WO1994012192A1

WO1994012192A1 - Antiviral agents

Info

Publication number: WO1994012192A1
Application number: PCT/AU1993/000606
Authority: WO
Inventors: Helmut Weigold; Angeline Ingrid Bartholomeusz; Sebastian Mario Marcuccio; George Holan
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1992-12-01
Filing date: 1993-11-29
Publication date: 1994-06-09
Anticipated expiration: 1995-06-01
Also published as: CN1093906A

Abstract

Heteropolytungstates having anti-flaviviral activity having the general formulae (I) to (VII): (I) An[XW12O40] wherein X is selected from P?v, SiIV, GeIV, CoII, CoIII, ZnII, CuII, BIII, HI¿2, Al?III, FeIII, VV, GaIII, MnIV, CIV¿. (II) A¿n?[X2W18O62] wherein X is P?V¿; (III) A¿n?[XW11O39] wherein X is selected from P?V, SiIV, GeIV, BIII, AlIII, GaIII, FeIII, CoIII¿; (IV) A¿n?[XW9O34] wherein X is selected from P?V, SiIV, GeIV¿; (V) A¿n?[X2W17-mMmO61] wherein X is P?V¿, M is MoVI, and m is 0, 1, 2, 4, or 5; (VI) A¿n?[X2W15-mMmO56] wherein X is P?V¿, M is V?V or MoVI¿, and m is 0 or 1; (VII) A¿n?[XM2W9O39] wherein X is P, and M is Zr; and wherein in each of the general formulae (I) to (VII), A is a cation, and n is the number of cations necessary for electrical neutrality of the molecule; or dimers, hydrates or pharmaceutically acceptable derivatives thereof. Pharmaceutical compositions and methods for the treatment or prophylaxis of a flaviviral-associated infection which involve the use of these compounds are also disclosed.

Description

ANTIVIRAL AGENTS

The present invention relates to heteropolytungstates and pharmaceutically acceptable derivatives thereof, and to the use of these compounds and

pharmaceutical compositions containing them for the treatment or prophylaxis of infections by viruses which are confirmed or probable members of the family Flaviviridae, for example infections such as yellow fever, dengue fever, Australian encephalitis, Japanese encephalitis and Hepatitis C.

The family Flaviviridae is a newly-recognised large group (in excess of 70 species) of small, enveloped viruses that contain a single strand of positive-sense RNA of 10 kilobases. Flaviviruses are well known to be the causative agents of a number of human diseases including the most important arthropod-borne viral afflictions of mankind - dengue, yellow fever, and Japanese encephalitis. In addition, eight flaviviruses are known to cause disease in domestic or wild animals of economic importance. Yellow fever and dengue fever are widespread and well known as mosquito borne diseases of tropical countries. There are between 30 and 60 million flavivirus infections per year including one million Japanese encephalitis infections. The extent of Hepatitis C is not known with any degree of certainty because an infection can exist for many years without the patient being aware of the symptoms. Hepatitis C produces a much higher rate of chronic liver infection than Hepatitis B which is a recognised hazard in many countries. About 50% of patients develop chronic infections, compared with 5 to 10% of those infected with Hepatitis B. Chronic infection causes cirrhosis of the liver, impairs liver function, and 20-30 years later causes liver failure. It has been estimated that the rate of infection approaches and may exceed 1% of the population in Australia. There is no proven cure or vaccine for Hepatitis C. Effective vaccines are available for some flaviviruses only, eg for yellow fever, Japanese encephalitis and tick-borne encephalitis. Treatment of dengue fever and Australian encephalitis relies on the patient's own immune defences; infections can be fatal.

An antiviral drug to control infections with flaviviruses is thus highly desirable. Drugs which control or inhibit replication have proven to be effective in the control of some other viruses, however, because of the difficulty, of inhibiting viruses while leaving the non-infected cells unimpaired, few antiviral drugs are currently in widespread clinical use. As a consequence of this difficulty and recent recognition of flaviviruses as a unique group with a unique replication strategy, no attention has yet been paid to antiviral compounds to control flaviviral infections. All members of the family Flaviviridae possess a unique replication strategy which is inhibited by the compositions of this invention. The non-structural genes NS3 and NS5 which have been proposed to be involved in replication share a great deal of sequence similarity between species, and hence an inhibitor of replication should be active against all flaviviruses.

