WO2004105779A2 - Green tea and oplyphenol inhibitors of bacterial proteases - Google Patents

Abstract

The use of green tea extract and/or polyphenols as protease inhibitors, in particular as metalloprotease inhibitors, in the treatment of bacterial infections associated with protease activity, in particular for the treatment or prevention of anthrax. Preferred polyphenols are those extractable from green tea, in particular catechins and especially catechin gallate and (-) -epigallocatechin gallate.

Description

GREENTEAANDPOLYPHENO INHIBITORS OFBACTERIALPROTEASES

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

This invention relates to inhibitors of bacterial proteases. BACKGROUND OF THE INVENTION

Proteases are implicated in a wide variety of disease states, including cancer (Biol. Chem., 383 (2002), 101-105) and certain bacterial infections (Science 280 (1998), 734; Nature 418 (2002), 229). There is thus a need for protease inhibitors.

One particular bacterium whose pathogenicity involves a protease is Bacillus anthracis (BA), the causative agent of anthrax. The BA 87kDa lethal factor (LF) is secreted by the bacterium together with the protective antigen (83 kDa, PA) and an edema factor (89 kDa, EF) (Annu. Rev. Microbiol.

55 (2001), 647). PA binds to a ubiquitous type I membrane protein, and is rapidly nicked by membrane bound proteases to a 63kDa form which oligomerises and binds LF (Curr. Top.

Microbiol. Immunol. 271 (2002), 61). Following endocytosis within acidic compartments, the PA oligomer undergoes an acid-driven conformational rearrangement, which mediates the transfer of LF from the lumen of a late endocytic compartment to the cytoplasm (Curr. Top. Microbiol. Immunol.

271 (2002), 61). Here LF displays its metallo-proteolytic activity directed toward the N-terminus of the MAPK-kinase family of proteins (Science, 280 (1998), 734; Nature 418(2002), 386; Int. J. Med.

Microbiol. 290(2000) 421-427). LF+PA (lethal toxin, LeTx) is cytotoxic to macrophages (J. Biol. Chem. 261 (2001), 7123), while in LPS-stimulated macrophages it also induces apoptosis (Nature 414 (2001), 229). There is evidence that LeTx action on macrophages plays a major role in the development of systemic disease (Annu. Rev. Microbiol. 55 (2001), 647). LF consists of four domains, where the C-terminal domain is a metallo-(zinc-dependent)-endopeptidase (Nature 414 (2001), 229). The lethal activity of LeTx depends on PA (for mediating cell entry) and on the metallo-proteolytic activity of LF (for toxicity; the Glu687-Ala mutant, devoid of proteolytic activity, is non-toxic).

There is thus a need for inhibitors of Bacillus anthracis lethal factor.

DISCLOSURE OF THE INVENTION

Green tea extract has been found to inhibit the activity of metallo-proteases involved in bacterial pathogenesis, and in particular to inhibit the activity of B.anthracis lethal factor. The inhibitory activity is also displayed by polyphenols present within the extract. Green tea extract and its polyphenol components are thus useful in the treatment or prevention of diseases or conditions arising from bacterial infections, and in particular arising from B.anthracis infection.

The invention therefore provides a method for the treatment or prevention of a disease or condition associated with bacterial infection, comprising the step of administering a therapeutically effective amount of a green tea extract to a patient. The invention also provides the use of a green tea extract in the manufacture of a medicament for the treatment or prevention of a disease or condition associated with bacterial infection. The disease or condition is preferably anthrax.

In addition, the invention provides a method for the treatment or prevention of a disease or condition associated with bacterial infection, comprising the step of administering a therapeutically effective amount of a polyphenol, or a pharmaceutically acceptable derivative thereof, to a patient. The invention also provides the use of a polyphenol, or a pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for the treatment or prevention of a disease or condition associated with bacterial infection. The disease or condition is preferably anthrax.

The invention also provides a method for inhibiting the proteolytic cleavage of a mitogen activated protein kinase kinase, comprising the step of administering a therapeutically effective amount of (i) a green tea extract and/or (ii) a polyphenol, or a pharmaceutically acceptable derivative thereof, to a patient. The method may be performed in vivo or in vitro.

The invention also provides a composition comprising a bacterial protease and a polyphenol, preferably in the form of a complex. Such a complex may be used as an immunogen. The protease is preferably B. anthracis lethal factor.

The invention also provides a crystal of a protease and a polyphenol. Such crystals can be used for X-ray diffraction studies of protease inhibition e.g. to provide atomic structural information in order to aid rational design of further inhibitors. The protease is preferably B.anthracis lethal factor.

