WO2006037958A2 - Treatment of helminth infection by inhibition of tyrosinase - Google Patents

Abstract

This invention relates to the finding that the inhibition of tyrosinase activity, for example using Kojic acid or related compounds, reduces egg production by helminths and may therefore be useful in the treatment of helminthiasis.

Description

Treatment of Helminth Infection by Inhibition of Tyrosinase

This invention relates to agents and methods useful in the treatment of helminth infection, in particular to schistosome infection and schistosomiasis.

There are three major groups of helminths containing members that have man as their main host: digenean flukes (trematodes) , tapeworms (cestodes), and roundworms (nematodes) .

The most significant helminth parasites in humans, in terms of morbidity and mortality, are digean flukes of the schistosome family. The three major schistosome species that infect man are the African species Schistosoma haematobium and Schistosoma mansoni and the Asian species, Schistosoma japonicum.

Within definitive vertebrate hosts, schistosome sexual reproduction leads to prolific egg production that allows tine parasite to maintain genetic heterozygosity, and ensures survival through highly efficient gamete transmission.

However, for the host, one major adverse consequence of this process results from inflammatory reactions directed against the eggs that, instead of being transmitted to the outside environment with faeces or urine, become trapped in tissues such as the liver and intestines. These host-mediated immunological responses, if uncontrolled, precipitate a series of pathological reactions that can ultimately lead to the death of infected individuals (Hoffmann KF et al. Adv Parasitol 2002; 52:265-307) .

Currently, it is estimated that at least 200 million human infections exist in 74 tropical and sub-tropical countries, leading to chronic debilitating disease and up to 300 000 human deaths per year (Fenwick A, et al. Trends Parasitol 2003; 19(11) : 509-15) . Although an effective anti-schistosome drug praziquantel has been available for 25 years, its mechanism of action remains unclear and the number of human infections stubbornly refuses to decline. Because of the stable prevalence of disease and the emerging possibility of drug resistance, there exists an urgent need to identify novel chemotherapeutic and immunoprophylactic targets useful for future disease intervention programmes.

The present inventors have discovered that inhibition of tyrosinase activity leads to reduced egg production by helminths and the targeting of helminth tyrosinases may therefore be useful in reducing the symptoms and mortality associated with helminthiasis.

One aspect of the present invention provides a method of treating helminthiasis in an individual comprising; administering a tyrosinase inhibitor to said individual.

Other aspects of the invention provide a tyrosinase inhibitor for use in the treatment of helminthiasis and the use of a tyrosinase inhibitor in the manufacture of a medicament for the treatment of helminthiasis.

Helminthiasis may include infection with any helminth which produces eggs in order to transmit its gametes. Examples of helminths which may be treated in accordance with the present methods include: roundworms (nematodes) such as Ascaris lumbricoides, Enterobius vermicularis, Trichuris trichuria, Trichinella spiralis, Strongyloides stercoralis, Ostertagia circumcincta and Cooperia spp; hookworms such as Necator americanus and Ancylostoma duodenale; filarial worms (nematodes) such as Dirofilaria immitis_r Wuchereria bancrofti, Brugia malayi, and Onchocerca volvulus; digenean flukes (trematodes) including Schistosoma spp such as Schistosoma haematobium, Schistosoma mansoni and Schistosoma japonica, Opisthorchis sinensis, Clonorchis sinensis, Fasciola spp, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogauai and Paragonimus sp. and tapeworms such as Taenia saginata and Taenia solium.

In some preferred embodiments, the helminth may be a Schistosoma spp such as Schistosoma haematobium, Schistosoma mansoni and

Schistosoma japonica. Infection with a helminth of a Schistosoma spp is generally termed schistosomiasis.

Inhibitors of helminth tyrosinases may include kojic acid (KA: 5- hydroxy-2- (hydroxymethyl) -4H-pyran-4-one) , vitamin C, cysteine, arbutin, glutathione, hydroquinone (see, for example US4526179 JP27909/86, JP-59157009) , hydroquinone ethers, retinoids (see, for example EP341664A1, WO99/15148, US4959393, US6132740, WO 00/56702), 4-n-butylresorcinol, 4-isoamyl resorcinol, ascorbic acid, 2, 5-hydroxyphenylcarboxylic acid derivatives (see, for example US5449518) , chalcones, chamaecin, colloidal sulphur benzoic acids (see, for example US5580549, JP 10081626), hydroxyphenyl oxamates (see, for example US6159482) , phenolic amides (see, for example WO99/32077), oxydesberatrols (see, for example JP10072330) , flavonoids (see, for example JP10101543) , copper chelators such as diethyldithiocarbamic acid (DETC) , and modifications, analogues and derivatives of these compounds.

In preferred embodiments, the tyrosinase inhibitor is a kojic acid compound. Kojic acid compounds include kojic acid and analogues, derivatives and modified forms of kojic acid. Various modified kojic acid compounds are known in the art (see, for example US4278656, US4369174, JP5221846, JP8134090, JP5320025) .

Analogues, derivatives and modified forms of kojic acid (i.e. kojic acid compounds) may have the general formula I:

wherein:

X is an oxygen atom or a sulphur atom; Y is an oxygen atom, a sulphur atom, or an NH group; Z is an oxygen atom, a sulphur atom, or an NH group; R₁, R₂, R₃ and R₄ are independently selected from H and the substituents listed below; and

L is optionally substituted C_x._η alkylene, C₅_₆ arylene, Cχ-₇ alkylene-C₅_₆ arylene or C₅-₆ arylene-Ci_₇ alkylene.

Preferably, X is an oxygen atom. Preferably, Y is an oxygen atom. Preferably, Z is an oxygen atom.

Preferably, L is Ci_₄ alkylene. More preferaby, L is methylene.

Preferably, R_x is selected from H, halo, cyano, carboxy, formyl, optionally substituted Ci_₇ alkyl, C₅-₂o aryl, C₃_₂₀ heterocyclyl, Ci- 7 alkylene-C₅_₂₀ aryl, C₁^ alkylene-C₃_₂₀ heterocyclyl, acyl, ester, amido, amino and sulfone.

More preferably, R_x is selected from H, optionally substituted Ci_₇ alkyl, optionally substituted phenyl, and optionally substituted Ci-₇ acyl. Preferred unsubstituted C₁-.₇ alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl and tert- butyl. Preferred substituted C₁-₇ alkyl groups include haloalkyl groups, e.g. -CX¹ ₃, -CHX'₂, -CH₂X', -CH₂CH₂X', -CH₂CHX'₂, and -CH₂CX'₃ wherein X' is selected from F, Cl, Br or I . Preferred C₁- ₇ acyl groups are -C(=O)Me, -C(=O)Et, -C(=O)Pr, -C(=O)Bu, and -C(=O)Ph.

It is most preferred that R₁ is H. Preferably, R₃ is selected from H, halo, cyano, carboxy, formyl, optionally substituted Ci_₇ alkyl, C₅-₂o aryl, C₃_₂₀ heterocyclyl, Ci-. 7 alkylene-C₅_₂o aryl, Ci_₇ alkylene-C₃-₂o heterocyclyl, acyl, ester, amido, amino, sulfone, an amino acid or polypeptide chain wherein Z is bonded, to the terminal carbon atom of the amino acid or polypeptide chain, and

-C(=O)R₅ wherein R₅ is an amino acid or polypeptide chain wherein the carbon atom of said C(=0) group is bonded to the terminal nitrogen atom of the amino acid or polypeptide chain.

More preferably, R₃ is selected from H, optionally substituted Cχ_₇ alkyl, optionally substituted phenyl, and optionally substituted Ci-₇ acyl. Even more preferably, R₃ is H.

Preferably, R₂ and R₄ are independently selected from halo; OH; -OMe, -OEt, -O(tBu); -OCH₂Ph; -SH; -SMe, -SEt, -S(tBu), - SCH₂Ph; -C(=O)H; -C(=0)Me, -C(=O)Et, -C(=O) (tBu) and -C(=0) Ph; -C(=O)0H; —C(=O)OMe, -C(=O)OEt and -C (=0) 0(tBu) ; -C(=O)NH₂, -C (=0)NHMe, -C (=0)NMe₂, -C (=0)NHEt; -NHC(=0)Me, -NHC (=0)Et,

-NHC (=0) Ph, -NH₂, -NHMe, -NHEt, -NH(iPr), -NH (nPr) , -NMe₂, -NEt₂, -N(iPr)₂, -N(nPr)₂, -N(nBu)₂ and -N(tBu)₂; -CN; -NO₂; -Me, -Et, - nPr, -iPr, -nBu and -tBu; -CF₃, -CHF₂, -CH₂F, -CCl₃, -CBr₃, -CH₂CH₂F, -CH₂CHF₂, -CH₂CF₃; -OCF₃, -OCHF₂, -OCH₂F, -OCCl₃, -OCBr₃, -OCH₂CH₂F, -OCH₂CHF₂, -OCH₂CF₃; -CH₂OH, -CH₂CH₂OH, -CH(OH)CH₂OH;

-CH₂NH^-CH₂CH₂NH₂, -CH₂CH₂NMe₂; and substituted or unsubstituted phenyl.

It is preferred that one of R₂ and R₄ is H. More preferably, both R₂ and R₄ axe H.

For convenience, many chemical moieties are represented using well known abbreviations, including but not limited to, methyl (Me) , ethyl. (Et) , n-propyl (nPr) , iso-propyl (iPr) , n-butyl (nBu) , sec—butyl (sBu) , iso-butyl (iBu) , tert-butyl (tBu) , n- hexyl (nHex) , cyclohexyl (cHex) , phenyl (Ph) , biphenyl (biPh) , benzyl (Bn), naphthyl (naph), methoxy (MeO), ethoxy (EtO), benzoyl (Bz) , and acetyl (Ac) .

For convenience, many chemical compounds are represented using well-known abbreviations, including but not limited to, methanol (MeOH), ethanol (EtOH), iso-propanol (i-PrOH) , methyl ethyl ketone (MEK) , ether or diethyl ether (Et₂O) , acetic acid (AcOH) , dichloromethane (methyLene chloride, DCM) , acetonitrile (ACN) , trifluoroacetic acid (T¹FA) , dimethylformamide (DMF) , tetrahydrofuran (THF) , and dimethylsulfoxide (DMSO) .