Heteropolytungstate compounds have been known for over 100 years. Most of their applications stem from their redox chemistry and also their high ionic weights and charges. Their redox chemistry has lead to their use as catalysts for the oxidation of organic substrates such as, for example, propylene to acrylic acid, ethylene to acetaldehyde. In the biological field heteropolytungstates have found use as electron dense stains for electron microscopy, as analytical reagents for proteins and several have also been shown to inhibit viral DNA and RNA polymerases. (J. C. Cherman, et al, Biockem. Biophys. Res. Commun, 1975, 65, 1229; M. Hervé, et al, ibid, 1983, 116, 222.)

The heteropolytungstates within the scope of this invention include the Keggin and Dawson (also known as the Wells-Dawson) type structures and compounds based on these structures in which one or more of the tungsten atoms are removed and, in the majority of cases, exchanged by other metal atoms.

Vacancies in the structures are most often created by the extraction of WO⁴⁺ or W₃O₆ ⁶⁺ from the Keggin (XW₁₂O₄₀ ^n-) or Dawson (P₂W₁₈O₆₂ ^6-) species. Isomers of these unsaturated (lacunary) polyanions are possible, a consequence of the location of the vacancy. (R. Massart R. Contant, J. M. Fruchart, J. M. Ciabrini, M. Fournier, Inorg. Chem. 1977, 16, 2916; T. L. Jorris, M. Kozik, N. Casan-Pastor, P. J. Domaille, R. G. Finke, W. K. Miller and L. C. W. Baker, J. Am. Chem. Soc. 1987, 109, 7402; T. J. R. Weakley, Polyhedron 1987, 6, 931; R. Contant and J.-P. Ciabrini, J. Chem. Res. (S), 1977, 222; R. G. Finke, M. W. Droege and P. J.

Domaille, Inorg. Chem.„ 1987, 26, 3886; M. T. Pope, "Heteropoly and Isopoly Oxometalates", Springer- Verlag, Berlin, 1983.) The position of the vacancy in P₂W₁₇O₆₁ ^10- is defined by the prefix α₁- for a belt vacancy or α₂- for a cap vacancy. The rotation of W₃-oxide triads in the structures leads to a number of isomers. Thus a 60' rotation of a W₃ triad cap can convert, for example, an α- isomer to the β- isomer. In the trivacant polyanions of the type XW₉O₃₄ ^n-, A- or B- forms are obtained, depending upon whether a corner-linked W₃ oxide triad is lost (A- form) or an edge-linked W₃ oxide triad has been removed(B- form). Unsaturated heteropolyanions can behave as ligands by bonding, at their vacant site, with metal ions. These metal ions, when not sterically crowded, can carry ligands such as water, organic coordinating species or organometallic groups. Organometallic moieties can also react with exposed oxygen atoms on, for example, trisubstituted Keggin or Dawson structures (R. G. Finke and M. W.

Droege,J. Am. Chem. Soc, 1984, 106, 7274 and R. G. Finke, B. Rapko and P. J. Domaille, Oiganometallics 1986, 5, 175). An oxygen atom on the Keggin structure can also be alkylated with reagents such as trimethyloxonium salts (W. H. Knoth and R. L. Harlow,J. .Am. Chem. Soc.1981, 103, 4265). Some of the oxygen atoms on heteropolytungstates can also be exchanged for fluorine atoms (F. Chauveau, P. Doppelt and J. Lefebvre, Inorg. Chem. 1980, 19, 2803; T. L. Jorris, M. Kozik and L. C. W. Baker, Inorg. Chem. 1990, 29, 4584). Other heteropolyanion species are formed by reaction of two W₅O₁₈H^5- ions with metal ions such as the lanthanoids (R. D. Peacock and T. J. R.

Weakley, J. Chem. Soc. A, 1971,1836). Heteropolyanions having PW₇

phosphotungstate groups, generally bridged by phosphate group(s), are known (J. Fuchs and R. Palm, Z. Natwforsch. 1988, 43b, 1529 and R. Acerete, J.

Server-Carrio, A. Vegas and M. Martinez-Ripoll,J. Am. Chem. Soc, 1990, 112, 9386).

The central atom in the compounds can vary widely, especially in the case of the simpler Keggin type structures. The central atom in the Dawson type structures is most often phosphorus.

Heteropolytungstate species are often more stable in solution than the corresponding heteropolymolybdates. Heteropoly compounds of other metals, such as niobium and vanadium, have also been made but often are stable only over a more limited pH range.

It has now been unexpectedly discovered that heteropolytungstate polyanions containing a "central" species (designated X in the examples of structural types listed below) are active against viruses belonging to the

Flaviviridae family. In particular they inhibit the replication of such viruses stopping the development of an infection.