Green tea extract Green tea is a well known social and medicinal beverage. All teas (green, black, and oolong) are derived from the leaves of Camellia sinensis, and the difference in the three teas is in how the plucked leaves are prepared. All tea leaves are withered, rolled and heated, but black teas go through an oxidative process known as fermentation before the final heating process, oolong teas are partially fermented, and green tea is not fermented. Green tea contains volatile oils, vitamins, minerals, and caffeine, but the main pharmacological constituents are polyphenols. As a herbal remedy, therefore, green tea is often treated to give 'green tea extract' (GTE), which is high in polyphenols. GTEs are widely available and their biological effects have been studied in detail, including in human clinical trials (e.g. Young et al. (2002) Br J Nutr 87(4):343-55; Pisters et al. (2001) JClin Oncol 19(6):1830-8). The GTE may be a decoction extract of green tea, a water extract of green tea, or an ethanol extract of green tea. It may or may not be decaffeinated.

Polyphenols

GTEs are known to contain high concentrations of monomeric polyphenols of the catechin group and derivatives thereof, including (+)-catechin ('C'), (-)-epicatechin (ΕC), (+)-gallocatechin ('GC'), (-)-epigallocatechin (ΕGC), (-)-epicatechin gallate ('ECG') and (-)-epigallocatechin gallate (ΕGCG') (J. Periodontal 64 (1993), 630-636). Polyphenols are promising as potential compounds for use in therapy, as they show few side effects. They have been studied for cancer prevention in particular. Certain classes of polyphenols are generally known to have microbicidal activity. It has been shown that tea polyphenols have a bactericidal effect against pathogenic bacteria (Phytochemicals and Phytopharmaceuticals, 1999, 214-221 ; Function food for disease prevention I, 1998, 217 - 224.).

As used herein, the term 'polyphenol' means a compound possessing two or more benzene rings, each benzene ring bearing one or more hydroxy groups or functional derivatives thereof. Polyphenols may also have at least one further hydroxy group or functional derivative thereof not bound directly to a benzene ring. Hydroxy group functional derivatives include esters, ethers (e.g. silyl ethers or alkyl ethers) and acetals. The term 'polyphenol' includes polyphenol derivatives wherein one or more oxygen atoms are independently substituted by S, Se or NR, where R is H, straight or branched Cι_-lυ alkyl or C_3-ι₀ cycloalkyl, preferably H.

Polyphenols useful for the invention are preferably flavanoid polyphenols, particularly those extractable from Camellia sinensis and/or from green tea. Particular polyphenols of interest are the catechins, including catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, catechin gallate, epigallocatechin gallate. Particularly preferred catechins are (+)-catechin (C), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-catechin gallate (CG), (-)-epigallocatechin gallate (EGCG). Especially preferred are CG and EGCG. EGCG is particularly preferred. Extensive data on EGCG is available from the National Cancer Institute

EGCG (CAS registry number 989-51-5) is variously known as: epigallocatechin gallate; epigallocatechin 3-gallate; (-)-epigallocatechin-3-O-gallate; 3,4,5-trihydroxybenzoic acid, (2R-cis)- 3,4-dihydro-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-2H-l-benzopyran-3-yl ester; and (2R, 3R)-2- (3,4,5-trihydroxyphenyl)-3,4-dihydro-l(2H)-benzopyran-3,5,7-triol 3-(3,4,5-trihydroxybenzoate). Preferred polyphenols are those in which a hydroxy group is derivatised as a gallate ester. Examples of such polyphenols are^' catechin gallate and epigallocatechin gallate.

The structures of various polyphenols are shown in figure 8.

The term 'pharmaceutically acceptable derivative' as used herein, means any pharmaceutically acceptable salt, addition compound, or any other compound which upon administration to a recipient is capable of providing, whether directly or indirectly, a polyphenol of the invention or a pharmaceutically acceptable metabolite.

The term 'pharmaceutically acceptable metabolite' as used herein, means a metabolite or residue of a polyphenol of the invention which gives rise to a biological activity exhibited by the polyphenols of the invention. Pharmaceutically acceptable derivatives include derivatives produced by covalent modification of the polyphenols of the present invention. The term 'pharmaceutically acceptable derivative' further includes not only pharmaceutically acceptable salts of the polyphenols of the invention but also pharmaceutically acceptable salts of any derivatives produced by covalent modification of the polyphenols of the invention.

The term 'pharmaceutically acceptable salt', as used herein, refers to a salt prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids and bases.

Examples of inorganic acids suitable for use in this invention include, but are not limited to hydrochloric, hydrobromic, hydroiodic, sulfuric, and phosphoric acids. Appropriate organic acids for use in this invention include, but are not limited to aliphatic, aromatic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic, stearic, sulfanilic, algenic, and galacturonic.

Examples of inorganic bases suitable for use in this invention include metallic salts made from aluminium, calcium, lithium, magnesium, potassium, sodium, and zinc. Appropriate organic bases may be selected, for example, from N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine (N-methylglucamine), and procaine.

