharmaceutically acceptable acid addition salts and pharmaceutically acceptable cationic salts. A "pharmaceutically acceptable cationic salt" includes, but is not limited to, cationic salts such as the alkali metal salts, for example, sodium or potassium, as well as alkaline earth metal salts, for example magnesium, ammonium salts a nd the like. A "pharmaceutically acceptable acid addition salts" includes, but is not limited to salts such as hydrochloride, hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogenphosphate, acetate, succinate, citrate, methanesulfonate (mesylate) and p- toluenesulfonate (tosylate) salts.

In certain embodiments, amorphous forms of the statins may be administered to a subject. Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may b e recovered by evaporation of the solvent.

Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example include those mentioned by Berge et al, in J. Pharm. Sci. 66, 1 -19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, thfluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate/fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p- toluenesulphonate salts.

The compounds and/or agents of the invention may be prepared by chemical synthesis techniques. It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound and/or agent of the invention. This may be achieved by conventional techniques, for example as described in "Protective Groups in Organic Synthesis" by T W Greene and P G M Wuts, John Wiley and Sons lnc.(1991 ), and by P.J. Kocienski, in "Protecting Groups", Georg Thieme Verlag (1994).

It is possible during some of the reactions that any stereocentres present could, under certain conditions, be epimerised, for example if a base is used in a reaction with a substrate having an optical centre comprising a base-sensitive group. It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection/deprotection regimes and the like as these are well known in the art.

The statin compounds or caffeine compounds or related salts for use in the invention may be separated and purified by conventional methods.

As such, as used herein, the term "therapeutically effective amount" means the amount of a composition of the invention which is required to reduce the severity of and/or ameliorate a viral mediated infection or at least one symptom thereof.

As used herein, the term "prophylactically effective amount" relates to the amount of a composition of the invention which is required to prevent the initial infection, replication or recurrence of infection of a subject with a viral pathogen.

As used herein, the term "treatment" and associated terms such as "treat" and "treating" means the prevention of the infection by, or the prevention or reduction of the progression, severity and/or duration of a viral infection mediated by a viral pathogen in a subject, wherein said reduction or amelioration results from the administration of a composition of the invention. The term 'treatment' therefore refers to any regimen that can benefit a subject. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviative or prophylactic effects. References herein to

"therapeutic" and "prophylactic" treatments are to be considered in their broadest context. The term "therapeutic" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylactic" may be considered as reducing the severity or the onset of a particular condition. "Therapeutic" may also reduce the severity of an existing condition.

The statin compounds or caffeine compounds of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutically active agents.

The statin compounds or caffeine compounds, such as the statin hydroxy acid salt or derivatives and metabolites of caffeine may be synthesized or isolated from natural sources. Mevinolinic acid (lovastatin hydroxy acid salt) is a known constituent of red yeast rice. Caffeine may be obtained as an extract from plants rich in this chemical. Alternatively the compounds may be synthesised synthetically.

The combined medicament composition comprising statins or precursors thereof along with caffeine or a salt, analogue, derivative or metabolite thereof may be administered alone, but will preferably be administered as part of a pharmaceutical composition which will generally also comprise a suitable pharmaceutical excipient, diluent or carrier, said excipient, diluent, buffer stabiliser or carrier being selected depending on the intended route of administration. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.

The statin/caffeine combined medicament composition of the invention may be administered to a subject in need of treatment via any suitable route. The precise dose will depend upon a number of factors, including the precise nature of the form of the statin to be administered.

Routes of administration may include, but are not limited to, parenterally (including subcutaneous, intramuscular and intravenous, by means of, for example a drip patch), oral, rectal, nasal, gastric, topical (including buccal and sublingual), infusion, vaginal, intradermal, intraperitoneally, intracranially, intrathecal and epidural administration. In certain embodiments the composition is administered via oral or nasal inhalation. For administration via the oral or nasal inhalation routes, preferably the active ingredient will be in a suitable pharmaceutical formulation and may be delivered using a mechanical form including, but not restricted to an inhaler, nebuliser device or a nasal spray. Further, where the oral or nasal inhalation route is used, administration by a SPAG (small particulate aerosol generator) may be used.

For intravenous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection and Lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

Further forms of oral administration include addition of the composition to animal feed and/or drinking water for administration to animals in the treatment and/or prophylaxis of viral infection in animals.

The statin/caffeine composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.

Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Lippincott Williams & Wilkins; 20th edition

(December 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H. C. et al. 7th Edition ISBN 0- 683305-72-7, the entire disclosures of which is herein incorporated by reference.

The statin along with caffeine or a salt, analogue, derivative or metabolite thereof is preferably administered to a subject in a "therapeutically effective amount", this being an amount sufficient to show benefit to the subject. In particular, the benefit may be the treatment or partial treatment or amelioration of at least one symptom associated with the viral infection. Where the context demands, a "therapeutically effective amount" is an amount which induces, promotes, stimulates or enhances the development of an antiviral response by the subject. In relation to dosage, the actual amount of a composition of the invention administered to a subject, as well as the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. It may be necessary to adjust the dose of a compound when it is administered in the form of a salt, such as a laureate, the salt forming moiety of which has an appreciable molecular weight. Calculation of the dosage amount for other forms of the free base form such as salts or hydrates is easily accomplished by performing a simple ratio relative to the molecular weights of the species involved.