As indicated herein, th.e analogues, derivatives and modified forms of kojic acid, including the compounds of formula I, may be unsubstituted or substituted (i.e. optionally substituted) by one or more functional groups. Examples of substituents include the R_x-R₄ groups shown in formula I. Thus, the phrase "optionally substituted", as used herein, pertains to a group, as above, which may be unsubstitiαted or which may be substituted by one of the following substituent groups or one of the groups listed above:

Unless otherwise specified, the term "substituted" means a parent group which bears one or more substituents. The term "substituent" is used herein in the conventional sense and refers to a chemical moiety wriich is covalently attached to, appended to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a. variety of parent groups are also well known.

By way of example , the substituent ( s ) , often referred to herein as Ri , R₂ , R₃ and R₄ , are independently selected from: Ci_₇alkyl ( including , e . g . , unsutDstituted C^alkyl , Ci_₇haloalkyl , Ci_₇hydroxyalkyl , Ci-_TCarboxyal kyl , Ci_₇aminoalkyl , C₅_₂₀aryl- Cχ-₇alkyl) ; C₃_₂₀heterocyclyl; C₅-₂oaryl (including, e.g., C₅-₂ocarboaryl, C₅_₂oheteroaryl, Ci-₇a__kyl-C₅-₂₀aryl and C₅_₂ohaloaryl) ) ; an optionally substituted C₁-7 alkyl group; a C₃-₂o heterocyclyl group; a C₅_₂o aryl groiαp; an optionally substituted heterocyclic ring having from 4 to 8 ring atoms; or one or more of the following substituent groups: Halo: -F, -Cl, -Br, and -I; Hydroxy: -OH; Ether: -OR, Ci_₇ alko;xy: -OR, wherein R is a C₁^ alkyl group; Ci_₂ alkdioxylene; Oxo (keto, -one) : =0; Imino (imine) : =NR; Formyl: -C(=O)H; Acyl (keto) : -C(=0)R; Ester: -C(=O)OR; Acyloxy: -OC (=0)R; Amido: -C(=O)NR¹R²; Acylamido:

-NR¹Ct=O)R²; Thioamido: -Ct=S)NR¹R²; Tetrazolyl; Amino: -NR¹R²; Amidine: -C(=NR)NR₂; Nitro: -NO₂; Nitroso: -NO; Azido: -N₃; Cyano: -CN; Isocyano: -NC; Cyanato: -OCN; Isocyanato: -NCO; Thiocyano: -SCN; Isothiocyano (isofchiocyanato) : -NCS; Sulfhydryl: -SH;

Thioether: -SR; Disulfide: -SS-R; SuIfone: -S(=O)₂R; SuIfine: -S(=O)R; Sulfonyloxy: -OSf=O)₂R; Su.Ifinyloxy: -OS(=O)R; SuIfamino: -NR¹S(=O)₂OH; Sulfonamino: -NR¹Sf=O) ₂R; SuIfinamino: -NR¹Sf=O)R, Sulfamyl: -S (=0)NR¹R²; Phosphoramidite: -OP (OR¹) -NR² ₂; and Phosphoramidate: -OP (=0) (OR¹) -NR² ₂, wherein R is a C₁-V alkyl, C₃_₂₀ heterocyclyl, or C₅-₂₀ aryl group.

Furthermore, the substituent (s) , often referred to herein as R₁, R₂, R₃ and R₄, may also be selected from an amino acid or a polypeptide chain, wherein the substituent is bonded to the compound of formula (I) via the terminal carbon atom of the amino acid or polypeptide chain, or via the terminal nitrogen atom of the amino acid or polypeptide chain. The synthesis of amino acid and peptide derivatives of kojic acid is described in JP5221846 and JP5320025.

The term "halo", as used herein, pertains to the monovalent moiety -Y, wherein Y is a halogen atom. Examples of halo groups include -F, -Cl, -Br, and -I. Alkyl: The term "alkyl, " as used herein, pertains to a monovalent moiety obtained by removing a hydrocjen atom from a carbon atom of a hydrocarbon compound having frrom 1 to 7 carbon atoms (unless otherwise specified) , which may b>e aliphatic or alicyclic, and which may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated) . Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, cycloalkyenyl, cylcoalkynyl, etc.

Aryl: The term "aryl," as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 20 ring atoms (unless otherwise specified) . Preferably, each ring has from 5 to 7 ring atoms .

In this context, the prefixes (e.g., C₅_₂o_/ C₅-₇, C₅_₆, etc.) denote the number of ring atoms, or range of number of: ring atoms, whether carbon atoms or heteroatoms. For example, the term "C₅_₆aryl," as used herein, pertains to an aryl group having 5 or 6 ring atoms. Examples of groups of aryl groups include C₅-₂₀aryl, C₅-i₅aryl, C₅_₁₂aryl, C₅-₁₀aryl, C₅-₇aryl, C₅_₆aryl, C₅aryl, and C₆aryl .

The ring atoms may be all carbon atoms, as in "carboaryl groups."

Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e., phenyl) (C₆), naphthalene (Ci₀), azulene (Ci₀) , anthracene (Ci₄) , phenanthrene (Ci₄) , naphthacene (C₁₈) , and pyrene (C₁₆) .

Examples of aryl groups which comprise fused r±ngs, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (C₉), indene (C₉), isoindene (C₉), tetraline (1,2, 3, 4-tetrahydronaphthalene (Ci₀) , acenaphthene (Ci₂), fluorene (Ci₃) , phenalene (C_i3) , acephenanthrene (C₁₅) , and aceanthrene (C_x6) . Alternatively, the ring atoms may include one or more heteroatoms, for example oxygen, nitrogen, and sulphur, as in

"heteroaryl groups".

C₃_₂o heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a C₃_₂o heterocyclic compound, said compound having one ring, or two or more rings (e.g., spiro, fused, bridged) , and having from 3 to 20 ring atoms, atoms, of which from 1 to 10 are ring heteroatoms, and wherein at leas^~t one of said ring(s) is a heterocyclic ring. Preferably, each rixig has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. "C₃_₂₀" denotes ring atoms, whether carbon atoms or heteroatoms.

In one preferred embodiment, the substituent (s) , for example those referred to herein as R₁, R₂, R₃ and R₄, are independently selected from:

-F, -Cl, -Br and -I; -OH;

-OMe, -OEt, -O(tBu) and -OCH₂Ph;

-SH;

-SMe, -SEt, -S(tBu) and -SCH₂Ph;

-C(=O)H; -C(=O)Me, -C(=O)Et, -C(=O) (tBu) and -C (=0) Ph;

-C(=O)OH;

-C(=O)OMe, -C(=O)OEt and -C(=0)0(tBu) ;

-C(=O)NH₂, -C (=0)NHMe, -C (=0)NMe₂ and -C(=0)NHEt;

-NHC(=0)Me, -NHC(=O)Et, -NHC (=0) Ph, succinimidyl and maleimidyl; -NH₂, -NHMe, -NHEt, -NH(iPr), -NH(nPr), -NMe₂, -NEt₂, -N(iPr)₂,

-N(nPr)₂, -N(nBu)₂ and -N(tBu)₂;

-CN;

-NO₂;

-Me , -Et, -nPr, -iPr, -nBu and -tBu; -CF₃, -CHF₂, -CH₂F, -CCl₃ , -CBr₃, -CH₂CH₂F, -CH₂CHF₂ and -CH₂CF₃ ; -OCF₃, -OCHF₂, -OCH₂F, -OCCl₃, -OCBr₃, -OCH₂CH₂F, -OCH₂CHF₂ and -OCH₂CF₃;

-CH₂OH, -CH₂CH₂OH and -CH(OH)CH₂OH; -CH₂NH₂,-CH₂CH₂NH₂ and -CH₂CH₂NMe₂; and, substituted or unsubstituted phenyl.

Examples of phenyl substituents include, but are not limited, to those discussed below under the heading "substituents".

If the phenyl group has less than the full complement of substituents, they may be arranged in any combination. For example, if the phenyl group has a single substituent other than hydrogen, it may be in the 2-, 3-, or 4-position. Similarly, if the phenyl group has two substituents other than hydrogen, they may be in the 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3, 5-positions. If the phenyl group has three substituents other than hydrogen, they may be in, for example, the 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,5,6-, or 3, 4, 5-positions. If the phenyl group has four substituents other than hydrogen, they may be in, for example, the 3,4,5,6-, 2,4,5,6-, 2,3,5,6-, 2,3,4,6-, or 2, 3, 4, 5-positions. Preferred substituted phenyl groups are singly or doubly substituted at the 2, 3 or 4-positions. Preferred substituents are halo groups, alkyl groups or ester groups.

In one preferred embodiment, the substituent (s) , often referred to herein as R₁, R₂, R₃ and R₄, are independently selected from: -F, -Cl, -Br, -I, -OH, -OMe, -OEt, -SH, -SMe, -SEt, -C(=0)Me, - C(=O)OH, -C(=0)0Me, -CONH₂, -CONHMe, -NH₂, -NMe₂, -NEt₂, -N(nPr)₂, -N(IPr)₂, -CN, -NO₂, -Me, -Et, -CF₃, -OCF₃, -CH₂OH, -CH₂CH₂OH, -CH₂NH₂, -CH₂CH₂NH₂ and -Ph.

In one preferred embodiment, the substituent (s) , often referred to herein as Ri, R₂, R₃ and R₄, are independently selected from: hydroxy; ether (e.g., Ci-₇alkoxy) ; ester; amido; amino; and, C^alkyl (including, e.g., unsubstituted Ci_₇alkyl, Ci_₇haloalkyl, C₁_₇hydroxyalkyl , Ci-₇carboxyalkyl , C^aminoalkyl , C₅-₂₀aryl- C_!-₇alkyl ) .