The polyphenols of the invention may exist in a number of diastereomeric and enantiomeric forms. Diastereomeric and enantiomeric forms of the polyphenols of the invention may be differentiated by the direction in which they rotate plane-polarised light. A dextrorotatory (d) substance rotates plane- polarised light in a clockwise or positive (+) direction. A levorotatory (1) substance rotates plane- polarised light in a counterclockwise or negative (-) direction. The present invention encompasses pure diastereomers and enantiomers as well as mixtures, including racemic mixtures, of diastereomers and enantiomers.

As used herein, 'treatment' includes prophylactic treatment. As used herein, a 'patient' means an animal, preferably a mammal, preferably a human in need of treatment.

The amount of the active polyphenol or pharmaceutically acceptable derivative thereof administered should be a therapeutically effective amount where the composition is used for the treatment of a disease or condition and a prophylactically effective amount where the composition is used for the prevention of a disease or condition. The term 'therapeutically effective amount' used herein refers to the amount of compound needed to treat or ameliorate a targeted disease or condition. The term 'prophylactically effective amount' used herein refers to the amount of compound needed to prevent a targeted disease or condition. The exact dosage will generally be dependent on the patient's status as the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to therapy. The precise amount can be determined by routine experimentation, but may ultimately lie with the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg (mass of drug compared to mass of patient) to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

The polyphenols or pharmaceutically acceptable derivatives thereof of the invention may be administered as a medicament by mucosal or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), oral, intranasal, rectal, vaginal and topical (including buccal and sublingual) administration. The polyphenols or pharmaceutically acceptable derivatives thereof of the invention are preferably formulated with one or more anti-oxidant compounds to protect ECGC from oxidation. Since GTE includes naturally occurring anti-oxidant molecules, it is therefore preferred that the invention comprises the use or administration of GTE.

For parenteral administration, the polyphenols will generally be provided in injectable form. For oral administration, the polyphenols of the invention or pharmaceutically acceptable derivatives thereof will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose. Corn starch and alginic acid are suitable disintegrating agents. Suitable binding agents include starch and gelatin. Suitable lubricating agents include magnesium stearate, stearic acid or talc. The tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absoiption in the gastrointestinal tract. Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredients are mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

Compositions for use with the invention may comprise 'pharmaceutically acceptable carriers', such as sugars or salts. They may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. A thorough discussion of pharmaceutically acceptable carriers and excipients is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition (ISBN: 0683306472).

Diseases and Conditions The invention is useful for the treatment or prevention of a disease or condition associated with bacterial infection, and in particular for diseases or conditions associated with bacterial protease activity e.g. those in which infection or pathogenesis is mediated by a bacterial metalloprotease, and particularly those mediated by a zinc metalloprotease.

The bacterial infection may be due to any type of bacterium. The invention is particularly useful for with Gram positive bacteria, such as those in the genus Bacillus, including B.anthracis (BA). The polyphenols the invention have been found to be powerful inhibitors of the BA lethal factor, and therefore the prevention or treatment of anthrax is a preferred aspect of the invention.

Whereas the only known biological substrates of LF are members of the mitogen activated protein kinase kinase (MAPKK) family, the polyphenols of the invention have been found to be potent inhibitors of LF. Inhibition of LF activity can be monitored in various ways. Enzymatic activity can be followed in vitro by standard enzymology techniques (e.g. using the assay disclosed by Cummings et al. (2002) Proc Natl Acad Sci USA 99(10):6603-6), by biochemical screening methods (e.g. using the p-anilide peptide substrates disclosed by Tonello et al. (2002) Nature 418:386) and can be monitored in vivo by assessing toxicity in animal models. Cellular assays for toxicity are also available. Optimisation

The invention further extends to a method of modifying a precursor polyphenol to provide a polyphenol or a pharmaceutically acceptable polyphenol derivative having increased bioavailability and/or pharmacokinetics relative to the precursor polyphenol.

Preferred derivatives which are more cell membrane permeable than the precursor polyphenols of the invention are included in the term 'pharmaceutically acceptable derivative'. Preferably, such cell membrane permeable derivatives are peracetylated derivatives. Glycosylated derivatives of the polyphenols of the invention are also included within the term 'pharmaceutically acceptable derivative'. Glycosylated derivatives are advantageous as they show improved solubility compared to the unglycosylated polyphenols of the invention. By 'glycosylated derivative' is meant a polyphenol of the invention wherein one or more glucose moieties are attached to one or more phenol rings.

Other preferred derivatives are polyphenols which fall within formula (I), (II) or (III) below.