In certain embodiments, the caffeine composition is administered at a higher dose than the statin compound. In certain embodiments, the effective dose of the caffeine composition is up to four times higher than that of the statin compound. In certain embodiments, the effective dose of the caffeine composition is up to eight times higher than that of the statin compound. Accordingly, in one embodiment, the pharmaceutical composition of the invention comprises four to eight times more caffeine than statin compound.

A "subject" in the context of the present invention includes and encompasses mammals such as humans, primates and livestock animals (e. g. sheep, pigs, cattle, horses, donkeys); laboratory test animals such as mice, rabbits, rats and guinea pigs; and companion animals such as dogs and cats. It is preferred for the purposes of the present invention that the mammal is a human. The term "subject" is interchangeable with the term "patient" as used herein.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes' or 'including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

As used herein, terms such as "a", "an" and "the" include singular and plural referents unless the context clearly demands otherwise. Thus, for example, reference to "an active agent" or "a pharmacologically active agent" includes a single active agent as well as two or more different active agents in combination, while references to "a carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.

EXAMPLES

Example 1 : Safety profile of statin (PTPM7) (lovastatin) alone and in combination with Caffeine (C53) against Influenza infection (H3N2) in the cotton rat (S. hispidus)

Materials and Methods

1. Day 0 - Divide 140 young adult cotton rats (6-8 weeks old) into 14 groups of 10 animals. Ear tag, prebleed and treat as indicated. Group Animal

A PTPM74 μg

B PTPM716μg

C PTPM750 μg

D PTPM74 μg / Wuhan 10⁷

E PTPM716μg/Wuhan10⁷

F PTPM750μg/Wuhan10⁷

G PTPM74μg/C53160μg

H PTPM716μg/C53160μg

I PTPM750μg/C53160μg

J PTPM74 μg / C53160 μg / Wuhan 10⁷

K PTPM716 μg / C53160 μg / Wuhan 10⁷

L PTPM750 μg / C53160 μg / Wuhan 10⁷

M Mock /Wuhan 10⁷

N Mock/ Uninfected

Treat each group with 0.1 ml of indicated preparation intranasally.

2. Day 1 - Repeat treatment for all groups.

3. Day 2 - Repeat treatment for all groups.

4. Day 3 -Infect groups D, E, F, J, K, L and M intranasally with 0.1 ml of Influenza A / Wuhan at 10⁷ TCID₅₀.

5. Day 4 - Euthanize 5 animals from each group. Remove the nose and the lung bloc in groups A, B, C, G, H, I and N for histopathology. Remove the nose and lung block in groups D, E, F, J, K, L and M for viral quantification and lung histopathology. 6. Day 7 - Repeat step 5 for the remaining animals.

Results

The results of this example are shown in Figure 1.

Example 2 - Prophylactic treatment with statin alone in the cotton rat (S. hispidus) against Influenza A/Wuhan (H3N2)

Materials and Methods

1. Day 0 - Divide 70 young adult cotton rats (S. hispudus) 6-8 weeks old into 7 groups of 10 animals each. Pre-bleed and ear tag all animals and treat with indicated preparations:

Group Animal #

A Untreated/ Uninfected 67639-648 B Mock treated / Wuhan 10⁷ 67649-658

C Statin dose 50 μg / Wuhan 10⁷ 67659-668

D Statin dose 100 μg / Wuhan 10⁷ 67669-678

E Statin dose 200 μg / Wuhan 10⁷ 67679-688

F Zanamivir 0.5 mg/ml / Wuhan 10⁷ 67689-698 G Oseltamivir 1.75 mg/ml / Wuhan 10⁷ 67699-708

lntranasally inoculate 0.1 ml of indicated preparations in groups C-F and gastric gavage group G with 0.2 ml.

2. Day 1 - Repeat intranasal inoculation of groups C-F and gastric gavage Group G.

3. Day 2 - Repeat intranasal inoculation of groups C-F and gastric gavage Group G. 4. Infect groups B-G intranasally with Influenza A / Wuhan at 10⁷TCID_5O at 0.1 ml volume.

5. Day 4 - Euthanize 5 animals from each group and remove the lung bloc. Trisect for viral quantitation, histopathology and cytokine / chemokine analysis (TNF-α and IFN-γ). Remove the Nasal turbinates for viral quantitation.

6. Day 5 - Repeat step 5 for the remaining animals in all groups.

Results

The results of this example are shown in Figure 2.

Example 3 - evaluation of statin and caffeine combination against H5N1. H3N2 and H1 N1 virus infection in BALB/C mice- Materials and Methods Virus Allantoic fluid from SPF chicken embryos infected with influenza A / Duck / Beijing / 2000 (H5N1 ) was prepared under non-pyrogenic conditions by Specific Pathogen-Free Avian Supply (SPAFAS) and stored at - 80⁰C. H5N1 virus was then purified by ultracenthfugation and suspended in Dulbecco's phosphate-buffered saline with calcium and magnesium (Sigma, St. Louis, MO) plus 0.2% albumin (Sigma). The starting titre of the purified H5N1 virus, free of endotoxin and mycoplasma contamination, was 1 x 10⁸ / ml_ medial tissue culture infectious doses (TCID₅₀) as determined by 50% endpoint dilution in triplicate in Madin-Darby canine kidney cells incubated for 72 hours in the presence of trypsin. Prior to inoculation the virus was diluted 1 :2 in Hank's balanced salt solution containing Ca²⁺ and Mg²⁺ ions (Gibco-BRL). Mice were inoculated intranasally with the virus preparation with 2 x 10⁷ TCID₅₀ in a volume of 100 μl_ / mouse following light ethylether inhalation anaesthesia.