Accordingly, the present invention provides a method

for the treatment or prophylaxis of a flavivirus-associated infection, which comprises the use of an effective amount of at least one heteropolytungstate compound selected from:

(a) a compound of the general formulae I to VII: (i) A_n[XW₁₂O₄₀] I wherein X is selected from P^V,Si^IV,Ge^IV,Co^II,Co^III-Zn^II,Cu^II,

B^lll,H^l ₂,Al^lll,Fe^lll,V^V,Ga^lll,Mn^lV ,C^IV; (ii) A_n[X₂W₁₈ O₆₂] II wherein X is P^V;

(iii) A_n[XW₁₁O₃₉] III wherein X is selected from

P^V,Si^IV,Ge^IV,B^llI,Al^lII,Ga^lII,Fe^lII,Co^lll;

(iv) A_n[XW₉ O₃₄] IV wherein X is selected from P^V,Si^IV,Ge^IV,

(v) A_n[X₂W _17-mM_mO₆₁] V wherein X is P^V,

M is Mo^VI, and

m is 0, 1, 2, 4, or 5;

(vi) A_n[X₂W _15-mM_mO₅₆] VI wherein X is P^V,

M is V^V or Mo^VI, and

m is 0 or 1;

(vii) A_n[XM₂W₉O₃₉] VII wherein X is P, and

M is Zr;

and herein in each of the general formulae I to VII, A is a cation, and

n is the number of cations necessary for electrical neutrality of the molecule;

(b) a dimer of a said compound of the general formula I to VII; or

(c) a hydrate or pharmaceutically acceptable derivative thereof. The compounds of the general formulae I to VII are polyanions with associated cations (A) for electrical neutrality. Preferably, the cation (A) is a proton, an alkali metal ion, an alkaline earth metal ion, an ammonium ion or an alkylammomum ion of the formula R_4-nH_nN⁺, wherein R is an alkyl group of from 1 to 6 carbon atoms and n is 1, 2, or 3.

These compounds crystallize with a variable number of molecules of water of crystallization dependent upon the conditions of product recovery and subsequent treatment, and all such hydrates are included within the scope of the invention.

By "a pharmaceutically acceptable derivative" is meant any

pharmaceutically acceptable salt, or any other compound which, upon

administration to the recipient, is capable of providing (directly or indirectly) a compound of the invention or an active metabolite or residue thereof.

The method of the present invention is particularly directed to the treatment or prophylaxis of a flavivirus-associated infection in a patient in need of such treatment or prophylaxis, and the method comprises the administration to said patient of said effective amoimt of the at least one heteropolytungstate compound as broadly described above.

The patient may be a human, or an animal such as a domestic or wild animal, particularly an animal of economic importance.

An "effective amoimt" of the heteropolytungstate compound as used in accordance with this invention is an amount effective to inhibit flavivirus replication. The present invention also extends to the use of at least one

heteropolytungstate selected from

(a) a compound of the general formula I to VII as broadly described above, (b) a dimer of a said compound of the general formula I to VI; or

(c) a hydrate or pharmaceutically acceptable derivative thereof, in the

manufacture of a medicament for the treatment or prophylaxis of a flavivirus- associated infection.

In another aspect, the present invention also provides a pharmaceutical composition for the prophylaxis or treatment of a flavivirus-associated infection, which comprises at least one heteropolytungstate selected from

(a) a compound of the general formula I to VII as broadly described above; (b) a dimer of a said compound of the general formula I to VII; or

(c) a hydrate or pharmaceutically acceptable derivative thereof, in association with one or more pharmaceutically acceptable carriers or diluents.

The pharmaceutical compositions of the present invention may comprise an effective amoimt of one or more compounds selected from general formulae I to IV in association with one or more pharmaceutically acceptable carriers or diluents, and optionally other antiviral or other therapeutic agents. Each carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. It should be understood that in addition to the ingredients particularly mentioned, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavouring agents.

Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Finally, in accordance with the present invention, there is provided a heteropolytungstate compound selected from

(a) a compound of the general formula I to VII as broadly described above;

(b) a dimer of a said compound of the general formula I to VII; or

(c) a hydrate or pharmaceutically acceptable derivative thereof.

Presently preferred compounds in accordance with this invention include the compounds listed in Example 1 below.

The compounds of general formulae I to VII may be prepared by the literature methods or adaptions thereof, varying reactants and conditions as required to obtain the target compound. General review articles, describing the preparation, structure and properties of many of the compounds, include P.