The invention further provides compounds having the structures represented by the formulae (I), (II) and (III):

formula (III)

and pharmaceutically acceptable derivatives thereof, wherein:

X is independently O, S, Se or NR¹;

Y¹ is CA₂, O, S, Se, NR¹, C=O, C=S, C=Se or C-NR¹;

Y² is CA₂, O, S, Se, NR¹, C=O, C=S, C=Se or C=NR^!;

Y³isO, S, SeorNR¹;

Y⁴isCAorN;

Z¹ is CA₂, O, S, Se, NR¹, 0=0, C=S, C=Se or C=NR^I;

Z² is CA₂, O, S, Se, NR¹, CO, C=S, C=Se or C=NR¹;

A is independently H, halogen, -NO₂, -CN, -CO₂H, -CO2R¹, -SO₃H, -SOR¹, -SOaR¹, -SO3R¹, -OCO2R¹, -C(O)H, -C(OR}, -OC(0)R', -NR^] ₂, -OC(O)NR^]2, -NCR^CCO)^, -C(S)NR¹ ₂, - NR¹C(S)R¹, -SOa R'z,

-R¹ or -XR¹;

R¹ is independently H, or optionally substituted C_1-2o hydrocarbyl or heterohydrocarbyl;

O.

L is a pharmaceutically acceptable anion; nisO, 1,2, 3, 4 or 5; m is 0 or 1; p is 0 or 1, qisO, 1, 2, 3 or 4; risO, 1,2, 3 or 4; provided that the compound is not EGCG. Compounds of formula (I), (II) or (III) have excellent resistance to oxidation in vivo.

The term 'halogen' is used herein to refer to any of fluorine, chlorine, bromine and iodine. Preferred halogens are fluorine or chlorine, more preferably fluorine.

The term 'hydrocarbyl' means a monovalent group consisting of carbon and hydrogen. Hydrocarbyl groups thus include alkyl, alkenyl and alkynyl groups, cycloalkyl (including polycycloalkyl), cycloalkenyl and aryl groups and combinations thereof, e.g. alkylcycloalkyl, alkylpolycycloalkyl, alkylaryl, alkenylaryl, cycloalkylaryl, cycloalkenylaryl, cycloalkylalkyl, polycycloalkylalkyl, arylalkyl, arylalkenyl, arylcycloalkyl and arylcycloalkenyl groups.

Unless indicated explicitly otherwise, where combinations of groups are referred to herein as one moiety, e.g. arylalkyl, the last mentioned group contains the atom by which the moiety is attached to the rest of the molecule.

Preferably, when R¹ is hydrocarbyl, R¹ is: optionally substituted Cι.g alkyl, alkenyl or alkynl; optionally substituted C₃.1₀ cycloalkyl or cycloalkenyl; optionally substituted C_5-ι₂ aryl; optionally substituted Cι.₈alkylC_3-ιocycloalkyl, Cι.salkenylC_3-ιocycloalkyl or C_1-8alkynlC_3-ιocycloalkyl; optionally substituted C_3-1ocycloalkylCι-₈alkyl, C_3-ιocycloalkylCι_-8alkenyl or C_3-ιocycloalkylCι.. ₈alkynl; optionally substituted C₃.ιocycloalkenylCι_-8alkyl, C_3-ιocycloalkenylC_1-8alkenyl or C_3-ιocycloalkenylCι_-8alkynl; optionally substituted Cι.₈alkylC_3-ιocycloalkenyl, C_1-8alkenylC_{3- l}ocycloalkenyl or C_1-8alkynlC_3-ι₀cycloalkenyl; optionally substituted Cι_-8alkylC_5-ι₂aryl, Cι_-salkenylC_5- ι₂aryl or C_1-8alkynlC_5-1 aryl; or optionally substituted Cs-πarylCi-salkyl, C_5-i₂arylCi_-8alkenyl or Cs. ₁₂arylCι_-galkynl.

The terms 'alkyl', 'alkenyl' or 'alkynl' are used herein to refer to both straight and branched chain forms.

The term 'alkyl' means a saturated hydrocarbyl group. Preferred alkyl are Cι_-4 alkyl such as methyl, ethyl, n-propyl, i-propyl or t-butyl groups. The term 'alkenyl' means a hydrocarbyl group having at least one carbon-carbon double bond and preferably no carbon-carbon triple bonds. Preferred alkenyl are C_2-4 alkenyl.

The term 'alkynl' means a hydrocarbyl group having at least one carbon-carbon triple bond and preferably no carbon-carbon double bonds. Preferred alkynl are C_2- alkynl.

The term 'aryl' means an aromatic group, such as phenyl or naphthyl. In general, the aryl groups may be monocyclic or bicyclic fused ring aromatic groups. Preferred aryl are C_6-ιo aryl.