Mice were anaesthetized with ether and exposed to 100 μl_ of the virus by intranasal instillation. In all prevention models, the drug was administered 4 hours before viral infection. In the treatment models, the drug was given at both 24 hours and 48 hours after viral infection. All groups contained 10 mice.

H5N1 : 130 female BALB/C mice (6-8 weeks old, 20-22 g) were randomized into 13 groups (A to M).

H3N2 and H1 N1 : 120 female BALB/C mice (4-6 weeks old, 18-20 g) were randomized into 12 groups (A to L).

Table 1. H5N1 Groups.

Table 2. H3N2 Groups.

All mice were observed daily for changes in mental health, physical appearance and deaths, if any. Mice were sacrificed on day 7 and on day 16 (5 and 14 days post infection) in both H3N2 and H1 N1 studies. This occurred on day 10 and day 14 (9 and 13 days post infection) in the H5N1 experiment. These mice were dissected and tissue examined. Parameters for evaluation of antiviral activity included mental health, physical appearance, mortality, virus load and lung pathology changes determined over the duration of the experiment.

H5N1 Group

Mice were randomized into 13 groups (10 mice per group). Groups A, C, E, G and I were administered intranasally or intragastrically / orally with drugs. Then mice were infected intranasally with 100 μl of Influenza A / Duck / Beijing / 2000 (H5N1 ) at 2^χ10⁷ TCID₅₀ after 4 hours. These 5 groups were the prevention models. Groups B, D, F, H, J and K were infected intranasally with 100 μl of Influenza A / Duck / Beijing / 2000 (H5N1 ) at 2^χ10⁷ TCID₅₀ and were administered with drugs either intranasally or intragastrically 24 hours and 48 hours post infection. These six groups were treatment models. Group L was infected intranasally with 100 μl of Influenza A / Duck / Beijing / 2000 (H5N1 ) at 2^χ10⁷ TCID₅₀ and received no drugs. Group M did not receive any virus or drugs. This group acted as a normal control.

H3N2 Group Mice were randomized into 12 groups (10 mice per group). Groups A, C, E, G and I were administered intranasally or intragastrically with drugs. Mice were then infected intranasally with 100 μl_ of Influenza A / Guangzhou / 2005 (H3N2) at 2 X 10⁶⁵ TCID₅₀ after 4 hours. These 5 groups were the prevention models. Groups B, D, F, H and J were infected intranasally with 100 μl_ of Influenza A / Guangzhou / 2005 (H3N2) at 2 X 10⁶⁵TCID₅₀ Groups B, D, F, H and J were administered intranasally or intragastrically with drugs after 24 hours post infection. This was repeated at 48 hours post infection. These 5 groups were the treatment models. Group L did not receive any virus or treatment. This group acted as a normal control. Group K was infected intranasally with 100 μL of Influenza A / Guangzhou / 2005 (H3N2) at 2 X 10⁶⁵ TCID₅₀ and received no drugs.

H1 N1 Group

Mice were randomized into 12 groups (10 mice per group). Groups A, C, E, G and I were administered intranasally or intragastrically with drugs. Then mice were infected intranasally with 100 μl of Influenza A / Guangzhou / 2005 (H1 N1 ) at 2^χ10⁶ TCID₅₀ after 4 hours. These five groups were the prevention models. Groups B, D, F, H and J were infected intranasally with 100 μl of Influenza A / Guangzhou / 2005 (H1 N1 ) at 2^χ10⁶ TCID₅₀ These groups were then administered with drugs either intranasally or intragastrically 24 and 48 hours later. These groups were the therapy models. Group K was infected intranasally with 100 μl of Influenza A / Guangzhou / 2005 (H1 N1 ) at 2^χ10⁶ TCID₅₀ As group K acted as a virus control group, no drugs were administered at any stage. Group L received no virus and no drugs, acting as a normal control group.

In all three experiments, symptoms were observed three times per day over the course of the experiment. All dead mice (both sacrificed and spontaneous deaths) were dissected and observed for general pathological changes and lungs were removed for RT-PCR and lung pathology tests.

Statin

The statin compound used was lovastatin hydroxy acid salt.

RT-PCR Analysis

Sun Yat-sen Universities RT-PCR is an assay that employs a pair of in- house designed primers and dual labeled fluorescent probes that specifically target conservative region of the HA gene of H3N2. The test was performed according to the protocol as described in Influenza A / H3 Detection kit, which was purchased from Guangzhou Huayin Biomed S&T Co., Ltd, China. This assay is a one-step-PCR system. Briefly, total RNA was extracted from lung tissue by sequential extraction with TriZol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Then viral RNA is used for amplification in the RT-PCR assay.

RT-PCR assay was carried out with a Light Cycler (ABI Prism 7500, USA). The RT-PCR reactions (total volume 25 μl_) contained 20 μL RT-PCR reaction mix, 0.2 μL Taq DNA polymerase, 0.5 μL reverse transcriptase and 5 μL RNA templates. The thermal profile used was initiated at 37°C for 25 minutes, followed by 94°C for 2 minutes, followed by 5 cycles of PCR at 94°C for 15 seconds, 55°C for 15 seconds and 72°C for 20 seconds and followed by 40 cycles of PCR at 94°C for 5 seconds and 55°C for 35 seconds.