Souchay, "Ions Mineraux Condensés", Masson, Paris, 1969; M. T. Pope,

"Heteropoly and Isopoly Oxometalates", Springer-Verlag, Berlin, 1983; T. J. R. Weakley, Structure and Bonding, Springer-Verlag, Berlin, 1974, 18, 131; M. T. Pope and A. Müller, Angew. Chem. Int. Ed. Engl. 1991, 30, 34. Novel compounds of the invention are prepared by the methods disclosed herein, and these methods of synthesis also form part of the present invention. The compounds of the invention useful as active anti-flaviviral agents are listed as formulae I to VII below, along with appropriate methods of preparation for each sub-type:

I A_n[XW₁₂O₄₀]

X = P^V, Si^IV Ge^IV, Co^II, Co^lII, Zn^II, Cu^II, B^lII, H^l2, Al^lII, Fe^lII, V^V, Ga^lII,

Mn^IV, C^IV. These compounds were made using known literature methods including: A. Teze and G. Herve, Inorg. Synth, 1990, 27, 93-96. (Ed. A. P. Ginsberg)

Whiley-Interscience (X = Si); C. Rocchiccioli-Deltcheff, M. Fournier, R. Frank and R. Thouvenot, Inorg. Chem., 1983, 22, 207-216 and references therein (X = P, Si, Ge, B); L. C. W. Baker and T. P. McCutcheon, J. Am. Chem. Soc, 1956, 78, 4503 and subsequent papers (X = Co). I^I A_n[X₂W₁₈ O₆₂]

X = P^V.

The compounds were synthesized by standard methods such as given in the references: R. Contant, Inorg. Synth, 1990, 27, 105. (Ed. A. P. Ginsberg)

Whiley-Interscience; R. G. Finke, M. W. Droege and P. J. Domaille, Inorg. Chem., 1987, 26, 3886; lII A_n[XW₁₁O₃₉]

X = P^V, Si^IV, Ge^IV, B^lIIAl^lII, Ga^lII, Fe^lII, Co^lII

These compounds were prepared following standard procedures; eg for X = Si or Ge the methods of A. Tezé and G. Herve, Inorg. Synth., 1990, 27, 89-92. (Ed. A. P. Ginsberg) Whiley-Interscience; J. inorg. nud. Chem, 1977, 39, 999 were used; the compound X = P was prepared by the method of R. Contant, Can. J. Chem., 1987, 65, 570.

IV A_n[XW₉O₃₄]

X = P^V, Si^IV, Ge^IV,

The compounds X = Si were prepared following the method of A. Teze and G. Hervé, Inorg. Synth, 1990,.27, 129.(Ed. A. P. Ginsberg) Whiley-Interscience. The compounds X = P were prepared following the methods of R. Massart, R.

Contant, J. M. Fruchart, J. M. Ciabrini, M. Fournier, Inorg. Chem, 1977, 16, 2916 and R. G. Finke, M. W. Droege and P. J. Domaille, ibid, 1987, 26, 3886. Those containing Ge were made by the method of G. Herve and A. Tézé, Inorg. Chem, 1977, 16, 2115; ibid 1977, 16, 2115.

V A_n[X₂W _17-mM_mO₆₁]

X = P^V,

M = Mo^IV

m = 0, 1, 2, 4, 5.

The compounds m = 2 were obtained by the reactions of M. Abbessi, R Contant, R. Thouvenot,and G. Hervé, Inorg. Chem., 1991, 30, 1695. Those compounds, m = 1, 4 or 5, were made according to R. Contant and J.-P. Ciabrini, J. inorg. nud. Chem, 1981, 43, 1525. The species having m = 0 were made following the method of R. Contant, Inorg. Synth, 1990, 27, 107.(Ed. A. P. Ginsberg)

Whiley-Interscience.

X = P^V

M = V^V, Mo^IV

m = 0 or 1

The compounds m = 0 were made according to R. G. Finke, M. W. Droege and P. J. Domaille, Inorg. Chem, 1987, 26, 3886 or R. Contant, Inorg. Synth, 1990,.27, 108.(Ed. A. P. Ginsberg) Whiley-Interscience,.and those with m = 1 were obtained by the reactions of M. Abbessi, R Contant, R. Thouvenot,and G. Herve, Inorg. Chem., 1991, 30, 1695.