The term 'heterohydrocarbyl' means a hydrocarbyl group in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O or N. Heterohydrocarbyl groups thus include heteroalkyl, heteroalkenyl and heteroalkynyl groups, cycloheteroalkyl (including polycycloheteroalkyl), cycloheteroalkenyl and heteroaryl groups and combinations thereof, e.g. heteroalkylcycloalkyl, alkylcycloheteroalkyl, heteroalkylpolycycloalkyl, alkylpolycycloheteroalkyl, heteroalkylaryl, alkylheteroaryl, heteroalkenylaryl, alkenylheteroaryl, cycloheteroalkylaryl, cycloalkylheteroaryl, heterocycloalkenylaryl, cycloalkenylheteroaryl, cycloalkylheteroalkyl, heteroalkylalkylcyclo, polycycloalkylheteroalkyl, polycycloheteroalkylalkyl, arylheteroalkyl, heteroarylalkyl, arylheteroalkenyl, heteroarylalkenyl, alkylarylcyclohetero, heteroarylcycloalkyl, arylheterocycloalkenyl and heteroarylcycloalkenyl groups.

Preferably, when R¹ is heterohydrocarbyl, R¹ is: optionally substituted Cι_-8 heteroalkyl, heteroalkenyl or heteroalkynl; optionally substituted C_3-lυ cycloheteroalkyl or cycloheteroalkenyl; optionally substituted C_5-ι heteroaryl; optionally substituted Cι,₈heteroalkylC_3-1ocycloalkyl, Cι_-8heteroalkenylC_{3- l}ocycloalkyl or C_1-8heteroalkynIC₃..₁ocycloalkyl; optionally substituted Cι.₈alkylC_3-ιocycloheteroalkyl, Cι_-8alkenylC3-ιocycloheteroalkyl or Cι_-8alkynlC_3-ι₀cycloheteroalkyl; optionally substituted C_3- ιocycloalkylCι_-8heteroalkyl, C_3-ιocycloalkylCι_-8heteroalkenyl or C_3-ιocycloalkylCι.₈heteroalkynl; optionally substituted C_3-1ocycloheteroalkylC_1-8alkyl, C_3-ιocycloheteroalkylC_1-8alkenyl or C_3- ι₀cycloheteroalkylCι_-8alkynl; optionally substituted Ci_-8heteroalkylC_3-10cycloalkenyl₅ Cι„ ₈heteroalkenylC_3-ιocycloalkenyl or Cι_-8heteroalkynlC_3-1ocycloalkenyl; optionally substituted Cμ ₈alkylC_3-ι₀cycloheteroalkenyl, C_1-8alkenylC_3-10cycloheteroalkenyl or Cι_-8alkynlC_{3- l}ocycloheteroalkenyl; optionally substituted C_3-1ocycloalkenylC_1-8heteroalkyl, Qj.iocycloalkenylCi. sheteroalkenyl or C₃.ιocycloalkenylC_)-8heteroalkynl; optionally substituted C_3- ιocycloheteroalkenylC_1-8alkyl, C_3-1ocycloheteroalkenylC]_-8alkenyl or C_3-ιocycloheteroalkenylCι. ₈alkynl; optionally substituted Cι_-8lιeteroalkylC_3-ι₂aryl, Cι.₈heteroalkenylC_5-ι₂aryl or Ci. ₈heteroalkynlC₅_i₂aryl; optionally substituted

Cι_-8alkenylC_5-ι₂heteroaryl or

optionally substituted C_5-12heteroarylCι_-8alkyl, C_5-i₂heteroarylCi_-8alkenyl or Cs-πheteroarylCi-salkynl; or optionally substituted Cs.πarylCi-sheteroalkyl, C_5-ι₂arylCι. sheteroalkenyl or C_3-ι₂arylCι_-8heteroalkynl. Where reference is made to a carbon atom of a hydrocarbyl group being replaced by an O, S, Se or N atom, what is intended is that:

— CH— — N —

I is replaced by I

— CH= is replaced by — N=; or

-CH₂- is replaced by -O- -S- or -Se-. The term 'heteroalkyl' means an alkyl group in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O or N.

The term 'heteroalkenyl' means an alkenyl group in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O or N. The term 'heteroalkynyP means an alkynyl group in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O or N.

The term 'heteroaryl' means an aryl group in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O or N. Examples of heteroaryl are pyridyl, pyrrolyl or furanyl.