Fluorescent signals were obtained once per cycle at the end of the extension step with detectors corresponding to the FAM (530 nm) channels. Data acquisition and analysis of RT-PCR assay were performed using the Rotor-Gene analysis software, Version 6.0 (Corbett research supporting programme). The sample was defined as positive if the threshold cycle (Ct) of the specimen is less then or equal to 30 and the sample was defined as negative if the Ct exceeded 30.

Histopathology of lung tissue

On days 10 and 14 (9 and 13 days post infection), 5 mice were sacrificed from each group in the H5N1 experiment. On days 7 and 16 (5 days and 14 days post virus infection), 5 mice were sacrificed in each group from H3N2 and H1 N1 experiments. A lung pathology assay was performed. A 5 mm lung piece from the ventral end of each left lobe was collected for histopathology. Formalin-fixed, paraffin-embedded lung tissue blocks from the lungs were examined by routine staining with hematoxylin and eosin. The extent of inflammation was graded without knowledge of the variables by two independent investigators. Grading was performed semi- quantitatively according to the relative degree of inflammatory infiltration. The grading was as follows: '-' no inflammation, "+" mild infiltration of inflammatory cells clustered around bronchioles, interstitial capillary extension and congestion and slight exudation in a small amount of alveolus, "++" increased infiltration of large quantities of inflammatory cells clustered around bronchioles, exudation of bronchiole cavity, increased exudation in alveolus and partial consolidation of the lung, "+++" infiltration of large quantities of mononuclear cells in lung tissue, most or all of lung consolidation and bronchus obstruction with inflammatory exudates.

Statistics

Fisher's Exact Test was used to analyze the H5-specific RNA positive rate difference among groups. Chi square test were used to compare the H5- specific RNA positive rate between groups. In all tests, an alpha level of p < 0.05 was taken as an indication of statistical significance. Results

H5N1 Group

All mice were considered mentally normal on day 1 (Table 4). On day 2, all groups were mentally normal. However, one mouse in group E had signs of mental depression and horripilation. This mouse died along with another mouse from the same group. These animals were removed, dissected and examined. No visible pathological change was visible. Day 3 marked an obvious onset of symptoms in group L. This was the virus group and all animals were sluggish, mentally depressed and showed signs of horripilation. Horripilation was also observed in groups B, D, F, G, I and J. Groups A, C, E, H, K and M were mentally normal. However, a further death was discovered in group E. All dead mice were removed, dissected and examined for pathological damage. Groups A, C, E, H, K and M were mentally normal over day 4 to day 7. Horripilation was still seen in groups B, D, F, G, I, J and L, with mental depression observed in group L also. On day 4, another death occurred in group F. Upon examination, no visible pathological change was evident. Over days 8 and 9, groups A, C, E, H, K and M were mentally normal and groups B, D, F, G, I and J have exhibited a decrease in horripilation. Hair returned to normal. Mental depression and horripilation was still present in group L. Day 10 marked a decrease in all symptoms in all groups except group L. All groups were considered mentally normal except the virus group. 5 mice were sacrificed from each group with the exception of group E and F where 3 and 4 mice were sacrificed respectively. An examination of tissues was carried out. The hearts of mice in group L were grey/white in colour, indicative of necrosis and the lungs of this group were an abnormal deep red colour and inflammation was obvious. The heart of one animal in group F was also a grey/white colour and the lungs of another individual of the same group was an abnormal white colour. In groups B, C, D, G and J, the animals' hearts showed evidence of necrosis. There was no pathological damage in groups A, E, H, I, K and M. These tissues were removed for RT-PCR and lung pathology analysis. Days 11 to 13, all mice showed signs of recovery except group L, which had signs of depression and horripilation. These symptoms continued in this group until day 14 when all remaining animals were sacrificed and dissected. There was no visible pathological change evident in any group except group L, where lungs were inflamed and red and hearts were a grey/white colour, again indicating cell death.

Table 4. General observations (H5N1 ).

*Visible Pathological Damage: grey/white hearts indicative of necrosis and abnormal deep red inflamed lungs

Results for PCR analysis for H5N1 are shown in Table 5 and these are summarised in Table 6.

Table 5. PCR analysis (H5N1).

The percentage of mice showing positive viral detection (H5N1 Group) using PCR analysis, following treatment with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric) or TAMIFLU ™, is shown in Figure 3.

Results of the histopathology analysis for H5N1 Group are shown in Table 7. These are summarised in Table 8.

'-' indicates no damgage, '+' slight damage, '++'moderate damage, '+++' severe damage

Table 8. Histopathology summary (H5N1 ).

'-' indicates no damage, '+' slight damage, '++'moderate damage, '+++' severe damage

The percentage of animals with pathological damage is shown in Figure 4 for mice infected with H5N1 and treated with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric) or TAMIFLU™.

Figure 5 shows combined results for the PCR and histopathology analysis for mice treated with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric) or TAMIFLU™.

The significance of these results is highlighted when the comparison with TAMIFLU™ is considered. Not only is the statin/caffeine combination extremely effective at inhibiting the virus, it has also demonstrated that it is iinn ffaacctt aass gg<ood as, and in some cases better, than TAMIFLU™, even at low doses.