VII A_n [XM₂W₉O₃₉]

X = P

M = Zr

The product was obtained on reaction of [(η⁵-C₅H₅)₂ZrCl]₂O (1 part) with

A-α-Na₈HPW₉O₃₄. nH₂O (1 part) at room temp, followed by treatment of the reaction solution with excess KCl. The product was taken up several times in hot water and precipitated with KCl and then recrystallized from a dilute KCl solution. Recrystallization in the absence of KCl results in the compound separating as a gel. After washing with several aliquots of water, the product was air dried to a "glassy" solid. Analyses found and that calculated (in brackets) for

K₇Zr₂W₉PO₃₉H₄19H₂O were:%P 1.10 (1.00); %W 51.4 (53.17); %Zr 5.98 (5.86); %K 8.49 (8.79); H₂O 10.9 (11.0); titration of the H⁺ exchanged (on an Amberlite IR-120 [H⁺] column) gave end points at 3.9 and 7.0 equivalents of base (KOH). Titration of the product (not proton exchanged) with KOH gave an end point below 0.5 equivalents, suggesting that one of the potassium ions on the product may be partially replaced by a proton. The extent of such an exchange would, presumably, be influenced by the pH of the recrystallization solution. Infrared bands: PO₄ ^3- at 1096,1060 and 1033 cm^-1; W= O_t 950 and 942 cm^-1; W-O_c-W 880 cm^-1; W-O_e -W 802 cm^-1.

In the compounds of general formulae I to VII, when a transition metal atom(s) replace(s) one or more tungsten atoms in the structure, the oxygen on the transition metal atom(s) may be either doubly protonated (H₂O), singly protonated (OH), or completely deprotonated (O). The acidity of these protons, and the compounds that are obtained, as is known to one skilled in the art of

heteropolytungstate chemistry, depends on the nature of the transition metal atom, its oxidation state, the basicity of the polyanion formed and the basicity of the solution from which the compounds were isolated. In the compounds of the invention not all oxygen atoms are necessarily oxo groups and the charge (and hence the number of counter cations (A)) on the polyanion will depend on the number of protons attached to the oxygen atom(s). Furthermore, compounds containing groups such as, for example, MOH, may dimerize by an intermolecular condensation reaction. Dimers, where formed, of the compounds listed, are also included in the invention. Many of the compounds of the invention can occur in a number of isomeric forms. In fact, it is at times difficult to obtain isomerically pure compounds. All isomers or isomer mixtures are included in this invention. Many of the compounds can undergo one or more electron reductions.

The reduced compounds are also included in this invention.

The charge on the polyanions can vary, depending upon the extent of protonation of the polyanions, as noted earlier, and upon the oxidation states of the metal atoms. The number of associated counter cations (A) will vary correspondingly. A may be a proton, an alkali metal ion, an alkaline earth metal ion, or ammonium or alkyl ammonium ion of type R_4-nH_nN⁺, where R is an alkyl group of from 1 to 6 carbon atoms, and n is 1, 2 or 3. The required cation is generally introduced into the compound either by use of an ion exchange resin or by precipitation with excess of a salt of that cation.

It is to be noted that, as one skilled in the art of heteropolyanion chemistry would know, not all combinations of the elements given in general formulae I to VII are isolable.

Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable hquid carrier. Compositions for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile hquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage compositions are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

The compounds according to the invention may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary

compositions include those adapted for:

(a) oral administration, external application, for example drenches (e.g.

aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous,

intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced into the udder via the teat;

(c) topical application, e.g. as a cream, ointment or spray applied to the skin; or

(d) intravaginally, e.g. as a pessary, cream or foam.

It should be understood that in addition to the ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavouring agents. The compositions according to the invention may be administered for therapy by any suitable route, including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). Preferably, administration will be by the oral route, however it will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the composition and the chosen active ingredient.

In general a suitable dose of the active ingredient will be in the range of 3.0 to 120 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg per kilogram body weight per day and most preferably in the range 15 to 60 mg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient per unit dosage form Ideally the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 1 to about 100 μM, preferably about 2 to 50 μM. This may be achieved, for example, by the

intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 0.1 to about 100 mg/kg of the active ingredient. Desirable blood levels may be maintained by a

continuous infusion to containing about 0.4 to about 15 mg/mg of the active ingredient.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way. The term "active

ingredient" as used in Examples 3 to 5 means one or more compounds of selected from formulae I to VII or a pharmaceutically acceptable derivative thereof. EXAMPLE 1

The compounds in Table 1 were prepared by the methods described above and dissolved in doubly distilled water for testing as described in Example 2 below:

EXAMPLE 2: Antiviral Activity

The compounds listed in Example 1 were tested for their ability to inhibit RNA synthesis in an in vitro polymerase assay (Chu and Westaway, 1985, 1987; Brun and Brinton, 1986). In this assay, flavivirus RNA comprising the genomic 44S RNA, a double-stranded replicative form (RF) and a partially-double-stranded replicative intermediate (Rl) are detected by the incorporation of [α-³²P]GTP.