Where reference is made to a substituted group, the substituents are preferably from 1 to 3 in number and selected from Ci-βalkyl, Cι_-6alkoxy, thio, Cι-₆alkylthio, carboxy, carboxy(Cι-₆)alkyl, formyl, Ci. ₆alkylcarbonyl, C^alkylcarbonylalkoxy, nitro, trihalomethyl, hydroxy, Cι_-6alkylhydroxy, hydroxy(Cι_₆)alkyl, amino, Cι_-6 alkylamino, di(Cι_-6 alkyl)amino, aminocarboxy, C_1-6 alkylaminocarboxy, di(Cι_-6 alkyl)aminocarboxy, aminocarboxy(Cι.₆)alkyl, Cι_ δalkylaminocarboxy (C 1 _-6)alkyl, di(C i.₆alkyl)aminocarboxy(Cι _-6)alkyl, C ι -₆alkylcarbonylamino, C_5-8 cycloalkyl, Cs-scycloalky^C^alkyl, Ci-ealkylcarbony^Ci-όalky^amino, aryl, aryl(Cι_-6alkyl), (Ci. ₆alkyl)aryl, halo, Cr^alkylhalo, sulphamoyl, tetrazolyl and cyano. Most usually, substituents will be selected from Cι-₆alkyl_s Cι_-6 alkoxy, thio, Cι_6 alkylthio, carboxy, carboxy(C_1-6)alkyl, formyl, C_1-6 alkylcarbonyl, Cι_-6 alkylcarbonylalkoxy, nitro, trihalomethyl, hydroxy, C_1-6 alkylhydroxy, hydroxy(Cι.₆)alkyl, amino, Cι_-6alkylamino, di(Cι_-6alkyl)amino, aminocarboxy, Cι_-6alkylaminocarboxy, di(C_1-6alkyl)aminocarboxy, aminocarboxy(Cι_-6)alkyl, C_1-6 alkylaminocarboxy(Cι.₆)alkyl, di(C_1-6alkyl)aminocarboxy(Cι.₆)alkyl, Cι_-6 alkylcarbonylamino, C_5-8 cycloalkyl, Cs_-8 cycloalkyl(Cι.₆)alkyl,

sulphamoyl, tetrazolyl and cyano.

Preferred Compounds

Preferred compounds of formulae (I), (II) or (III) are protease inhibitors.

Other preferred compounds of formulae (I), (II) or (III) are polyphenols or pharmaceutically acceptable derivatives thereof.

Preferably, when A is a substituent of Ar¹, Ar² or Ar³, A is not H.

Compounds of formula I are preferred.

With regard to the aromatic ring Ar¹, r is preferably 2 or 3, more preferably 2. When r = 2, the ring Ar¹ is preferably substituted as follows:

and when r = 3, the ring Ar¹ is preferably substituted as follows:

With regard to the aromatic ring Ar², q is preferably 2. The ring Ar² is preferably substituted as follows:

With regard to the aromatic ring Ar³, n is preferably 3. The ring Ar³ is preferably substituted as follows:

For each ring Ar¹, Ar² or Ar³ independently, A is preferably R¹ (more preferably alkyl (e.g. methyl) or arylalkyl (e.g. benzyl)), or XR¹ (more preferably OH, alkyl-O- (e.g. methoxy) or arylalkyl-O- (e.g. benzyl-O-)). XR¹ is preferred. Preferably, for each ring Ar¹, Ar² and Ar³ each X is O and R¹ is H.

Preferably, each X is O.

Preferably, Y¹ is CA₂, O, S, Se or NR¹, more preferably CR'₂, O, S, Se or NR¹, more preferably O or S, still more preferably O.

Preferably, Y² is CR'₂₎ O, S, Se, NR¹, C=O, C=S, C=Se or C=NR^!, more preferably CR^] ₂ or O, more preferably CH₂ or O, still more preferably CH₂.

Preferably, Y³ is O or S, more preferably O.

Preferably, Y⁴ is CR¹ or N, more preferably CR¹, still more preferably CH.

Preferably, Z¹ is CA₂, 0, S, Se or NR¹, more preferably CR^] ₂, O, S, Se or NR¹, more preferably O or S, still more preferably O. Preferably, Z² is C=O, C=S, C=Se or C=NR¹, more preferably C=0 or C=S, still more preferably CO. Θ

L is preferably the anion of a pharmaceutically acceptable acid. Examples of anions of pharmaceutically acceptable inorganic acids are chloride, bromide, iodide, sulphate or phosphate.

Examples of anions of pharmaceutically acceptable organic acids are formate, acetate, propionate, succinate, glycolate, glucuronate, maleate, furate, glutamate, benzoate, anthranilate, salicylate. phenylacetate, mandelate, embonate, methanesulfonate, ethanesulfonate, pantothenate, benzenesulfonate, stearate, sulfanilate, alginate and galacturonate.

Preferred embodiments of formula I are:

(i) m = l, Z¹ is O, p = l and Z² is C=O;

(ii) m - 0, p = 1 and Z² is C=O; and (iii) m =0 and p = 0

In these embodiments (i)-(iii), A in respect of aromatic ring Ar³ is preferably R¹ (more preferably alkyl (e.g. methyl) or arylalkyl (e.g. benzyl)), or XR¹ (more preferably OH, alkyl-O- (e.g. methoxy) or arylalkyl-O- (e.g. benzyl-O-)).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : LF activity in the presence of different catechins.

Figure 2: EGCG and GTE prevent cell death by LeTx.

Figure 3: Western blot showing that cleavage of MEK3 is inhibited by EGCG. Figure 4: Inhibition of LF activity by increasing concentrations of EGCG. Figure 5: Decrease in cell viability by decreasing concentrations of EGCG. Figure 6: Effect of pre-treatment with EGCG on toxicity of LeTx. Figure 7: Effect of post-treatment with EGCG on toxicity of LeTx. Figure 8: Structures of polyphenols. Figure 9: Inhibition of MEK2 and MEK3 cleavage by EGCG.