In particular, the PCR results for H5N1 have revealed that when administered intranasally as a prevention drug, the statin/caffeine combination is more effective than TAMIFLU™ when administered in the same manner (the virus was detected in 30% of samples, in comparison to 50% of TAMIFLU™ samples). The statin/caffeine combination as a therapeutic when administered intranasally has shown that it is just as effective as TAMIFLU™ (intranasal administration treatment) as both groups resulted in 40% of samples being positive for the virus. It is also important to emphasize that the doses of TAMIFLU™ were higher than that of the statin combination (200μg versus 50μg) and that increasing the doses of the statin combination may therefore increase its efficacy. TAMIFLU™ has proven superior as an intragastric administration treatment. However, preliminary results for H5N1 studies demonstrated that the statin/caffeine combination was extremely effective when this route of administration was used as a prophylaxis (only 10% of samples were positive for virus).

Histopathology results have indicated that the most successful groups were those that were administered with the statin/caffeine combination intranasally as a prevention drug and TAMIFLU™ intragastrically as a treatment. Both groups had less pathological damage than other groups (6 animals per group with no damage). The statin/caffeine combination proved superior to TAMIFLU™ when intranasally administered. The therapeutic effect of the statin/caffeine combination was greater then that of TAMIFLU™. TAMIFLU™ treated groups had a higher percentage of animals with damage to tissue than that of the groups treated with the statin/caffeine combination (Figures 3 to 5 and Tables 5 to 8).

In summary, according to the symptoms, general pathologic changes and RT-PCR analysis, the preventative effect of the statin combination is superior to the therapeutic effect. The effect of 50 μg of statin and 200 μg of caffeine is superior to the effect of 25 μg of statin and 200μg of caffeine. The preventative effects of this statin/caffeine combination is superior to the therapeutic effect of TAMIFLU™. However, the therapeutic effect of the statin/caffeine combination is weaker than that of TAMIFLU™.

H3N2 Group

All mice were considered mentally normal on days 1 and 2 (Table 9). Day 3 marked an obvious onset of symptoms in group K (virus group). Mice appeared sluggish, depressed and horripilation was evident. Horripilation was also observed in groups D and F. All remaining groups were considered normal (A, B, C, E, G, H, I, J and L). On days 4, 5 and 6, horripilation eased in groups D and F. Groups A, B, C, E, H, I, J and L were mentally normal. Group K still exhibited mental depression and horripilation. On day 7, animals in group K remained sluggish and depressed and horhpliation was still evident. All other groups were recorded as being normal with no symptoms evident. On the same day, 5 mice from each group were sacrificed and examined. The hearts of all 5 mice from group K were grey and white, indicative of necrosis and the lungs were considered abnormal as they were a deep red colour and inflammation was clear. Groups A, B, D, F and H exhibited abnormal whitish lungs. No visible pathological change was evident in groups C, E, G, I, J and L. From day 8 to day 15, symptoms remained unchanged in group K, although horripliation eased on day 11. All other groups remained mentally normal with normal coat colour and texture. On day 16, all mice were observed as being mentally normal except groups K. All remaining mice were sacrificed and dissected. The hearts of all mice in group K were grey/white in colour (necrosis) and lungs were an abnormal red colour. In other groups, no visible pathologic change was evident. Lung pathology results revealed the following: in normal control group L, 5 days post infection, no inflammation was observed. In the virus group K, two of the five mice were observed as having moderate pathology changes with increased infiltration of large quantities of inflammatory cells clustered around bronchioles, exudation in bronchiole activity and increased exudation in alveolus. Severe pathology changes were observed in three of five mice, with infiltration of large quantities of mononuclear cells.

In groups B, C, D, F, H and I, most animals had slight pathological damage with interstitial capillary extension and congestion and slight exudation in small amount of alveolus. A few were observed as having moderate pathological damage. In groups A, E, G and J, slight pathological damage was recorded with only infiltration of small quantity of inflammatory cells observed. No moderate or severe pathological damage was observed.

14 days post infection the lung pathology changes lessened. Only in virus group K, the lungs of mice were recorded as having slight or moderate pathology change. In groups which received treatment, only slight pathological damage was observed and this was only in a small minority of mice.

Table 9. General observations (Group - H3N2).

Visible Pathological Damage: grey/white hearts indicative of necrosis and abnormal deep red inflamed lungs

Results for PCR analysis for H3N2 are shown in Table 10 and these are summarised in Table 11.

The percentage of mice showing positive viral detection (H3N2) using PCR analysis, following treatment with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), Ribavirin or TAMIFLU ™, is shown in Figure 6.

Results of the histopathology analysis for H3N2 are shown in Table 12. These are summarised in Table 13.

Table 12. Histopathology analysis (Group - H3N2).

'-' indicates no damage, '+' slight damage, '++' moderate damage, '+++' severe damage Table 13. Histopathology summary (Group - H3N2).

indicates no damage, '+' slight damage, '++'moderate damage, '+++' severe damage

The percentage of animals with pathological damage is shown in Figure 7 for mice infected with H3N2 and treated with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), Ribavirin or TAMIFLU TM

Figure 8 shows combined results for the PCR and histopathology analysis for mice treated with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), Ribavirin or TAMIFLU ™.

As for H5N1 , the significance of these results is highlighted when the comparison with TAMIFLU TM is considered.

