WO2014060581A1 - Use of hydroxytirosol and derivatives thereof as quorum quenchers - Google Patents

Abstract

Herein is described the quorum quenching activity of compounds of formula (I) or (II) such as hydroxytyrosol (HT), hydroxytyrosol acetate (HTA), 3,4-dihydroxyphenylacetic acid (DOPAC) and derivatives thereof. It is also described their use in the treatment of bacterial infections or a disorder associated with biofilm formation in a subject, associated with quorum sensing caused by pathogenic bacteria as well as their use in the manufacturing of food, foodpakage, medical devices and pharmaceutical compositions.

Description

USE OF HYDROXYTIROSOL AND DERIVATIVES THEREOF AS QUORUM

QUENCHERS

FIELD OF THE INVENTION

The present invention relates to inhibitors of bacterial quorum sensing in multicellular community bacteria and their use in microbial intervention strategies associated with quorum sensing caused by pathogenic bacteria, such as the treatment of bacterial infections or disorders associated with biofilm formation.

BACKGROUND

Polyphenols are a wide family of compounds found in fruits and vegetables, wine, tea, cocoa, and extra-virgin olive oil, which exhibit strong antioxidant activity by scavenging different families of Reactive Oxygen Species (ROS). Polyphenols of plant origin have been reported to have a variety of biological effects, including antioxidant, anticarcinogenic, anti-inflammatory and antimicrobial activities (Cowan, M.M., Clin. Microbiol. Rev., 1999, 12: 564-582). In particular, hydroxytyrosol (HT) has been suggested as one of the most effective members of the polyphenol family. Hydroxytyrosol can be found in leaves and fruits of olive, extra virgin olive oil and it is specially abundant in olive oil mill wastewaters from where it can be recovered (Fernandez-Bolafios, J.G. et al., Curr.Org.Chem., 2008, 12, 442-463.). Olive oils differ in the total polyphenol content, in fact, wide ranges have been reported (50-1000 g/g). Levels of individual phenols are difficult to establish due to natural variability and strong dependence on oil age and history after production. Median values have been reported, for hydroxytyrosol, for example, it has been found to be about 1 .9 μg g (Boskou, D. et al., "Olive Oil, Chemistry and Technology", 2^nd edition, ACCS Press, pages 79-80).

Several studies have evidenced the broad spectrum of beneficial effects that hydroxytyrosol shows such as cancer chemoprevention by inducing apoptosis, cardioprotection and anti-atherogenic activity, skin photoprotection and antiinflammatory activity (Cicerale, S. et al., R. Int. J. Mol. Sci., 2010, 1 1 , 458-479; Fernandez-Bolafios, J.G. et al., 2008; Granados-Principal, S. et al., Nutr. Rev., 2010, 68, 191 -206; Obied, H.K., et al., Food Chem. Tox., 2007, 45, 1238-1248; Rietjens, S.J.,et al., J. Agric. FoodChem., 2007, 55, 7609-7614). However, studies investigating the antimicrobial effects of olive oil phenolic compounds are conflicting. Many authors have reported the high antimicrobial potential of olive oil phenolic compounds, including HT (Medina, E. et al., J. Agric. Food Chem., 2007, 55, 9817-9823; Medina, E.et al., J. Food Prot., 2007, 70, 1 194-1 199; Medina, E. et al., J. Agric. Food Chem., 2006, 54, 4954-4961 ; Medina, E. et al., J. Food Prot., 2009, 72, 261 1 -2614; Bisignano, G. et al., J. Pharm. Pharmac, 1999, 51 , 971 -974.; Bubonja-Sonje, M. et al., 201 1 , Food Chem., 201 1 , 127, 1821 -1827). Conversely, other research studies have demonstrated little or no antimicrobial activity of synthetic HT (Serra, A . et al., Inn. Food Sci. Emer. Tech., 2008, 9, 31 1-319; Karaosmanoglu, H., et al., J. Agric. Food Chem., 2010, 58, 8238- 8245; Obied et al., 2007). Herein is shown that the antimicrobial activity of HT and its derivatives is very limited and they cannot be considered as effective antimicrobial agents as very high concentrations are needed (about >1000 μg mL).

On the other hand, in the last years, many research studies have focused in the search of new anti-pathogenic agents to control microbial infections via the inhibition of Quorum Sensing (QS) (Al-Hussaini, R. and Mahasneh, A.M., Molecules, 2009, 14, 3425-3435; Brackman, G. et al., Res.Microb., 2009, 160, 144-151 ; Huber, B. et al., Zeitschrift Fur Naturforschung C, 2003, 58, 879-884; Vikram, A. et al., Int. J. Food Microb., 2010, 140, 109-1 16; Truchado, P. et al., Food Control, 2012, 24, 78-85.).