MODESFORCARRYING OUT THE INVENTION EGCG was obtained from Calbiochem (code 324880). Decaffeinated GTE was obtained from SOFAR (Trezzano Rosa, Milan, Italy).

Inhibition ofLFlytlc activity

The ability of catechins EGC, EC, CG and EGCG to inhibit the lytic activity of LF was investigated.

LF (InM) was pre-incubated with lμM catechin for 5 minutes at 37 °C, then added to 5μM AcGYβARRRRRRRRVLRpNA substrate [Tonello et al, supra] in 25mM Na₂HPO₄, 15 mM NaCl, pH 7.4, and incubated for 5 minutes at 25°C. The release of p-nitroaniline by LF was monitored at

405nm with a spectrophotometer (ε₄os = 9920 M^"1 cm^"1). Results are shown in Figure 1. In the presence of CG and EGCG, lytic activity was reduced to less than 10% compared to activity in the absence of any catechin (the control). The inhibitory constants lie in the range 0.1-lμM, with EGCG having an IC₅₀ of around lOOnM (Figure 4).

Macrophage cell viability The effect of the catechins, and of GTE, on cell viability was also studied.

RAW264.7 macrophage cells were grown in DMEM supplemented with 10% FCS and antibiotics, and incubated in 5% CO₂ in air at 37°C. The cells were seeded onto 96-well plates at 2xl0⁴/well in the FCS-DMEM medium, treated for 4 hours with 400ng/ml LF (which has been pre-incubated for 15 minutes at 37°C with lOμM catechin or with GTE, and then mixed with PA). As shown in Figure 2, cells maintained for 24 hours in the presence of the LF which had been pre-incubated with lOμM EGCG (IμM final concentration) were protected against toxicity was seen (94% viability). Excellent results were also seen with GTE. Thus EGCG and GTE prevent macrophage cell death which is otherwise caused by LeTx, with IC₅o<50nM (Figure 5).

In separate experiments, RAW264.7 cells were seeded onto 96-well plates at 5xl0³/well and were treated with various concentrations of EGCG (in DMEM) twice daily for 5 days. LeTx (400ng/ml LF, 80ng/ml PA) was then added and cell viability was determined 4 hours later by CellTiter 96™ assay (Promega). As shown in Figure 6, pre-treatment with lOμM or higher EGCG protected cells.

In further experiments, EGCG was added after LeTx. RAW264.7 cells were seeded onto 96-well plates at 2xl0⁴/well as above, as LeTx was added. EGCG was then added after various intervals at 50μM or lOOμM, and cell viability was determined 4 hours later. Results are in Figure 7.

The most effective inhibition was obtained when adding EGCG before LeTx. When given together with or after the toxin, the inhibitor was less powerful, but was nonetheless very effective, since cytolysis of macrophages was 50% prevented by adding 50μM EGCG even after 90 minutes incubation with the toxin. These findings qualify EGCG as the most powerful inhibitor of LF known to date (cf. Table 2 of Hammond & Hanna (1998) Infect Immun 66:2374-78; Tang & Leppla (1999) Infect Immun 67:3055-60).

Control cells treated with EGCG alone always maintained 100% viability.

MAPK-kinase cleavage

The only known natural substrate of LF is the MAP kinase kinase (MAPKK) proteins, which are present in several different isoforms within the cell (Duesbery et al, 1998, Science 280:734-737; Vitale et al. 1998 Biochem Biophys Res Commun 248:706-711; Vitale et al. 2000 Biochem J 352:739-745). MAPKK cleavage within LF-sensitive macrophage cells in culture is associated with their death (Friedlander, 1986, J Biol Chem 261:7123-7126). Inhibitors of LF proteolytic activity have to be not only strong but also permeable to the cell plasma membrane to be effective in preventing MAPKK cleavage and macrophage death. The ability of EGCG to inhibit cleavage of MAPK-kinase 3 (MEK3; Tonello et al. (2002) Nature 418:386; Friedlander et al. (2001) J Biol Chem 261:7123ff) in macrophages was studied. Cells were mixed with pre-incubated LeTx, as above, and the presence of MEK3 was monitored by western blot. As shown in Figure 3, EGCG is an effective inhibitor of intracellular proteolytic activity even when sub-micromolar concentrations of pre-incubated LeTx were used. Cleavage of MEK3 was completely inhibited by EGCG at concentrations >100nM.