The H3N2 experiment exhibited more successful results for the statin/caffeine combination. The statin/caffeine combination (intragastric administration as a prophylaxis) is superior to TAMIFLU ™ (intranasal administration as a prophylaxis and also intragastric administration as a therapeutic). 0% of samples had viral RNA present in this statin/caffeine group. Equal results were revealed for prophylaxis intranasal administration for TAMIFLU ™ and the statin/caffeine combination. TAMIFLU ™ proved superior to the combination when intragastric administration was considered for treatment. Again, this may have been due to low doses of the combination and high doses of TAMIFLU ™. The histopathology results indicated that less pathological damage was observed in the statin and caffeine intranasal administration prevention group, when compared to TAMIFLU ™ administered intranasally as a prevention. 40% of animals from our combination group had no damage compared to only 10% of animals in the TAMIFLU ™ group (90% of these animals had pathological damage to tissue). The combination proved to be an equal competitor against TAMIFLU ™ as a therapeutic for intragastric administration. Both groups had 40% of animals with no pathological damage.

The results indicated that the statin/caffeine combination can inhibit virus replication of H3N2 in the lungs. According to the symptoms, general pathological change and RT-PCR results, the statin/caffeine combination has excellent preventative effects and has also shown some therapeutic effects. Compared to Ribavirin and TAMIFLU ™, the preventative effect of the statin/caffeine combination by intragastric administration is far superior and demonstrates excellent potential. RT-PCR results have detected 50% and 10% of virus in Ribavirin and TAMIFLU ™ treated samples respectively, compared to 0% detection in the statin/caffeine combination treated sample.

H1 N1 Group All mice were considered normal on day 1 and day 2, although two mice from group C died on day 2 (Table 14). These were dissected and no visible pathological change was observed. Day 3 marked an obvious onset of symptoms in group K, the virus group. Mice became depressed, sluggish and exhibited signs of horripilation. Horripilation was also observed in groups B, C, D, F and H, whereas mice in groups A, E, G, I, J and L were all mentally normal. From day 4 to day 6, horripilation eased in groups B, C, D, F and H. Groups A, E, G, I, J and L were mentally normal. However, mental depression and horripilation was evident in group K. On day 7, mental depression and horripilation was seen in group K and the animals were sluggish. All other groups were mentally normal and there were no physical symptoms of the virus. 5 mice from each group were sacrificed (3 mice from group C). These sacrificed mice were examined for pathological changes. In the virus group K, the hearts of the mice were grey/white and lungs were an abnormal white. In groups A and C, hearts of 2 mice had evidence of necrosis. In groups D, F and G, one animal in each group also exhibited signs of necrosis in heart tissue. Groups B, E, I, J and L showed no evidence of pathological change. From day 8 to day 15, animals of group K were sluggish, mentally depressed and showed signs of horripilation. On day 9, horriplation decreased to only three animals in group K and on day 12 no animals showed any evidence of this particular symptom. All other groups were mentally normal over this 8-day period. On the final day of observation, all groups were mentally normal and no pathological change was identified in any of the sacrificed animals, except group K which had grey/white hearts indicative of cell death and abnormal red lungs. Table 14. General observations (Group H1N1).

* Visible Pathological Damage: grey/white hearts indicative of necrosis and abnormal deep red inflamed lungs

Results for PCR analysis for H1 N1 are shown in Table 15.

The percentage of mice showing positive viral detection (H1 N1 ) using PCR analysis, following treatment with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), Ribavirin or TAMIFLU ™, is shown in Figure 9. Results of the histopathology analysis for H1 N1 are shown in Table 16. These are summarised in Table 17.

Table 17. Histopathology summary (Group - H1 N1 ).

'-' indicates no damage, '+' slight damage, '++' moderate damage, '+++' severe damage

The percentage of animals with pathological damage is shown in Figure 10 for mice infected with H1 N1 and treated with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), Ribavirin or TAMIFLU ™.

Figure 11 shows combined results for the PCR and histopathology analysis for mice treated with lovastatin hydroxy acid salt and caffeine (intranasal or intragastric), Ribavirin or TAMIFLU ™.

As for H5N1 and H3N2, the significance of these results is highlighted when the comparison with TAMIFLU ™ is considered. The same experiment was carried out using the H1 N1 virus, and again, the statin/caffeine combination has shown its effectiveness as an inhibitor of this virus.

The most successful result according to PCR analysis was the group administered with the combination as a prevention. Also, TAMIFLU ™ and Ribavirin were equally successful as an intragastric therapeutic drug and intranasal prevention drug respectively. The preventative effect of the combination (intragastric administration) is equal to the therapeutic effect of TAMIFLU ™ (intragastric administration also). TAMIFLU ™ (intranasal prevention) is not as effective as the statin/caffeine combination (intranasal administration, therapy and intragastric administration prevention).

Histopathology results have revealed similar success as H5N1 and H3N2 experiments. The combination equally competes with TAMIFLU ™ as a prophylaxis when administered intranasally. Both groups had 30% of animals with no pathological damage. However, TAMIFLU ™ is superior as a therapeutic when administered intragastrically. 50% of animals in this group had no pathological damage when compared to only 30% of animals in the combination group.

The results indicate that the statin/caffeine combination can inhibit the replication of H1 N1 virus in the lung. According to the RT-PCR results, the therapeutic effect of the combination by intranasal administration is superior to that of the TAMIFLU ™ prevention effect when administered intranasally. When the statin/caffeine combination is administered intragastrically, its prevention effect is equal to that of the TAMIFLU ™ intragastric therapeutic effect.

Conclusion These results have shown excellent potential. It is also important to bear in mind that the doses of statin used in these experiments were 50 μg whereas TAMIFLU ™ was administered in doses of 200μg. This warrants further investigation as increasing the doses of statin to match that of TAMIFLU ™ could possibly result in the combination having an increased therapeutic effect.