A. Preparation of virus-infected Vero cell extracts

Vero cells were infected at a multiplicity of infection of 7 for Type 2 dengue (DEN-2) virus (New Guinea C strain; Sabin and Schlesinger, 1945) or Kunjin (KUN) virus (strain MRM 61 C; Boulton and Westaway, 1972). Extracts containing RNA-dependent RNA polymerase (RDRP) activity derived from DEN-2 virus-infected cells were prepared at 30 to 36 h p.i., when polymerase activity was at a maximum. Similarly, extracts of KUN virus-infected cells were prepared at the time of maximum polymerase activity at 24 h p.i. (Chu and Westaway, 1985).

The cells were pelleted by centrifugation and resuspended in 10 mM sodium acetate at a concentration of 2x10⁷ cells /ml. They were then disrupted by passaging 20 times through a 21 gauge needle followed by 20 times through a 26 gauge needle. The disrupted cells were centrifuged at 800 g for 7 min to obtain a supernatant fraction and a pellet of the nuclear-associated material. All RDRP assays were performed using the supernatant fraction, hereafter referred to as the cell extract, which was stored at -70°C and used after only one cycle of

freeze/thawing.

B. RNA-dependent RNA polymerase assay

The RDRP activity in the cell extract was assayed as previously described with the following modifications (Chu and Westaway, 1985). In each RDRP assay the virus-infected cell extract contained 4.5-6 mg/ml of protein. The compound to be tested dissolved in double distilled water and RNasin (0.5 units /ml, Promaga) were added to the cell extract for 10 min prior to the addition of the other components of the RDRP assay. The final reaction mixture (total volume of 50 μl) contained 50 mM Tris-HCl pH 8.0, 10 mM magnesium acetate, 7.5 mM potassium acetate, 10 mM 2-mercaptoethanol, 6 μg actinomycin D (AND), 5 mM phosphoenolpyruvate, 3 units/ μl pyruvate kinase, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 25 μM GTP, 5 μCi [a-³²P] GTP (Amersham, specific activity 410 Ci/mmol), 0.5 units/ml RN asin, 30 μl of infected cell extract and the test compound (from 0.5 to 100 μM). The reaction was stopped after 30 min at 37°C by the addition of EDTA to a final concentration of 10 mM. An equal volume of TNE-SDS (50 mM Tris-acetate pH 7.6, 0.1 M sodium acetate, 1 mM EDTA and 2% SDS) was added to disrupt membranes. The RNA was then extracted with phenol and precipitated by ethanol.

C. Electrophoresis of RNA

RNA samples were mixed with an equal volume of sample buffer containing 7 M urea in TBE (89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA) and 0.5% bromophenol blue, and were separated by electrophoresis through 3%

polyacrylamide gels containing 7 M urea in TBE. The gels were fixed in 10% acetic acid, dried and radiolabelled bands detected by autoradiography.

Results

The compounds tested inhibited the synthesis of both DEN-2 and KUN RF RNA. There was also a decrease in the amount of Rl detected with increasing

concentration of drug. The 50% and peak inhibitory concentrations are given in Table 2.

a Cone, at which > 75% inhibition of RNA synthesis is achieved EXAMPLE 3: Tablet Formulations

The following formulation A may be prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression. mg/tablet

Formulation A

(a) Active ingredient 250 250

(b) Lactose B.P. 210 26

(c) Povidone B.P. 15 9

(d) Sodium starch glycollate 20 12

(e) Magnesium stearate 5 3

-------- ---------

500 300

The following formulation B, may be prepared by direct compression of the admixed ingredients.

Formulation B mg/capsule

Active ingredient 250

Pregelatinised starch NF15 150

-----------

400

Formulation C (Controlled release formulation)

This formulation may be prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression.

mg/tablet

(a) Active ingredient 500

(b) Hydroxypropylmethylcellulose 112

(methocel K4M Premium) (c) Lactose B.P. 53

(d) Povidone B.P.C. 28

(e) Magnesium stearate 7

---------

700

EXAMPLE 4: Capsule Formulations

Formulation A

A capsule formulation may be prepared by admixing the ingredients of Formulation B in Example 3 above and filling into a two-part hard gelatin capsule. Formulation B (infra) may be prepared in a similar manner.