Similar experiments were carried out to monitor cleavage of MAPK-kinase 2. Figure 9 shows the % of cleaved kinase, and reveals that EGCG is a powerful inhibitor of the intracellular cleavage of both MAPKK-2 and MAPKK-3. Animal model

The best animal model for testing the activity of LeTx is the Fisher 344 rat, which dies 40-60 min following intravenous injection of the toxin (Ezzel et al. (1984) Infect. Immun. A5:761ff). This model was used to study the protective effect of EGCG or GTE. LeTx (12μg LF + 30μg PA) was injected into the caudal vein of rats either without pre-incubation (control) or with 5 minutes of pre-incubation with 50μM EGCG, lOOμM EGCG or lOOμM GTE (which had been decaffeinated and titrated 50% in EGCG prior to use). Control rats showed symptoms about 1 hour after injection, but symptoms were delayed up to 5 hours in rats pre-treated with EGCG or GTE. Thus the toxicity of LeTx is removed by pre-incubation with EGCG or GTE.

Using the caudal vein for injection in the rat does not allow precise control of the volume injected, and repeated injections are difficult. A more reproducible injection route is via the femoral veins uncovered under anesthesia (using isoflurane). Thus 12 rats were injected with 200μl EGCG in the right vein and, after 5 minutes, 200μl LeTx (12μg LF, 30μg PA) in the left, to give circulating EGCG concentrations of about 50μM or lOOμM (assuming a total blood volume of 25ml). Of the 6 rats in the 50μM group, 4 developed symptoms 1-2 hours after the controls; in 2 animals of the second group the delay exceeded 5 hours, and 2 rats recovered completely. Similar effects were seen if GTE was injected instead of EGCG. The partial protective effect may be due to reduced availability of EGCG due to its metabolism, or by absorption by proteins and tissues affecting biodistribution, etc. Moreover, EGCG is labile and sensitive to oxidation.

It will be understood that the invention is described above by way of example only and modifications may be made while remaining within the scope and spirit of the invention.

Claims

I . A method for the treatment or prevention of a disease or condition associated with bacterial infection, comprising the step of administering a therapeutically effective amount of a green tea extract to a patient.

2. The use of a green tea extract in the manufacture of a medicament for the treatment or prevention of a disease or condition associated with bacterial infection.

3. A method for the treatment or prevention of a disease or condition associated with bacterial infection, comprising the step of administering a therapeutically effective amount of a polyphenol, or a pharmaceutically acceptable derivative thereof, to a patient.

4. The use of a polyphenol, or a pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for the treatment or prevention of a disease or condition associated with bacterial infection.

5. The method or use of any preceding claim, wherein the bacterial infection is a Bacillus anthracis infection.

6. The method or use of any preceding claim, wherein the disease or condition is anthrax.

7. A composition comprising a bacterial protease and a polyphenol.

8. The composition of claim 7, wherein the protease and a polyphenol are in a complex.

9. The composition of claim 7, wherein the bacterial protease is B.anthracis LF or LeTx.

10. A method for inhibiting the proteolytic cleavage of a mitogen activated protein kinase kinase, comprising the step of administering a therapeutically effective amount of a green tea extract to a patient.

II. A method for inhibiting the proteolytic cleavage of a mitogen activated protein kinase kinase, comprising the step of administering a therapeutically effective amount of a therapeutically effective amount of a polyphenol, or a pharmaceutically acceptable derivative thereof, to a patient.

12. A compound of formula (I), (II) or (III):

formula (I)

formula (II)

formula (III)

and pharmaceutically acceptable derivatives thereof, wherein:

X is independently O, S, Se or NR¹;

Y¹ is CA₂, O, S, Se, NR¹, C=O, C=S, C=Se or C=NR^];

Y² is CA₂, O, S, Se, NR¹, C=O, C=S, C=Se or C=NR^!;

Y³ is O, S, Se or R¹;

Y⁴ is CA orN;

Z¹ is CA₂, O, S, Se, NR¹, C=O, C=S, C=Se or C=NR^];

Z² is CA₂, O, S, Se, NR¹, C=O, C=S, C=Se or C=NR*;

A is independently H, halogen, -N0₂, -CN, -CO₂H, -CO₂R^], -SO₃H, -SOR¹, -SOzR¹, -SOsR¹, -OCOsR¹, -C(O)H, -C(O)R¹,

-C(S)NR^! ₂, - NR¹C(S)R¹, -SOaN ^, -NR'SQjR¹, -N(R¹)C(S)NR¹ ₂, -N^SO^R¹^ -R¹ or -XR¹;

R¹ is independently H, or optionally substituted Cι,₂o hydrocarbyl or heterohydrocarbyl;

Θ

L is a pharmaceutically acceptable anion; n is 0, 1, 2, 3, 4 or 5; m is 0 or 1 ; p is 0 or 1, q is 0, 1, 2, 3 or 4; r is O, 1, 2, 3 or 4; provided that the compound is not EGCG.

13. A crystal of a protease and a polyphenol.

14. The crystal of claim 13, wherein the protease is B.anthracis lethal factor.