In one preferred embodiment , the substituent ( s ) , often referred to herein as R₁ , R₂ , R₃ and R₄ , are independently selected from :

-OH;

-OMe , -OEt , -O ( tBu) and -OCH₂Ph;

-C (=0 ) 0Me , -C (=O ) OEt and -C ( =0 ) 0 ( tBu ) ;

-C(=O)NH₂, -C (=0)NHMe, -C (=0)NMe₂ and -C (=0)NHEt; -NH₂, -NHMe, -NHEt, -NH(iPr) -NH(nPr), -NMe₂, -NEt₂, -N(iPr)₂,

-N(nPr)₂, -N(nBu)₂ and -N(tBu)₂;

-Me, -Et, -nPr, -iPr, -nBu, -tBu;

-CF₃, -CHF₂, -CH₂F, -CCl₃, -CBr₃, -CH₂CH₂F, -CH₂CHF₂, and -CH₂CF₃;

-CH₂OH, -CH₂CH₂OH, and -CH(OH)CH₂OH; and, -CH₂NH₂₇-CH₂CH₂NH₂ and -CH₂CH₂NMe₂.

Alkylene: The term "alkylene, " as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 7 carbon atoms (unless otherwise specified) , which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub¬ classes alkenylene, alkynylene, cycloalkylene, etc.

In this context, the prefixes (e.g., C^₄, Cχ-₇, C₂_₇, C₃_₇, etc.) denote the number of carbon atoms, or range of number of carbon atoms. For example, the term "C₁.-_? alkylene, " as used herein, pertains to an alkylene group having from 1 to 7 carbon atoms. Examples of groups of alkylene groups include Ci_₄ alkylene ("lower alkylene"), and C₁-_? alkylene.

Examples of linear saturated C_1-.-_? alkylene groups include, but are not limited to, -(CH₂)_n- where n is an integer from 1 to 7, for example, -CH₂- (methylene) , -CH₂CH₂- (ethylene) , -CH₂CH₂CH₂- (propylene), -CH₂CH₂CH₂CH₂- (butylene) , and -CH₂CH₂CH₂CH₂CH₂CH₂- (hexylene) .

Arylene: The term "arylene," as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of an aromatic compound, which moiety has from 5 to 10 ring atoms (unless otherwise specified) . Preferably, each ring has from 5 to 7 ring atoms, more preferably from 5 to 6 atoms.

In this context, the prefixes (e.g., C₅-₁₀, C₅-₇, C₅_₆, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C₅_₆arylene, " as used herein, pertains to an arylene group having 5 or 6 ring atoms. Examples of groups of arylene groups include C₅_₁₀arylene, C₅-₇arylene, C₅_₆arylene, C₅arylene, and C₆arylene.

The ring atoms may be all carbon atoms, as in "carboarylene groups" (e.g., C₅_₁₀carboarylene) .

Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroarylene groups" (e.g., C₅-₁₀ heteroarylene) .

Arylene-alkylene: The term "arylene-alkylene, " as used herein, pertains to a bidentate moiety comprising an arylene moiety, -Arylene-, linked to an alkylene moiety, -Alkylene-, that is, -Arylene-Alkylene-.

Alkylene-arylene: The term "alkylene-arylene, " as used herein, pertains to a bidentate moiety comprising an alkylene moiety, -Alkylene-, linked to an arylene moiety, -Arylene-, that is, -Alkylene-Arylene-.

The analogues, derivatives and modified forms of kojic acid include the compounds of formula I derivatised in various ways. As used herein "derivatives" of these compounds includes well known ionic, salt, solvate and protected forms of the compounds or their substituents mentioned herein. For example, a reference to carboxylic acid (-COOH) also includes the anionic

(carboxylate) form (-COO^") , a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N⁺HR¹R²) , a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0^" ) , a salt or solvate thereof, as well as conventional protected forms.

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z- forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms") .

Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space) . For example, a reference to a methoxy group, -OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., Ci_₇alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl) .

The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

keto enol enolate

Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D) , and ³H (T) ; C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; 0 may be in any isotopic form, including ¹⁵O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al. , 1977, "Pharmaceutically Acceptable Salts,", J. Pharm. Sci., Vol. 66, pp. 1-19.

For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO^"), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺) . Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃) ₄ ⁺.

If "the compound is cationic, or has a functional group which may be cationic (e.g., -NH₂ may be -NH₃ ⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inoxganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ettianesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g., active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono- hydrate, a di-hydrate, a tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle the active compound in a chemically protected form. The term "chemically protected form" is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, and the like) . In practice, well-known chemical methods are employed to reversibly render unreactive a functional group, which otherwise would be reactive, under specified conditions. In a chemically protected form, one or more reactive functional groups are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group) . By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting gαroup may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999) . A wide variety of such "protecting", "blocking" or "masking" methods are widely used and well known in organic synthesis. For example, a compound which has two nonequivalent reactive functional groups, both of which would be reactive under specified conditions, may be derivatized to render one of the functional groups "protected" and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group. After the desired reaction (involving the other functional group) is complete, the protected group may be "deprotected" to return it to its original functionality.

For example, a hydroxy group may be protected as an ether (-OR) or an ester (-OC(=O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl) , or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (-OC(=0)CH₃, -OAc) .

For example, an aldehyde or ketone group may be protected as an acetal (R-CH(OR)₂) or ketal (R₂C(OR)₂)_/ respectively, in which the carbonyl group (>C=0) is converted to a diether (>C (OR)₂)_/ by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amide (-NRC0-R) or a urethane (-NRCO-OR) , for example, as: a methyl amide (-NHCO-CH₃); a benzyloxy amide (-NHCO-OCH₂C₆H₅, -NH- Cbz); as a t-butoxy amide ( -NHCO-OC (CH₃) ₃, -NH-Boc) ; a 2-biphenyl- 2-propoxy amide (-NHCO-OC(CHa)₂C₆H₄C₆H₅, -NH-Bpoc) , as a 9- fluorenylmethoxy amide (-NH-Fmoc) , as a 6-nitroveratryloxy amide (-NH-Nvoc) , as a 2-trimethylsilylethyloxy amide (-NH-Teoc) , as a 2, 2,2-trichloroethyloxy amide (-NH-Troc) , as an allyloxy amide (-NH-Alloc) , as a 2 (-phenylsulphonyl) ethyloxy amide (-NH-Psec) ; or, in suitable cases (e.g., cyclic amin.es), as a nitroxide radical (>N-O) .

For example, a carboxylic acid group may be protected as an ester for example, as: an Ci_₇alkyl ester (e.g. , a methyl ester; a t- butyl ester); a Ci-vhaloalkyl ester (e.g. , a Ci-₇trihaloalkyl ester); a triCi-valkylsilyl-Cx-valkyl ester; or a C₅-₂oaryl-Ci-.7alkyl ester (e.g., a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide.

For example, a thiol group may be protected as a thioether (-SR) , for example, as: a benzyl thioether; an acetamidomethyl ether (- S-CH₂NHC (=0) CH₃) .

It may be convenient or desirable to prepare, purify, and/or handle the active compound in the form of a prodrug. The term "prodrug" as used herein, means a compound which, when metabolised (e.g., in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties -

For example, some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). During metabolism, the ester group (-C(=O)OR) is cleaved to yield the active drug. Such esters may be foitrmed by esterification, for example, of any of the carboxylic acid groups (-C(=O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required.

Examples of such metabolically labile esters include those of the formula -C(=O)OR wherein R is: Ci_₇alkyl (e.g., -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu) ; Ci-₇aminoalkyl (e.g., aminoethyl; 2- (N,N—diethylamino) ethyl; 2- ( 4-morpholino ) ethyl ) ; and acyloxy-C₁_₇alkyl (e.g., acyloxymethyl; acyloxyethyl; pivaloyloxymethyl; acetoxymethyl; 1-acetoxyethyl; 1— (1-methoxy-l- methyl) ethyl-carbonxyloxyethyl; 1- (benzoyloxy) ethyl; isopropoxy- carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl; c^/clohexyl- carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy- carbonyloxyethyl; (4-tetrahydropyranyloxy) carbonyloxymethyl; 1- (4-tetrahydropyranyloxy) carbonyloxyethyl; (4- tetrahydropyranyl) carbonyloxymethyl; and 1- (4-tetrahydropyranyl) carbonyloxyethyl) .

Also, some prodrugs are activated enzymatically to ;yield the active compound, or a compound which, upon further chemical reaction, yields the active compound (for example, as in ADEPT, GDEPT, LIDEPT, etc.) . For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

Administration of the tyrosinase inhibitor is prefexably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, an<d rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A tyrosinase inhibitor as described herein may be administered as a pharmaceutical composition. A pharmaceutical composition may include, in addition to the tyrosinase inhibitor, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those s killed in the art. Such materials should be non-toxic and should, not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Another aspect of the invention provides a method of producing a pharmaceutical for use in treating helminthiasis comprising; admixing a tyrosinase inhibitor with a pharmaceutically acceptable excipient, carrier, buffer or stabiliser.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include 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.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, 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, or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Suitable formulations in accordance with the invention include non-topical formulations. For example, the tyrosinase inhibitor may be in an oral or parenteral formulation. A composition comprising a tyrosinase inhibitor may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Another aspect of the present invention provides a method of treating helminthiasis in a plant comprising; applying a tyrosinase inhibitor to said plant or the locus thereof.

Examples of tyrosinase inhibitors are described in more detail above.

Examples of helminths that are pathogenic in plants and which may be treated in accordance with the present methods include Meloidogyne spp, Heterodera spp, and Globodera spp.

The locus of the plant relates to the environment or surroundings of the plant and may include the field in which the plants are growing, or where the seeds of cultivated plants are sown, and the place where the seed will be placed into the soil.

Any plant susceptible to helminth infection may be treated in accordance with the invention. Examples of suitable plants include tobacco, cucurbits, carrot, vegetable brassica, melons, capsicums, grape vines, lettuce, strawberry, oilseed brassica, sugar beet, wheat, barley, maize, rice, soyabeans, peas, sorghum, sunflower, tomato, potato, pepper, alfafa, oat, vetch, cowpea, clover, snap bean and dry edible bean.

The tyrosinase inhibitor may be applied to the plant by any convenient method, e.g. spraying.

The tyrosinase inhibitor may be formulated for use in treating helminthiasis. For example, an agrochemical composition be formulated as an instant granulate, a flowable formulation, an emulsion concentrate or a wettable powder and may be used in combination with agriculturally acceptable adjuvants. Such formulations or compositions may be produced in conventional manner, e.g. by mixing the active ingredients with appropriate adjuvants (e.g. diluents or solvents and optionally other formulating ingredients such as surfactants) . Also conventional slow release formulations may be employed where long lasting efficacy is intended.