Example 4 - Preventive effect in cells. Materials and Methods Confluent monolayers of MDCK cells, cultured in a 96-well tissue culture plate (1x10⁵ cells/cm²), were overlaid with medium (DMEM containing 10g/mL trypsin, 1 % low-melting agarose, the test compounds PTPM7 (lovastatin) at concentrations ranging from 0.097, 0.19, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50μg/ml to 100μl, 200 μg/ml C53 (caffeine)). After incubation at 37°C for 1 hour, the supernatant in the culture plate was removed, and the cells were washed twice with serum-free MEM, were infected with the Influenza / A / Guangzhou / 2005 (H3N2 or H1 N1 ) virus. Meanwhile, a positive control (medium containing TAMIFLU ™), a virus control (medium not containing the test compounds) and a blank control (no virus adsorption) were included. Each experiment was performed in a triplicate of wells. After incubating cultures for 2-3 days at 37°C in 5% CO2, the cell monolayer was fixed with a 4% formaldehyde solution for 30 minutes. Fixed cells were stained with a 1 % crystal violet solution. Then the CPE of the virus-infected culture were microscopically observed. The number of plaques was counted, and the percentage inhibition of antiviral activity was calculated relative to the control.

Results Table 18 shows the number of plaques per well following infection with H3N2 for the various concentration ranges, TAMIFLU ™ and the controls (no drug or no virus). These results are represented in the graph shown in Figure 12.

Table 19 shows the number of plaques per well following infection with H1 N1 for the various concentration ranges, TAMIFLU ™ and the contro (no drug or no virus). These results are graphed in Figure 13.

These results show that at doses of greater than 50 μg/ml, the lovastatin/caffeine combination was more effective that TAMIFLU ™ at preventing both the H3N2 and H1 N1 viruses.

Example 5 - Therapeutic effect in cells- Materials and Methods

Confluent monolayers of MDCK cells, cultured in a 96-well tissue culture plate (1x10⁵ cells/cm²), were infected with the Influenza / A / Guangzhou / 2005 (H3N2 or H1 N1 ) virus. After 1 hour of virus adsorption, the supernatant in the culture plate was removed, and the cells were washed twice with serum-free MEM, and overlaid with medium (DMEM containing 10 g/mL trypsin, 1 % low-melting agarose, the test compounds PTPM7 (lovastatin) at concentrations ranging from 0.097, 0.19, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50 μg/ml, 200 μg/ml C53 (caffeine)). Meanwhile, a positive control (the supernatant in the culture plate was removed and replaced with medium containing TAMIFLU ™), a virus control (medium not containing the test compounds) and a blank control (no virus adsorption) were included. Each experiment was performed in a triplicate of wells. After incubating cultures for 2-3 days at 37°C in 5% CO2, the cell monolayer was fixed with a 4% formaldehyde solution for 30 minutes. Fixed cells were stained with a 1 % crystal violet solution. Then the CPE of the virus-infected culture were microscopically observed. The number of plaques was counted, and the percentage inhibition of antiviral activity was calculated relative to the control.

Results

Table 20 shows the number of plaques per well following infection with H3N2 for the various concentration ranges, TAMIFLU ™ and the controls (no drug or no virus). These results are graphed in Figure 14.

Table 21 shows the number of plaques per well following infection with H1 N1 for the various concentration ranges, TAMIFLU ™ and the controls (no drug or no virus). These results are graphed in Figure 15.

These results show that at doses of greater than 50 μg/ml, the lovastatin/caffeine combination was more effective that TAMIFLU TM at treating both the H3N2 and H1 N1 viruses.

All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

1. A method for the treatment and/or prophylaxis of a viral infection, the method comprising the steps of:

- further providing a therapeutically effective amount of a composition comprising caffeine or a salt, analogue, derivative or metabolite thereof, and

- administering said compositions to a subject in need of treatment.

2. The method as claimed in claim 1 wherein the compositions are administered to the subject sequentially.

3. The method as claimed in claim 1 wherein the compositions are administered to the subject separately.

4. The method as claimed in claim 1 wherein the compositions are administered to the subject simultaneously.

5. The method as claimed in any of the preceding claims wherein the statin pro-drug is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin and atorvastatin.

6. The method as claimed in any of claims 1 to 4 wherein the least one statin is a statin hydroxy acid salt.

7. The method as claimed in claim 6 wherein the statin hydroxy acid salt is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, cehvastatin and atorvastatin.

8. The method as claimed in claim 7 wherein the statin hydroxy acid salt is lovastatin hydroxy acid salt.

9. The method as claimed in any of the preceding claims wherein the viral infection is selected from the group consisting of a type A Influenza virus, type B Influenza virus, Hepatitis C, Hepatitis B, Hepatitis G, Hepatitis H and Respiratory Syncytical Virus.

10. The method as claimed in claim 9 wherein the type A Influenza virus is selected from the group consisting of H1 N1 , H3N2 and H5N1.

11. The method as claimed in any of the preceding claims wherein the subject is a human.

12. The method as claimed in any of the preceding claims wherein at least one of the compositions is administered intranasally or intragastrically.

13. The method as claimed in any of the preceding claims further comprising a step of: - administering a therapeutically effective amount of an antiviral compound.