Formulation B

mg/capsule

(a) Active ingredient 250

(b) Lactose B.P. 143

(c) Sodium starch glycollate 25

(d) Magnesium stearate 2

---------

420

Formulation C (Controlled release capsule)

The following controlled release capsule formulation may be prepared by extruding ingredients a, b and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets may then be coated with

release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule.

mg/capsule

(a) Active ingredient 250

(b) Microcrystalline cellulose 125

(c) Lactose B.P. 125 (d) Ethyl cellulose 13

-------

513

EXAMPLE 5: Injectable Formulation

Formulation:

Active ingredient 0.200 g

Hydrochloric acid solution, 0.1M qs to pH 5.0-7.0

Sodium hydroxide solution, 0.1M qs to pH 5.0-7.0

Sterile water qs to 10 ml

The active ingredient may be dissolved in most of the water (35°-40°C) and the pH adjusted to between 5.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch may then be made up to volume with the water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.

References

BOULTON, R.W. AND WESTAWAY, E.G. (1972).

Comparisons of Togaviruses:Sindbis virus (Group A) and Kunjin virus (Group B).

Virology 49, 283-289.

CHU, P.W.G. AND WESTAWAY, E.G. (1985).

Replication strategy of Kunjin virus:evidence for recycling, role of the replicative form RNA as template in semiconservative and asymmetric replication. Virology 140, 68-79.

CHU, P.W.G. AND WESTAWAY, E.G. (1987).

Characterization of Kunjin virus RNA-dependent RNA polymerase:reinitiation of synthesis in vitro. Virology 157, 330-337.

GRUN, J.B. AND BRINTON, M.A. (1986).

Characterisation of West Nile virus RNA-dependent RNA polymerase and cellular adenylyl and uridylyl transferases in cell-free extracts. Journal of Virology 60,

1113-1124.

SABIN, A.B. AND SCHLESINGER, R.W. (1945).

Production of immunity to dengue with virus modified by propagation in mice.

Science 101, 640-642.

Claims

CLAIMS:

1. A method for the treatment or prophylaxis of a flavivirus-associated infection, which comprises the use of an effective amount of at least one

heteropolytungstate compound selected from:

(a) a compound of the general formula I to VII:

(i) A_n[XW₁₂O₄₀] I wherein X is selected from P^V,Si^IV,Ge^IV,Co^II,Co^III-Zn^II,Cu^II,

B^III,H^I ₂,Al^III,Fe^IIl,V^V,Ga^III,Mn^IV,C^IV;

(ii) A_n[X₂W₁₈O₆₂] II wherein X is P^V;

(iii) A_n[XW₁₁O₃₉] III wherein X is selected from P^V,Si^IV,Ge^IV,B^llI,Al^lII,Ga^lII,Fe^lII,Co^lll;

(iv) A_n[XW₉ O₃₄] IV wherein X is selected from P^V,Si^IV,Ge^IV;

(v) A_n[X₂W _17-mM_mO₆₁] V wherein X is P^V,

M is Mo^VI, and

m is 0, 1, 2, 4, or 5;

(vi) A_n[X₂W _15-mM_mO₅₆] VI wherein X is P^V,

M is V^V or Mo^VI, and

m is 0 or 1;

(vii) A_n[XM₂W₉O₃₉] VII wherein X is P, and

M is Zr;

and wherein in each of the general formulae I to VII, A is a cation, and

n is the number of cations necessary for electrical neutrality of the molecule;

(b) a dimer of a said compound of the general formula I to VII; or

(c) a hydrate or pharmaceutically acceptable derivative thereof.

2. A method as claimed in claim 1, wherein the cation A of the compound of the general formula I to VII is a proton, an alkali metal ion, an alkaline earth metal ion, an ammonium ion or an alkylammonium ion of the formula R_{4- n}H_nN⁺, wherein R is an alkyl group of from 1 to 6 carbon atoms and n is 1, 2, or 3.