Another aspect of the invention provides a method of producing an agrochemical composition for use in treating helminthiasis in plants comprising; admixing a tyrosinase inhibitor with an agriculturally acceptable adjuvant or auxilliary.

Suitable formulation adjuvants and auxilliaries are well known in the art and are described, for example, in: Watkins, "Handbook of Insecticide Dust Diluents and Carriers", 2nd ed., Darland Books, Caldwell N. J., H. v. Olphen, "Introduction to Clay Colloid

Chemistry"; 2nd ed., J. Wiley & Sons, N.Y.; C. Marsden, "Solvents Guide"; 2nd ed., Interscience, N.Y. 1963; McCutcheon's "Detergents and Emulsifiers Annual", MC Publ. Corp., Ridgewood N.J.m Wiss. Verlagsgesell . , Stuttgart 1976; Winnacker-Kuchler, "Chemische Technologie" [Chemical Technology], Volume 7, C. Hauser Verlag Munich, 4th edition 1986; Wade van Valkenburg, "Pesticide Formulations", Marcel Dekker, N.Y., 1973; K. Martens, "Spray Drying" Handbook, 3rd ed. 1979, G. Goodwin Ltd. London.

Particularly formulations for use in spraying, e.g. water dispersible concentrates or wettable powders, may further comprise surfactants such as wetting and dispersing agents. Suitable wetting and dispersing agents are well known in the art.

A formulation may be a seed dressing formulation which may be applied to seeds. A seed dressing formulation may comprise the tyrosinase inhibitor and a diluent in suitable seed dressing formulation form, e.g. as an aqueous suspension or in a dry powder form having good adherence to the seeds. Such seed dressing formulations are known in the art. Seed dressing formulations may contain the tyrosinase inhibitor in encapsulated form, e.g. as slow release capsules or microcapsules.

An agrochemical formulation may also comprise other pesticidally active substances, for example insecticides, acaricides, herbicides and fungicides, and also with safeners, fertilizers and/or growth regulators, for example in the form of a ready-mix or tank mix.

In general, the formulations include from 0.01 to 90% by weight of active agent, from 0 to 20% agriculturally acceptable surfactant and 10 to 99.99% solid or liquid adjuvant(s) . Concentrated forms of compositions generally contain in between about 2 and 80%, preferably between about 5 and 70% by weight of active agent. Application forms of formulation may for example contain from 0.01 to 20% by weight, preferably from 0.01 to 5% by weight of active agent. Whereas commercial products will preferably be formulated as concentrates, the end user will normally employ dilute formulations.

For further details on the formulation of plant protection products, see for example G. C. Klingman, "Weed Control as a Science", John Wiley and Sons., Inc., New York, 1961, pages 81-96 and J. D. Freyer, S. A. Evans, "Weed Control Handbook", 5th ed., Blackwell Scientific Publications, Oxford, 1968, pages 101-103.

Other aspects of the invention relate to the use of helminth tyrosinases in the development of anti-helminth compounds. A method of identifying and/or obtaining a compound for the treatment of helminthiasis in an individual may comprise; contacting a helminth tyrosinase with a test compound, and; determining the activity of said tyrosinase in the presence of said test compound.

Tyrosinase activity in the presence of the test compound may be compared with activity in comparable reaction medium and conditions in the absence of a test compound. A reduction in activity in the presence of test compound relative to the absence is indicative that the test compound may be useful in the treatment of helminthiasis.

Tyrosinase activity may be determined by any convenient method. In general, tyrosinase activity may be assayed by measuring the accumulation of products or by-products, such as coupled reporter molecules or the disappearance or consumption of substrates. Various suitable tyrosinase assays are known in the art (see for example Winder AJ, Harris H. Eur J Biochem 1991; 198(2): 317-26). In some embodiments, the modified MBTH assay may be employed

(Winder et al (1991) supra) . Other suitable assays are described in Petris et al (2000) Human MoI. Gen. 9 19 2845-2851.

A helminth tyrosinase may be a tyrosinase from any helminth species whose gamete transmission is dependent on egg production. Suitable helminths are listed above. A helminth tyrosinase may show homology to S. mansoni tyrl (amino acid: AAP93838.1, nucleic acid: AY266330.1) or S. mansoni tyr2 (see SEQ ID NO: 1 and SEQ ID NO:2, also amino acid: AAW21822.1 GI:56682981, nucleic acid: AY675348.1 GI :56682980) .

For example, a helminth tyrosinase may comprise an amino acid sequence which shares greater than about 30% sequence identity with one or more of S. mansoni tyrl or S. mansoni tyr2, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 55%, greater than about 65%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%. The sequence may share greater than about 30% similarity with one or more of S. mansoni tyrl or S. mansoni tyr2, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity.

A suitable helminth tyrosinase may, for example, be a S. japonicum tyrosinase encoded by a nucleic acid comprising one or more of the sequences of database accession numbers BU791566, BU800153, BU801868, BU804015, BU803864, BU803670, BU802336, BU799939, BU799201, BU793439 and BU711606, Trichuris tyrosinase encoded by a nucleic acid comprising the sequences of database accession numbers BM277527 and/or BM277159, an Ancylostoma tyrosinase encoded by a nucleic acid comprising the sequences of database accession numbers CB272250 and/or CB189645, an Ostertagia/Cooperia tyrosinase encoded by a nucleic acid comprising the sequence of database accession number BQ099322, a Strongyloides tyrosinase encoded by a nucleic acid comprising the sequence of database accession number BE581134, a Heterodera tyrosinase encoded by a nucleic acid comprising the sequence of database accession number BI748071 and/or BI396860, an Globodera tyrosinase encoded by a nucleic acid comprising the sequence of database accession number AW505654 and/or AW505646 and/or a Meloidogyne tyrosinase.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Genetics Computer Group, Madison,

WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith- Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197) , or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used. Sequence identity and similarity may also be determined using Genomequest™ software (Gene-IT, Worcester MA USA) .

Sequence comparisons are preferably made over the full-length of the relevant sequence described herein.

Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.

In some embodiments, a helminth tyrosinase may be a schistosome tyrosinase, for example S. mansoni tyrl (AY266330) or tyr2 (AY675348: also shown in SEQ ID NO:1) or another tyrosinase set out above .

The helminth tyrosinase may be an isolated polypeptide or may be comprised in a cell, for example a host cell expressing a recombinant tyrosinase nucleic acid.

In other embodiments, the helminth tyrosinase may be in situ in a helminth, worm or comprised in a cell or extract thereof. An extract may be prepared, for example, by sonicating the helminth worm. A method of identifying and/or obtaining a compound for the treatment of helminthiasis in an individual may comprise; contacting a helminth with a test compound, and; determining the activity of tyrosinase in said helminth or an extract thereof in the presence of said test compound.

Suitable helminths are listed above.

The activity of tyrosinase in the helminth may be determined by producing an extract of the helminth, for example by sonication, and determining tyrosinase activity in the extract. Methods of determining tyrosinase activity are described elsewhere herein.

Prior to or as well as being screened for modulation of helminth tyrosinase activity, test substances may be screened for ability to bind with the helminth tyrosinase. This may be used as a coarse screen prior to testing a substance for actual ability to increase or enhance the activity of helminth tyrosinase activity. Methods for determining the binding of a test compound to a target polypeptide are well known in the art.

The amount of test substance or compound which may be added to a method described herein will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM.

A test compound suitable for use in the present methods may be a small chemical entity, peptide, antibody molecule or other molecule whose effect on helminth tyrosinase activity is to be determined. Natural or synthetic chemical compounds may be used, or extracts of plants which contain several characterised or uncharacterised components. Suitable test compounds may be selected from compound collections and designed compounds. Combinatorial library technology (Schultz, JS (1996) Biotechnol. Pαrog. 12:729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate helminth tyrosinase activity.

Other candidate compounds may be based on modelling the 3- dimensional structure of the helminth tyrosinase and using rational drug design to provide potential enhancer compounds with particular molecular shape, size and charge characteristics. Drug design is described in more detail below.

A suitable test compound may be a tyrosinase inhibitor, for example a tyrosinase inhibitor as set out above. In some preferred embodiments, the test compound may be kojic acid or an analogue, derivative or modification thereof (i.e. a kojic acid compound) .

The effect of a compound identified by a method described above may be assessed in a secondary scxeen. For example, the effect of the compound on helminth egg foriαation and production may be determined. Secondary screens may be performed in in vitro test systems or in vivo in animal models.

For example, in an in vitro test system, helminth worm pairs may be cultured in vitro and treated with a test compound, for example by supplementing the culture medium with the compound. The effect of the test compound on the formation of eggs by the worm pair may then be determined, for example by collecting and counting the eggs produced. Morpb-ological changes in the eggs may be detected using fluorescent or bright field microscopy.

In an in vivo test system, an an-Lmal model, for example a rodent such as a mouse, may be infected with helminth parasites and then treated with test compound, for example as a food or water supplement. The effect of tyrosinase inhibition may be determined relative to control-infected animals not treated with test compound. The effect of tyrosinase inhibition may be determined, for example by measuring faecal egg output and assessing the morphology and hatchability of these eggs. When the experiment is terminated, the animal may be sacrificed and the eggs present in the animal liver counted. Various immunological parameters such as cytokine and antibody levels may also Ibe measured. For example, levels of IL-4, IL-5, IL-13, IL-IO, IFN-gamma, and TNF- alpha and levels of total IgG and IgE, as well as IgG2a, IgG2b and IgGl subclasses may be measured. Liver patϊiology, for example granuloma volume around eggs, fibrosis, necrosis, etc. may also be assessed.

Control experiments may be performed as appropriate in the methods described herein. The performance of suitable controls is well within the competence and ability of a skilled person in the field.

A method as described herein may comprise identifying a test compound as an agent which inhibits the expres sion and/or activity of a helminth tyrosinase.

The identified compound may be isolated and/or purified. In some embodiments, the compound may be prepared, synthesised and/or manufactured using conventional synthetic techniques.