14. The method as claimed in claim 13 wherein the antiviral compound is selected from the group consisting of ribavirin, amantadine, rimantadine, oseltamivir and zanamivir.

15. A composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof for use in the treatment and/or prophylaxis of a viral infection.

16. A composition as claimed in claim 15 wherein the viral infection is influenza type A or influenza type B.

17. Use of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof in the preparation of a medicament for the treatment and/or prevention of viral infection.

18. Use as claimed in claim 17 wherein the statin pro-drug is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, cehvastatin and atorvastatin.

19. Use as claimed in claim 17 wherein the least one statin is a statin hydroxy acid salt.

20. Use as claimed in claim 19 wherein the statin hydroxy acid salt is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin and atorvastatin.

21. Use as claimed in claim 20 wherein the statin hydroxy acid salt is lovastatin hydroxy acid salt.

22. Use as claimed in any of claims 17 to 21 wherein the viral infection is selected from the group consisting of Hepatitis C, Hepatitis B, Hepatitis G, Hepatitis H and Respiratory Syncytical Virus.

23. Use as claimed in any of claims 17 to 21 wherein the viral infection is Influenza type A or Influenza type B.

24. Use as claimed in claim 23 wherein the type A Influenza virus is selected from the group consisting of H1 N1 , H3N2 and H5N1.

25. Use of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof in the preparation of a medicament for the prevention of damage to lung tissue caused by an immune response mounted against infection of a subject by a viral pathogen.

26. A method for preventing damage to lung tissue of a subject caused by an immune response following the infection of the subject by a viral pathogen, the method comprising the steps of:

- administering a therapeutically effective amount of a combined preparation comprising (a) at least one statin or statin-like compound, or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof, and (b) caffeine or a salt, analogue or a derivative thereof to the subject.

27. A composition for use in the prevention of lung tissue damage caused by an immune response mediated by a subject against a viral pathogen, said composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof.

28. A pharmaceutical composition for use in the treatment or prophylaxis of a viral infection comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof along with at least one diluent, carrier or excipient.

29. The composition as claimed in claim 28 wherein the least one statin is a statin hydroxy acid salt.

30. The composition as claimed in claim 29 wherein the statin hydroxy acid salt is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, cehvastatin and atorvastatin.

31. The composition as claimed in claim 30 wherein the statin hydroxy acid salt is lovastatin hydroxy acid salt.

32. The composition as claimed in claim 28 wherein the statin pro-drug is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin and atorvastatin.

33. The composition as claimed in any of claims 28 to 32 wherein the viral infection is selected from the group consisting of a type A Influenza virus, type B Influenza virus, Hepatitis C, Hepatitis B, Hepatitis G, Hepatitis H and Respiratory Syncytical Virus.

34. The composition as claimed in claim 33 wherein the type A Influenza virus is selected from the group consisting of H1 N1 , H3N2 and H5N1.

35. The composition as claimed in any one of claims 28 to 34 further comprising an antiviral compound.

36. The composition as claimed in claim 35 wherein the antiviral compound is selected from the group consisting of ribavirin, amantadine, rimantadine, oseltamivir (TAMIFLU ™) and zanamivir.

37. Use of a combined medicament comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof for the treatment of a viral infection in a subject.

38. Use as claimed in claim 37 wherein the least one statin is a statin hydroxy acid salt.

39. Use as claimed in claim 38 wherein the statin hydroxy acid salt is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin and atorvastatin.

40. Use as claimed in claim 39 wherein the statin hydroxy acid salt is lovastatin hydroxy acid salt.

41. Use as claimed in claim 37 wherein the statin pro-drug is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin and atorvastatin.

42. Use as claimed in any of claims 37 to 41 wherein the viral infection is selected from the group consisting of a type A Influenza virus, type B Influenza virus, Hepatitis C, Hepatitis B, Hepatitis G, Hepatitis H and Respiratory Syncytical Virus.

43. Use as claimed in claim 42 wherein the type A Influenza virus is selected from the group consisting of H1 N1 , H3N2 and H5N1.

44. Use as claimed in any of claims 37 to 43 wherein the subject is a human.

45. A method for lowering the level of cholesterol in a subject, the method comprising the steps of: - providing a therapeutically effective amount of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof,

- administering said compositions to a subject in need of treatment.

46. Use of a composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof in the preparation of a medicament for reducing cholesterol levels.

47. A composition comprising a statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof along with caffeine or a salt, analogue, derivative or metabolite thereof for use in the lowering of cholesterol levels in a subject.

48. A pharmaceutical composition for use in the reduction of cholesterol levels in a subject, said composition comprising at least one statin compound or a pharmaceutically active salt or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof along with a pharmaceutically acceptable excipient, carrier or diluent.

49. A method for the treatment and/or prophylaxis of Rhabdomyolysis, the method comprising the steps of:

- administering said compositions to a subject in need of treatment.

50. Use of a therapeutic composition comprising at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof in the preparation of a medicament for the treatment and/or prophylaxis of Rhabdomyolysis.

51. A composition comprising a statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof for use in the treatment or prevention or Rhabdomyolysis.

52. A pharmaceutical composition for the treatment and/or prophylaxis of Rhabdomyolysis, wherein said composition comprises at least one statin or statin-like compound or an analogue, metabolite, derivative, pharmaceutically active salt, precursor or pro-drug thereof and caffeine or a salt, analogue, derivative or metabolite thereof along with a pharmaceutically acceptable excipient, carrier or diluent.