3. A method as claimed in claim 1, wherein the compound of the general formula I to VII is selected from:

H₃PW₁₂O₄₀.nH₂O

Na₃PW₁₂O₄₀.nH₂O

H₄SiW₁₂O₄₀.nH₂O

Na₄SiW₁₂O₄₀.nH₂O

K₅BW₁₂O₄₀.nH₂O

(NMe₃H)₃VW₁₂O₄₀.nH₂O

K₆[CoW₁₂O .nH₂O

K₆[ZnW₁₂O₄₀].nH₂O

(NMe₃H)₆[H₂W₁₂O₄₀].nH₂

NaH₃[GeW₁₂O₄₀].nH₂O

K₆[P₂W₁₈O₆₂].nH₂O

K₇PW₁₁O₃₉.nH₂O

α-K₈SiW₁₁O₃₉.nH₂O K₇NaGeW₁₁O₃₉.nH₂O

A-Na₈HPW₉O₃₄.nH₂O

Δ-Na₈HPW₉O₃₄.nH₂O

Na₉H[A-β-SiW₉O₃₄].nH₂O

Na₁₀[GeW₉O₃₄].nH₂O

K₁₀[P₂W₁₇O₆₁].nH₂O

K₉Li[P₂W₁₅Mo₂O₆₁].nH₂O

α-Na₁₂P₂W₁₅O₅₆.nH₂O

Na₁₂P₂MoW₁₄O₅₆.nH₂O

K₇[Zr₂PW₉O₃₉H₄].nH₂O. or a pharmaceutically acceptable derivative thereof.

4. A method as claimed in any of claims 1 to 3, wherein said

heteropolytimgstate compound is administered in an amount effective to inhibit flavivirus replication.

5. A method as claimed in any of claims 1 to 4, wherein said

heteropolytungstate compound is administered in association with one or more pharmaceutically acceptable carriers or diluents.

6. A method as claimed in any of claims 1 to 5, wherein said flavivirus- associated infection is yellow fever, dengue fever, Australian encephalitis, Japanese encephalitis, or Hepatitis C.

7. A method as claimed in any of claims 1 to 6 for the treatment or prophylaxis of a flavivirus-associated infection in a patient in need of such treatment or prophylaxis, which comprises the administration to said patient of said effective amount of said at least one heteropolytungstate compound.

8. Use of at least one heteropolytungstate selected from (a) a compound of the general formula I to VII as defined in claim 1;

(b) a dimer of a said compound of the general formula I to VII; or

(c) a hydrate or pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for the treatment or prophylaxis of a flavivirus- associated infection.

9. A pharmaceutical composition for the prophylaxis or treatment of a flavivirus-associated infection, which comprises at least one heteropolytungstate selected from

(a) a compound of the general formula I to VII as defined in claim 1;

(b) a dimer of a said compound of the general formula I to VII; or

10. A pharmaceutical composition according to claim 9, wherein said compound of the general formula I to VII is selected from:

H₃PW₁₂O₄₀.nH₂O

Na₃PW₁₂θ₄₀.nH₂O

H₄SiW₁₂O₄₀.nH₂O

Na₄SiW₁₂O₄₀.nH₂O

K₅BW₁₂O₄₀.nH₂O

(NMe₃H)₃VW₁₂O₄₀.nH₂O

K₆[CoW₁₂O₄₀].nH₂O

K₆[ZnW₁₂O₄₀].nH₂O

(NMe₃H)₆[H₂W₁₂O₄₀].nH₂O

NaH₃[GeW₁₂O₄₀].nH₂O

K₆[P₂W₁₈O₆₂].nH₂O

K₇PW₁₁O₃₉.nH₂O

α-K₈SiW₁₁O₃₉.nH₂O

K₇NaGeW₁₁O₃₉.nH₂O

A-Na₈HPW₉O₃₄.nH₂O Δ-Na₈HPW₉O₃₄.nH₂O

Na₉H[A-β-SiW₉O₃₄].nH₂O

Na₁₀[GeW₉O₃₄].nH₂O

K₁₀[P₂W₁₇O₆₁].nH₂O

K₉Li[P₂W₁₅Mo₂O₆₁].nH₂O

α-Na₁₂P₂W₁₅O₅₆.nH₂O

Na₁₂P₂MoW₁₄O₅₆.nH₂O

11. A pharmaceutical composition according to claim 9 or claim 10 further comprising at least one other antiviral or therapeutic agent.

12. A heteropolytungstate compound selected from

(a) a compound of the general formula I to VII as defined in claim 1;

(b) a dimer of a said compound of the general formula I to VII; or

(c) a hydrate or pharmaceutically acceptable derivative thereof.

13. A compound according to claim 12, selected from:

K₇[Zr₂PW₉O₃₉H₄].nH₂O.

or a pharmaceutically acceptable derivative thereof.