Optionally, compounds identified as agents which inhibit the expression and/or activity of a helminth tyrosinase using an method described herein may be modified or subjected to rational drug design techniques to optimise activity or- provide other beneficial characteristics such as increased h.alf-life or reduced side effects upon administration to an individual. In some embodiments, a compound may be subjected to rational drug design techniques to optimise its activity and/or pharmaceutical or agrochemical properties for the treatment of helminthiasis.

A method of producing a compound for treating helminthiasis may comprise; providing a test compound, and; determining the interaction of said test compound with a helminth tyrosinase.

Helminth tyrosinases are described in more detail above.

Suitable test compounds include known tyrosinase inhibitors, examples of which are set out above, and compounds identified as helminth tyrosinase inhibitors using the methods described above. In some preferred embodiments, the test compound may be a kojic acid (KA) compound, including both unmodified KA and modified forms of KA (i.e. modified KA compounds) .

The structure of the test compound may be modified to optimise the interaction of the compound with helminth tyrosinase or to improve its pharmaceutical properties, for example to reduce side effects associated with the compound, increase the half-life of the compound in vivo, reduce the cost of synthesis of the compound, improve bio availability or increase the suitability of the compound for a particular method of administration. The modification of test compounds, such as KA, is described in more detail below. A preferred initial or starting test compound for use in the present methods is KA.

A method may further comprise; modifying the structure of the test compound, and; determining the interaction of the modified test compound with a helminth tyrosinase. The methods described above may be iterated in that an optimised or modified test compound may itself be the basis for further optimisation and/or modification.

In some preferred embodiments, the interaction of a test compound with a helminth tyrosinase may be determined in silico i.e. using computer-assisted techniques.

For example, a method for producing an anti-helminth compound may comprise: providing a structure comprising a three-dimensional representation of a helminth tyrosinase or a portion of a helminth tyrosinase; providing an test compound structure to be fitted to said helminth tyrosinase structure or selected coordinates thereof fitting the test compound structure to said helminth tyrosinase structure.

Fitting includes determining, by automatic or semi-automatic means, interactions between at least one atom of a test compound molecular structure and at least one atom of a helminth tyrosinase structure, and calculating the extent to which such an interaction is stable. Interactions include attraction and repulsion, brought about by charge, steric considerations and the like. Various computer-based methods for fitting are described further herein.

A modified test compound, for example a modified KA compound, may be fitted by computer to the structure of the helminth tyrosinase to ascertain how well the shape and the chemical structure of the compound will bind to the helminth tyrosinase. The interaction of the modified test compound with helminth tyrosinase may be determined relative to the interaction of unmodified test compound with helminth tyrosinase. For example, the interaction of a test compound with a helminth tyrosinase can be examined through the use of computer modelling using a docking program such as DOCK (Kuntz et al, J.MoI.Biol. 1982 , 161, 269-288, Makino et al, J. Comput. Chem. 1991, 18, 1812- 1825), AUTODOCK (Goodsell et al, Proteins 1990, 8, 195-202, Morris et al, J. Comput. Chem. 1998, 19, 1639-1662; Scripps Research Institute, La Jolla, Calif.), FlexX, (Rarey et al, J.MoI.Biol. 1996, 261, 470-489), ICM (Abagyan et al, J.Comput. Chem. 1994, 15, 488-506), MCSS (Molecular Simulations, San Diego, Calif.) , QUANTA (Molecular Simulations, San Diego, Calif.), Insight (Molecular Simulations, San Diego, Calif.), SYBYL (TRIPOS, Inc., St. Louis. Mo.) or LEAPFROG (TRIPOS, Inc., St. Louis, Mo. ) .

The structure or functionality of the compound may be adjusted or modified in the light of the structural information obtained by computer modelling about the interaction of the test compound to helminth tyrosinase e.g. to alter its interaction with helminth tyrosinase. The above steps may be repeated and re-repeated as necessary.

A method may further comprise the step of modifying or optimising the structure of the test compound. In particular, the structure of the test compound may be modified to optimise binding to the helminth tyrosinase structure.

A structure may be optimised by making modifications to the structure, for example, by adding molecular scaffolding, adding or varying functional groups, or connecting the molecule with other molecules (e.g. using a fragment linking approach) such that the chemical structure of the modulator molecule is changed while its original modulating functionality is maintained or enhanced. Such optimisation is regularly undertaken during drug development programmes to e.g. enhance potency, promote pharmacological acceptability, increase chemical stability etc. of lead compounds.

Modifications to the test compound structure will be those conventional in the art known to the skilled medicinal chemist, and will include, for example, substitutions or removal of groups containing residues which interact with the amino acid side chain groups of a helminth tyrosinase structure. For example, the replacements may include the addition or removal of groups in order to decrease or increase the charge of a group in a test compound, the replacement of a charge group with a group of the opposite charge, or the replacement of a hydrophobic group with a hydrophilic group or vice versa.

A modification may, for example, include the addition or substitution of one or more atoms or groups in the KA structure with one or more of the substituents defined above.

In many cases, substituents may themselves be substituted. For example a Ci_₇ alkyl group, a C₃-₂o heterocyclyl group, a C₅-₂o aryl group, or heterocyclic ring as described above may comprise one or more substituent groups .

It will be understood that these are only examples of the type of substitutions considered by medicinal chemists in the development of new pharmaceutical compounds and other modifications may be made, depending upon the nature of the starting compound and its activity.

In some embodiments, modifying a test compound, for example a tyrosinase inhibitor such as a KA compound, may comprise creating a new chemical compound based on that compound, for example by; modelling the pharmacophore as is well known in the art; searching databases of libraries of known compounds for derivatives or analogues (e.g. a compound which is listed in a computational screening database containing three dimensional structures of known compounds) ; or simulating test compounds having substitute moieties or certain structural features.

In some embodiments, modifying may include computational screening of one or more databases of compounds in which the three dimensional structure of the compound is known, with the structure of the compound to identify a modified compound and interacting the modified compound (e.g., docking, aligning, matching, interfacing) with the three dimensional structure of helminth tyrosinase protein by computer (e.g. as described by Humblet and Dunbar, Animal Reports in Medicinal Chemistry, vol. 28, pp. 275-283, 1993, M Venuti, ed., Academic Press) .

A method may further comprise identifying a modified test compound structure that is predicted to bind to the helminth tyrosinase protein with improved or optimised properties.

Improved binding properties may include decreased inhibition constants (Ki or Ki*) or dissociation constants relative to KA and/or changes to the type of inhibition (e.g. competitive, slow tight-binding, irreversible) .

The above-described processes may be iterated in that the optimised or modified compound may itself be the basis for further compound design.

The modified test compound may be obtained or chemically synthesised. Methods to synthesize suitable chemical compounds are known to those of skill in the art and depend upon the structure of the chemical being synthesized.

A synthesised test compound may be evaluated or tested in an in vivo or in vitro biological system in order to determine its activity and/or its pharmaceutical or pharmacological properties. For example, the compound may be evaluated by contacting it with a helminth tyrosinase, and; determining the binding of the modified test compound to the helminth tyrosinase and/or determining the inhibition of the helminth tyrosinase activity by the modified test compound.

Binding and/or inhibition may be determined relative to the binding of unmodified test compound to a helminth tyrosinase. Further optimisation or modification can then be carried out to arrive at one or more final compounds or in vivo or clinical testing.

Compound produced by the screening methods and/or drug design methods described above may be formulated into a composition, such as a medicament, pharmaceutical composition or drug, with a pharmaceutically acceptable excipient.

A method of producing a pharmaceutical composition for use in treating helminthiasis may comprise; identifying and/or obtaining a compound which inhibits the expression and/or activity of a helminth tyrosinase using a method described herein; and, admixing the compound identified thereby with a pharmaceutically acceptable carrier.

As described above, the compound may be modified to optimise the pharmaceutical properties thereof.

A method for preparing a pharmaceutical composition for the treatment of a helminthiasis may comprise; i) identifying and/or obtaining a compound which inhibits the expression and/or activity of a helminth tyrosinase, for example using a method as described herein, ii) synthesising the identified compound, and; -Lii) incorporating the compound into a pharmaceutical composition.

A. pharmaceutical composition may include, in addition to a compound identified as an inhibitor of helminth tyrosinase expression and/or activity, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Pharmaceutical compositions are described in more detail above.

In other embodiments, a compound produced by the screening methods and/or drug design methods described above may be formulated into an agrochemical composition with an agriculturally acceptable adjuvant.

Z\ method of producing an agrochemical composition for use in treating helminthiasis may comprise; identifying and/or obtaining a compound which inhibits the expression and/or activity of a helminth tyrosinase using a method described herein; and, admixing the compound identified thereby with an agriculturally acceptable adjuvant.

As described above, the compound may be modified to optimise the agrochemical properties thereof.

A method for preparing an agrochemical composition for the -treatment of a helminthiasis in a plant may comprise; ±) identifying and/or obtaining a compound which inhibits the expression and/or activity of a helminth tyrosinase, for example using a method as described herein, ii) synthesising the identified compound, and; iii) incorporating the compound into an agrochemical composition. An agrochemical composition may include, in addition to a compound identified as an inhibitor of helminth tyrosinase expression and/or activity, a agriculturally acceptable adjuvant, surfactant, buffer, stabiliser or other materials well known to those skilled in the art. Agrochemical compositions are described in more detail above.

The invention encompasses a compound identified and/or obtained using a method described above as an agent which may be useful in the treatment of helminthiasis, a pharmaceutical, veterinary or agrochemical composition, medicament, drug or other composition comprising smch a compound, a method comprising administration of such a composition to an individual, e.g. a human, non-human animal or plant, for treatment (which may include preventative treatment) of a helminthiasis, use of such a compound in manufacture of a composition for administration, e.g. for treatment of a helminthiasis, and a method of making a pharmaceutical, veterinary or agrochemical composition comprising admixing such a compound with an excipient, vehicle or carrier, for example a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

Helminthiasis, in particular schistosomiasis, is described in more detail above.

Other aspects of the invention relate to the S. mansoni tyrosinase 2 sequence (SMTYR2) which has been cloned and characterised by the inventors.

An aspect of the invention provides an isolated nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 80 % sequence identity to the sequence of SEQ ID NO: 1.

Preferably, the nucleic acid encodes a polypeptide comprising an amino acid sequence which shares greater than about 85% sequence identity with the sequence of Figure 1, greater than 90%, greater than about 95% or greater? than about 98%.

In some embodiments, the nucleic acid may encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1.

Sequence identity is commonly defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4.

Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi- Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.

Sequence comparison is preferably be made over the full-length of the relevant sequence described herein, except where otherwise stated.

Preferably, the polypeptide has tyrosinase activity, as described herein.

The nucleic acid coding sequence may be SEQ ID NO: 2 or it may be a mutant, variant, derivative or allele of SEQ ID NO: 2. The sequence may differ SEQ ZD NO: 2 by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of SEQ ID NO: 2, for example 5, 10, 20, 30, 40 or 50 nucleotides. Changes to a nucleotide sequence may result in an amino acid change at the protein le-vel, or not, as determined by the genetic code.

Thus, nucleic acid according to the present invention may include a sequence different from SEQ ID NO : 2 yet encode a polypeptide with the same amino acid sequence.

An isolated nucleic acid encoding a polypeptide as described herein may comprise a sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 2, greater than about 85%, greater than about 90%, or greater than about 95%.

In some preferred embodiments, the nucleic acid may comprise or consist of the nucleic acid sequence of SEQ ID NO: 2.

The present invention also extends to nucleic acid encoding a polypeptide as described above, the complement of which hybridizes with SEQ ID NO: 2 under stringent conditions. Suitable conditions include hybridi sation overnight at 42^DC in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55⁰C in 0.1 X SSC, 0.1% SDS (for sequences with 80%-90% or more identity), or hybridization overnight at 65^πC in 0.25M

Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextiran sulfate and a final wash at 60^DC in 0.1X SSC, 0.1% SDS (for sequences with greater than 90% identity) .

The present invention also includes fragments of such sequences, for example a fragment of the nucleotide sequence of SEQ ID NO:

2. In particular, the invention includes nucleic acids comprising or consisting of bases 240 to 1676 of SEQ ID NO: 2 or 302 to 1676 of SEQ ID NO: 2. Suitable fragments may consist of less than the full length sequence, for example ffrom 10, 20, 30, 40 or 50 nucleotides to 1400, 1500, 1600 or 1700 nucleotides. Such a fragment may encode a tyrosinase as described herein or may be useful as an oligonucleotide probe or primer.

Another aspect of the invention provides a polypeptide encoded by a nucleic acid as described herein, for example the nucleic acid sequence of SEQ ID NO: 2. Such a polypeptide may comprise or consist of the amino sequence of SEQ ID N0:l.

Preferably, a polypeptide has tyrosinase activity as described above.

The skilled person can use the techniques described herein and others well known in the art to produce large amounts of polypeptides and peptides, for instance by expression from encoding nucleic acid.

A polypeptide as described herein may include a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding th\e protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve one or more of insertion, addition, deletion or substitution of one or more amino acids, which may be without fundamentally altering the qualitative nature of the transferase activity of the wild type polypeptide.

An isolated nucleic acid as described herein may be comprised in a vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd ed±tion, Sambrook et al. , 1989, Cold Spring Harbor Laboratory Press- Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds . , John Wiley & Sons, 1992.

Systems for cloning and expression of a polypeptide in a varriety of different host cells are well known. Suitable host cells include bacteria systems. A common, preferred bacterial host is E. coll.

Another aspect of the present invention provides a host cell- containing heterologous nucleic acid encoding a polypeptide as described herein. Suitable host cells include microbial host cells such as E. coli.

The nucleic acid may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted loy inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. The nucleic acid may be on an extra-chromosomal vector within the cell.

The introduction of nucleic acid into a host cell, which ma^y (particularly for in vitro introduction) be generally refemred to without limitation as "transformation", may employ any available technique. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide is produced.

A method of producing a polypeptide may comprise:

(a) causing expression from nucleic acid which encodes a polypeptide in a suitable expression system to produce the polypeptide recombinantly; (b) testing the recombinantly produced polypeptide for tyrosinase activity.

Suitable nucleic acid sequences are described above.

A polypeptide may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid (for which see below) . Thus, a polypeptide may be provided free or substantially free from contaminants with which it is naturally associated (if it is a naturally-occurring polypeptide) . A polypeptide may be provided free or substantially free of other polypeptides.

Another aspect of the invention provides a method of making a cell comprising transforming a host cell with a nucleic acid as described herein.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below. Figure 1 shows the inhibition of schistosome tyrosinase diphenol oxidase activity by kojic acid.

Figure 2 shows the concentration of kojic acid that inhibits 50% of tyrosianse activity in worm extracts (black boxes) or from mushroom tyrosinase (open diamonds) . The star represents the concentration of kojic acid used in vitro culture experiments shown in Fig. 1 and Fig. 3.

Figure 3 shows egg production is inhibited in adult S. mansoni worm pairs cultured for 48 hrs by treatment with kojic acid.

Figure 4 shows RT-PCR analysis of tyrosinase expression in immature and mature S. mansoni worms

SEQ ID NO: 1 shows the amino acid sequence of S. mansoni tyrosinase 2.

SEQ ID NO: 2 shows the nucleotide sequence of S. mansoni tyrosinase 2. The signal peptide is encoded by bases 240 to 302. The polyA signal is at bases 1752 to 1757.

SEQ ID NO: 3 shows the amino acid sequence of S. mansoni tyrosinase 1.

SEQ ID NO: 4 shows the nucleotide sequence of S. mansoni tyrosinase 1.

Experimental Materials & Methods

Diphenol oxidase Assay

Single adult worms were sonicated in 500 μl cold 0.2M KH₂PO₄ (pH 6.7) and diphenol oxidase activity assayed with 4mM 3-methyl-2- benzothiazolinone hydrazone hydrochloride hydrate (MBTH, Sigma) and ImM 1-DOPA as a substrate. After incubation for 10 min at 3O⁰C, the reaction was stopped with 1% acetic acid. Absorbance was determined at 505 nm. One unit of diphenol oxidase causes a change in absorbance (A505) of 0.1 min.l at 30 ⁰C with pH 6.7 using 1-DOPA as a substrate (Eshete F et al. J Parasitol 1993;79 (3) :309-17) . Diphenol oxidase activity was additionally assayed on an individual worm basis.

RT-PCR

Miracidia, cercaria, 3-wk and 7-wk worms were homogenized in TRIZOL reagent (Invitrogen, UK) using a tissue dispersing tool (IKA Labortechnik, Germany) , and total RNA was isolated as recommended by the manufacturer. All RNA was treated with DNase I (Ambion Inc.) prior to reverse transcription to remove any potential genomic DNA contamination. Reverse transcription PCR was carried out using one microgram of parasite total RNA and oligo-dT primers. The PCR conditions and cycle number were strictly defined for each cytokine primer pair such that a linear relationship between input RNA and final PCR product was obtained. The RT-PCR primers for SmTYRl (5'-CTT CCG GAT GTA GAG GAT TTG-3' and 5'-CGG GAT ATG CGT TTG GAC TAG-3' ) , SmTYR2 (5'-CAC CAT AGA GTA AAT TCC TAC-3' and 5'-CAC ATA ATT TTG GTA TAA GAG¬ S' ), and alpha-tubulin (5'-GGC GGT GGT ACT GGT TCT GGG-3' and 5'- CAT TTA GCG CAC CAT CGA AGC-3' ) were synthesized at Sigma-Genosys (Cambridge, UK) .

Reverse transcriptase (-) negative controls were included to detect contaminating genomic DNA. Twenty-six cycles of PCR were used to amplify the SmTYRl- (301 bp) and alpha-tubulin (325 bp) fragments whereas 33 cycles were used to amplify the SmTYR2 fragment (226 bp) . All amplicons were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Images were captured by a digital camera and analyzed by gel documentation software (Kodak ID 2.0 electrophoresis documentation and analysis system 120, Eastman Kodak Co., New Haven, CT, USA and NIH Image (version 1.62)) . Amplification of alpha-tubulin served as an internal control for the amount of RNA and cDNA from each sample.

In vitro test system Seven-week old parasites were cultured at 37⁰C in DMEM (Sigma,

D6546) supplemented with 10% FCS, 2mM L-glutamine, and 100 μg/ml pen-strep for up to 48 hr in an atmosphere of 5% CO2. Ten worm pairs were cultured per ml of media in 24 well tissue culture plates and the media was changed every 24 hr.

For worm pairs treated with tyrosinase inhibitors, the culture media was supplemented with 1 mg/ml kojic acid. Eggs layed in culture were collected after 48 hr, counted using a sedgewick rafter, and subjected to bright field and fluorescent microscopy.

In vivo test system

Mice infected with 25 parasites are treated with various doses of kojic acid (3.0% w/v and 1.0% w/v) in their drinking water during weeks 5-8 post infection. Drinking water is changed every day until experiment terminates (week 8 post infection) .

The effect of tyrosinase inhibition is compared to control infected animals (not treated with kojic acid) .

Faecal egg output is measured twice weekly during weeks 5-8 post¬ infection and assess hatchability of these eggs. Liver egg counts are measured at 8 weeks post infection when the experiment is terminated.

Immunology parameters are measured at week 8 post infection specifically focusing on cytokine and antibody assays. Liver pathology is also assessed at 8-wk post infection (granuloma volume, fibrosis, necrosis, etc.) by commonly employed laboratory techniques. All of these experiments are compared against mock treated animals (infected but not treated with kojic acid) .

Results S. mansoni worm pairs were cultured as described above in the presence and absence of 1 mg/ml kojic acid. The diphenol oxidase activity of the S. mansoni tyrosinase was observed to be inhibited by kojic acid (figure 1) .

The concentration of kojic acid to inhibit 50% diphenol oxidase

(tyrosinase) activity in female worms was observed to to be about 0.5μM (figure 2) .

Eggs layed in culture were collected after 48 hr in culture in the presence or absence of 1 mg/ml kojic acid. Egg production was observed to be inhibited in adult S. mansoni worm pairs cultured treated with kojic acid (Figure 3) .

The expression of SmTYRl and SmTYR2 was determined in S. mansoni worms using RT PCR as described above. SmTYRl and SmTYR2 were observed to be expressed only in mature female worms (figure 4) . Note that M and C are immature larval stages of the parasite and 3 wk worms are sexually immature.

Egg morphology was observed to be affected by kojic acid mediated tyrosinase inhibition in in vitro cultured worm pairs. Forty- eight hour worm pairs cultured in the presence of kojic acid were found to produce abnormally shaped egg-like entities that exhibited minimal and patchy autofluorescence under UV illumination when compared to eggs produced by similarly cultured worm pairs in the absence of kojic acid. Eggs produced by tyrosinase-inhibited worm pairs were also smaller, lacked a mature egg-shell, and did not contain a species-specific spine.

This data shows that helminth tyrosinases are selectively expressed in mature female helminths and inhibition of these tyrosinases reduces or inhibits helminth egg production, which is known to cause pathological reactions in the host.

SEQUENCE LISTING

SEQ ID NO:1 MQHIWFIIILTFYLIIIINVQCLIPKLCVNNITAIGGSGICCPIP

NGSKYPCGGPGQGTCQKEYTQIDRIPFYLIMDDRMNWPSRFFNHFCKCENHYFGISCN ECWYGWEGKFCNKRKEYIRYNILSFSPKKRKMFVNWTRMLTTPTDYLILFEKDAIHS DPLWKPKFLDVDVQΎLFSFIHRYASRATLFHDNIDCIIRRHLDNNHEWGFLTWHRYY MLFWERHLRKIAIRLYGWTDFTIPYWDWVDSTRCDVCVNSLLGGYGQWVGQTRLIDPR SPFYMWPEYCSPPTTGSNCYSCHAGWPNFRVLTRYFESTAFPTTHNLLFTLSKDTFYL PQVKEDFDKCRGFHQALEGFCSFPGDNSTYSFMHNKVHNVVSGTFCCAATSPNDPLFL VHHTQIDRIFQLWFMYNRPRPTDYPNHGVALGNCRECNMVGFIPTIKHVQMFVNTKLI GYTYDNFNFGKRGFKGEKYLKCGPKYRV

SEQ ID NO:2

1 gaaatagaat agttaaacag attataacct tctatatata tctcattgaa tcggttgatt

61 tgattacatt tatttcaatt attaataaat tatatcacta tagagtaaat ttctacaaaa 121 ttccctcccc ccttttcttt ttatggacat atttaatgtg aataatattc attgaaaatt

181 aattaaatta ttcattataa ttaattatta taataaatta aatcaatatt attattatta

241 tgcaacatat atggtttatt attattttaa cattctattt aataataata aatgttcaat

301 gtcttatacc aaaattatgt gtaaataata ttacagcaat tggtggtagt ggtatttgtt

361 gtccaatacc taatggttcc aaatatccat gtggtggtcc aggtcaaggt acatgtcaaa 421 aagaatatac acaaattgat cgtataccat tttatttaat tatggatgat agaatgaatt

481 ggccatcaag attttttaat catttttgca aatgtgaaaa tcattatttt ggtatttcat

541 gtaatgaatg ttggtatggt tgggaaggaa aattttgtaa taaacgtaaa gaatatattc

601 gatataatat attatcattt tctccaaaaa aaagaaaaat gtttgttaat gttgtaacaa

661 gaatgcttac tactccaacg gattatttga tattatttga aaaagatgct atacattctg 721 atccattatg gaaacctaaa tttttagatg tagatgtaca atatttattc tcttttatac

781 atcgttatgc aagcagagct acattatttc atgataatat agattgtata atacgtagac

841 atttggataa taatcatgaa gtagttggat ttttaacttg gcatagatat tatatgttat

901 tttgggaaag acatctacgt aaaattgcaa ttcgactgta tggttggaca gatttcacaa

961 taccttattg g-gattgggtt gattctacaa ggtgtgatgt ttgtgttaat agtttattag 1021 gtggttatgg acaatgggtt gggcaaactc gtttaataga ccctagaagt ccattctaca

1081 tgtggccgga atactgttct cccccaacta cgggaagtaa ttgttatagt tgtcatgctg

1141 gttggccaaa ctttcgagta ttgacaagat atttcgaaag tacagcattc ccaacaactc

1201 ataacctttt ςyttcactctt tcaaaggata cgttttattt gcctcaagtg aaggaggatt

1261 ttgataaatg tcgtggtttt catcaagctc ttgaaggatt ttgttctttt cccggtgaca 1321 attcaactta ttcatttatg cataataaag tacataatgt agtgagtggt acattttgtt

1381 gtgcagctac ttcacccaat gatcctctat tcttagtaca tcatacacaa attgatagaa

1441 ttttccaatt atggtttatg tataatcgtc cacgtccaac ggattatcca aatcatggag

1501 ttgcacttgg aaattgtcgt gaatgtaata tggttggttt cattcctaca attaaacacg

1561 tacaaatgtt tgtaaataca aaactaatag gttatacata tgataatttt aattttggta 1621 aacgtggctt caagggagaa aaatacttga aatgtggtcc taaatatcgt gtctaattta

1681 ttatttactt gagatcataa caatacaaag catagaagct gaaattgaat acttttgatc

1741 aactttaaat gaataaatat tattccaaaa ataaaaaaaa ataaaaaaaa SEQ I D NO : 3

MIKTITFLYCIFILFNQFINIYGMIPEQCGYNLTRRIPVCCPISNINGEVCGGPKYGKCIQIWTP

REKIPSVFLIDDRIDWPKRYFTYFCQCYGNYFGSACDECWFGWKGRYCNKRSVRIRRDIRTLSNK ELHIFKTVIVLSQTWPSGYLLVDESMTSIWYVDPLRKLRLEHASVQYYITYLHRYGSRSTLYKNVKD

CEDYGILNFNHDGVCFPTWHRYYGLLWERLMSKIAMKVFGITDFATPYWDWTGLTYCDICTNRYI

GAPGRLSDLGRHISSRSPFSNLTEFCYEPIKDMLCSGCQKSRKRLTITREFKKGNLPDVEDLKYV

LSLSQYYVPGERLSPVCRSFNIALEGFCGRPGADPNSRWFHNKLHVLVDGSMCCTGTASNDPLFI

LHHTFIDKIFECWLKTYNPSPNAYPDKNVRPGHSSYSFVIGIIPLARNIEFFKPLSNFGVYYDNK LFGRFAHDGRPPYScsrmetitkgyy

SEQ ID NO: 4

TGTCAGAAATCTAGAAAGAGGTTAACAATTACACGGGAATTTAAAAAAGGGAATcttccggatgtagaggatttgAAATA

AAAGACATATAATCctagtccaaacgcatatcccgATAAAAATGTTCGTCCTGGACATTCAAGTTATTCATTTGTTATTG

AAAAΆAAAATGTTTAΆTTTAAAATCGTAATAAAAATAATAATTTTGGTAAAAAAAAAAAAAAΆΆ

Claims

Claims :

1. A method of treating helminthiasis in an individual comprising; administering a tyrosinase inhibitor to said individual.

2. A method according to claim 1 wherein the helminthiasis is schistosomiasis.

3. A method according to claim 2 wherein the schistosomiasis is caused by Schistosoma mansoni, Schistosoma japonica or Schistosoma haematobium.

4. A method according to any one of claims 1 to 3 wherein the tyrosinase inhibitor is a kojic acid compound having the formula I:

wherein:

X is an oxygen atom or a sulphunr atom; Y is an oxygen atom, a sulphur atom, or an NH group; Z is an oxygen atom, a sulphur atom, or an NH group; L is optionally substituted C₁-_T alkylene, C₅_₆ arylene, Ci-7 alkylene-C₅-₆ arylene or C₅_₆ arylene-Ci_₇ alkylene.

Ri is selected from H, halo, cyano, carboxy, formyl, optionally substituted Ci_₇ alkyl, C₅-₂o aryl, C₃-₂₀ heterocyclyl, Ci- 7 alkylene-C₅_2o aryl, Ci_₇ alkylene-C₃_2o heterocyclyl, acyl, ester, amido, amino and sulfone. R₃ is selected from H, halo, cyano, carboxy, formyl, optionally substituted Ci_₇ alkyl, C₅-₂0 aryl, C₃_₂o heterocyclyl, C_x- ₇ alkylene-C₅-2o aryl, Ci_₇ alkylene-C₃_₂o heterocyclyl, acyl, ester, amido, amino, sulfone, an amino acid or polypeptide chain wherein Z is bonded to the terminal carbon atom of the amino acid or polypeptide chain, and -C(=O)R₅ wherein R₅ is an amino acid or polypeptide chain wherein the carbon atom of said C(=O) group is bonded to the terminal nitrogen atom of the amino acid or polypeptide chain, and

R₂ and R₄ are independently selected from halo;

OH; -OMe, -OEt, -O(tBu); -OCH₂Ph; -SH; -SMe, -SEt, -S (tBu) , - SCH₂Ph; -C(=O)H; -C(=O)Me, -C(=O)Et, -C(=O) (tBu) and -C(=O)Ph; -C(=O)OH; -C(=O)OMe, -C(=O)OEt and -C (=0)0 (tBu) ; -C(=O)NH₂, -C(=0)NHMe, -C(=0)NMe₂, -C (=0)NHEt; -NHC(=O)Me, -NHC (=0) Et, -NHC(=O)Ph, -NH₂, -NHMe, -NHEt, -NH(iPr), -NH(nE>r), -NMe₂, -NEt₂, -N(IPr)₂, -N(nPr)₂, -N(nBu)₂ and -N(tBu)₂/ -CN; -NO₂; -Me, -Et, - nPr, -iPr, -nBu and -tBu; -CF₃, -CHF₂, -CH₂F, -CCl₃, -CBr₃, -CH₂CH₂F, -CH₂CHF₂, -CH₂CF₃; -OCF₃, -OCHF₂, -OCH₂F, -OCCl₃, -OCBr₃, -OCH₂CH₂F, -OCH₂CHF₂, -OCH₂CF₃; -CH₂OH, -CH₂CH₂OH, -CH(OH)CH₂OH; -CH₂NH₂, -CH₂CH₂NH₂, -CH₂CH₂NMe₂; and substituted or unsubstituted phenyl.

5. A method according to claim 4 wherein the kojic acid compound is kojic acid.

6. Use of a tyrosinase inhibitor in the manufacture of a medicament for the treatment of helminthiasis .

7. Use according to claim 6 wherein the helminthiasis is schistosomiasis.

8. Use according to claim 7 wherein the schistosomiasis is caused by Schistosoma mansoni or Schistosoma ja_ponica.

9. Use according to any one of claims 6 to 8 wherein the tyrosinase inhibitor is a kojic acid compound h_aving the formula:

wherein :

X is an oxygen atom or a sulphur atom; Y is an oxygen atom, a sulphur atom, or an NH group; Z is an oxygen atom, a sulphur atom, or an NH group; L is optionally substituted Ci_₇ alkylene, C₅_₆ arylene, Ci_₇ alkylene-C₅_₆ arylene or C₅_₆ arylene-Ci_₇ alkylene.

R₁ is selected from H, halo, cyano, carboxy, formyl, optionally substituted C^₇ alkyl, C₅-₂o aryl, C₃-₂o heterocyclyL , C₁- η alkylene-C₅_2o aryl, Ci_₇ alkylene-C₃-₂o heterocyclyl, acyl, ester, amido, amino and sulfone.

R₃ is selected from H, halo, cyano, carboxy, formyl, optionally substituted Ci_₇ alkyl, C₅_₂₀ aryl, C₃_₂₀ heterocyclyl., Ci- _η alkylene-C₅_2o aryl, Ci_₇ alkylene-C₃_₂₀ heterocyclyl, acyl, ester, amido, amino, sulfone, an amino acid or polypeptide chain wherein Z is bonded to the terminal carbon atom of the amino acid or polypeptide chain, and -C(=O)R₅ wherein R₅ is an amino acid our polypeptide chain wherein the carbon atom of said C(=0) group is bonded to the terminal nitrogen atom of the amino acid or polypeptide chain, and

R₂ and R₄ are independently selected from halo; OH; -OMe, -OEt, -O(tBu); -OCH₂Ph; -SH; -SMe, -SEt, -S(tBu), - SCH₂Ph; -C(=O)H; -C(=0)Me, -C(=O)Et, -C(=O) (tBu) and -C (=0) Ph.; -C(=O)OH; -C(=O)OMe, -C(=O)OEt and -C(=0)O(tBu) ; -C(=O)NH₂, -C(=0)NHMe, -C (=0)NMe₂, -C(=0)NHEt; -NHC(=0)Me, -NHC (=0) Et, -NHC(=O)Ph, -NH₂, -NHMe, -NHEt, -NH(iPr), -NH(nPr), -NMe₂, -NEt₂, -N(iPr)₂, -N(IiPr)₂, -N(nBu)₂ and -N(tBu)₂; -CN; -NO₂; -Me, -Et, - nPr, -iPr, -nBu and -tBu; -CF₃, -CHF₂, -CH₂F, -CCl₃, -CBr₃, -CH₂CH₂F, -CH₂CHF₂, -CH₂CF₃; -OCF₃, -OCHF₂, -OCH₂F, -OCCl₃, -OCBx₃, -OCH₂CH₂F, -OCH₂CHF₂, -OCH₂CF₃; -CH₂OH, -CH₂CH₂OH, -CH(OH)CH₂OH; -CH₂NH₂₇-CH₂CH₂NH₂, -CH₂CH₂NMe₂; and substituted or unsubstituted phenyl .

10. Use according to claim 9 wherein the kojic acid compound is kojic acid.

11. A pharmaceutical formulation for the treatment of helminthiasis comprising a tyrosinase inhibitor.

12. A pharmaceutical formulation according to claim 12 which is a non-topical formulation.

13. A pharmaceutical formulation according to claim 11 or claim 12 comprising a kojic acid compound.

14. A pharmaceutical formulation according to claim 13 comprising kojic acid

15. A method of producing a pharmaceutical formulation for use in treating helminthiasis comprising; admixing a tyrosinase inhibitor with a pharmaceutically acceptable excipient, carrier, buffer or stabiliser.

16. A method according to claim 15 wherein the tyrosinase inhibitor is a kojic acid compound.

17. A method according to claim 16 wherein the tyrosinase inhibitor is kojic acid.

18. Another aspect of the present invention provides a method of treating helminthiasis in a plant comprising; applying a tyrosinase inhibitor to said plant or the locus thereof.

19. A method of producing an agrochemical composition for use in treating helminthiasis in plants comprising; admixing a tyrosinase inhibitor with an agriculturally acceptable adjuvant or auxilliary.

20. A method according to claim 18 or claim 19 wherein the tyrosinase inhibitor is a kojic acid compound.

21. A method according to claim 20 wherein the kojic acid compound is kojic acid.

22. A method of identifying and/or obtaining a compound for the treatment of helminthiasis in an individual may comprise; contacting a helminth tyrosinase with a test compound, and; determining the activity of said tyrosinase in the presence of said test compound.

23. A method according to claim 22 wherein the helminth tyrosinase comprises an amino acid sequence which shares greater than about 30% sequence identity with S. mansoni tyrl or S. mansoni tyr2.

24. A method according to claim 22 wherein the helminth tyrosinase is 5. mansoni tyrl or S. mansoni tyr2.

25. A method according to any one of claims 22 to 24 wherein the helminth tyrosinase is comprised in a cell.

26. A method according to claim 25 wherein the cell is comprised in a helminth worm.

27. A method of identifying and/or obtaining a compound for the treatment of helminthiasis in an individual comprising; contacting a helminth with a test compound, and; determining the activity of tyrosinase in said helminth or an extract thereof in the presence of said test compound.

28. A method according to any one of claims 22 to 27 wherein the test compound is a tyrosinase inhibitor.

29. A method according to claim 28 wherein the test compound is a kojic acid compound.

30. A method according to claim 29 wherein the kojic acid compound is kojic acid.

31. A method according to any one of claims 22 to 30 identifying the test compound as an agent which inhibits the activity of a helminth tyrosinase.

32. A method according to claim 31 comprising determining the effect of the test compound on helminth egg production.

33. A method according to any one of claims 22 to 32 comprising isolating and/or purifying the identified compound.

34. A method according to any one of claims 22 to 33 comprising preparing, synthesising and/or manufacturing the identified compound.

35. A method according to claim 33 or claim 34 comprising modifying the test compound to optimise its pharmaceutical properties.

36. A method according to any one of claims 33 to 35 comprising formulating the test compound with a pharmaceutical excipient.

37. A method of producing a compound for treating helminthiasis may comprise; providing a test compound, and; determining the interaction of said test compound with a helminth tyrosinase.

38. A method according to claim 37 comprising modifying the structure of the test compound, and; determining the interaction of the modified test compound with a helminth tyrosinase.

39. A method according to claim 37 or claim 38 wherein the helminth tyrosinase comprises an amino acid sequence which shares greater than about 30% sequence identity with one or more of S. mansoni tyrl or S. mansoni tyr2.

40. A method according to claim 39 wherein the helminth tyrosinase is selected from the group consisting of S. mansoni tyrl, S. mansoni tyr2, S. japonicum tyrosinase 1 and S japonicum tyrosinase 2.

41. A method according to any one of claims 32 to 35 wherein the test compound is a tyrosinase inhibitor.

42. A method according to claim 41 wherein the test compound is a kojic acid compound.

43. A method according to claim 42 wherein the kojic acid compound is kojic acid

44. A method according to any one of claims 37 to 43 wherein the interaction of the test compound or modified test compound is determined in silico.

45. A method according to claim 44 wherein the interaction is determined by: (a) providing a structure comprising a three-dimensional representation of the helminth tyrosinase or a portion of the helminth tyrosinase;

(b) providing the structure of the test compound or modified test compound to be fitted to said helminth tyrosinase structure or selected coordinates thereof, and;

(c) fitting the test compound structure to said helminth tyrosinase structure.

46. A method according to claim 45 comprising the step of modifying or optimising the structure of the test compound to optimise binding to the helminth tyrosinase structure.

47. A method according to claim 46 comprising identifying a modified test compound structure which binds to the helminth tyrosinase protein with improved or optimised properties

48. A method according to claim 47 comprising obtaining or chemically synthesizing the modified test compound.

49. A method according to claim 48 comprising formulating the modified test compound into a composition with a pharmaceutically acceptable excipient.

50. A method of producing a pharmaceutical composition for use in treating helminthiasis may comprise; producing a compound which inhibits the activity of a helminth tyrosinase using a method according to any one of claims

37 to 49; and, admixing the compound identified thereby with a pharmaceutically acceptable carrier.

51. A method for preparing a pharmaceutical composition for the treatment of a helminthiasis may comprise; i) producing a compound which inhibits the activity of a helminth tyrosinase using a method according to any one of claims 37 to 49 ii) synthesising the identified compound, and; iϋ) incorporating the compound into a pharmaceutical composition.

52. A compound for the treatment of helminthiasis, said compound being identified and/or obtained using a method according to any one of claims 22 to 34 or produced by a method according to any one of claims 37 to 49.

53. A pharmaceutical composition comprising a compound according to claim 52.

54. A method of treating helminthiasis comprising administering a compound according to claim 52 to an individual in need thereof.

55. Use of a compound according to claim 52 in manufacture of a composition for treatment of helminthiasis.

56. A method of making a pharmaceutical composition comprising admixing such a compound according to claim 52 with a pharmaceutically acceptable excipient, vehicle or carrier.

57. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 80 % sequence identity to the sequence of SEQ ID NO: 1.

58. An isolated nucleic acid having the sequence of SEQ ID NO:2.

59. A vector comprising a nucleic acid according to claim 57 or claim 58.

60. A host cell comprising a vector according to claim 59.

61. An isolated polypeptide encoded by a nucleic acid according to claim 57 or claim 58.

62. A method of producing a polypeptide comprising:

(a) expressing a nucleic acid according to claim 57 or claim 58 in a suitable expression system to produce the polypeptide recombinantly; (b) testing the recombinantly produced polypeptide for tyrosinase activity.