Quorum Sensing is a communication system that allows bacteria to monitor their population density through the production and sensing of small signal molecules called autoinducers (Bassler, B.L., Cur. Op. Microb., 1999, 2, 582-587.; Brackman et al., 2009). Several authors have demonstrated that bacteria employ QS to control a variety of physiological processes, including bioluminescense, swarming, swimming, antibiotic biosynthesis, biofilm formation, sporulation and the production of virulence determinants (Cui and Harling, European Journal of Plant Pathology, 2005, 1 1 1 , 327- 339). Therefore, it is accepted that QS regulates the production of pathogenicity and virulence factors (Figure 1 ).

The finding that many pathogens rely on this cell-to-cell communication mechanism, known as quorum sensing or, antipathogenic or signal interference, to synchronize microbial activities essential for infection and survival in the host suggests a promising disease control strategy, i.e. quenching microbial quorum sensing or in short, quorum quenching. Work over the past few years has demonstrated that quorum-quenching mechanisms are widely conserved in many prokaryotic and eukaryotic organisms (Dong, Y.-H., Phil. Trans. R. Soc. B, 2007, vol. 362, no. 1483, 1201 -121 1 ).

Different bacterial species may produce different types of quorum-sensing signals, but they appear to adopt only two general mechanisms for detecting and responding to these signals. One general mechanism is represented by acylhomoserine lactone (AHL)-dependent quorum-sensing systems, and the other general mechanism is the autoinducing peptide (AIP) mediated mechanism.

The discovery that a wide spectrum of bacteria uses QS to control virulence factor production makes it an attractive target for antimicrobial therapy (Finch, R.G., Journal of Antimicrobial Agents and Chemotherapy, 1998, 42, 569-571 ). Recently, the inventors have reported the potential of urolithins, ellagitannin metabolites produced by colon microbiota, and an orange extract rich in blycosylated flavanones to inhibit QS- mediated infection processes such as biofilm formation and motility (Truchado, P., Journal of Agricultural and Food Chemistry, 2012, 60, 8885-8894).

Biofilm formation is also an important issue, especially due to the increasing use of implants, artificial heart valves and the like, which results in drug resistance and causes several persistent infections. Biofilms can be extremely resistant to removal and disinfection. Their complex and dense nature typically afford resistance to penetration by antimicrobials. Therefore, there is a need of new strategies to deal with bacterial infection issues that have no current effective solution. In this sense, disrupting QS may interfere with the ability of bacteria to form robust biofilms and thus render the bacteria more sensitive to antibacterial agents and the host's immune system.

Compounds that inhibit Quorum Sensing are capable of quenching this bacterial communication system without affecting other cellular functions. Therefore, they do not act as bactericidal or bacteriostatic agents, since they neither kill the bacteria nor inhibit its growth. However, though these compounds may not have bactericidal effect, they have important therapeutic applications due to their ability to attenuate virulence, drug resistance and/or biofilm formation.

WO 2009/1 14810 describes the use of ellagitannins as inhibitors of bacterial quorum sensing.

WO 2012/074912 describes methods for treating infections caused by bacteria that use 5'-methylthioinosine phosphorylase in a quorum sensing pathway comprising administering to a subject having the infection a sub-growth inhibiting amount of a 5'methylthioinosine phosphorylase inhibitor.

WO 201 1/001419 also describes compounds as inhibitors of quorum sensing in bacteria. Romero, M. et al. (Recent Patents on Biotechnology, 2012, 6, 2-12) makes a review of patents and patents applications related with Quorum Quenching.

Therefore, any quorum-quenching chemicals or enzymes that can effectively interfere with any of the key processes of the quorum sensing mechanisms could be potentially used for quenching quorum sensing and preventing microbial infections.

Further, the emergence of antibiotic resistance in microbial pathogens highlights why it is important to explore new ways to prevent and control infectious diseases.

BRIEF DESCRIPTION OF THE INVENTION

The inventors of the present invention have found that hydroxytyrosol (HT), hydroxytyrosol acetate (HTA) and 3,4-dihydroxyphenylacetic acid (DOPAC) have good anti-Quorum Sensing (anti-QS) activity, i.e. Quorum-Quenching activity. The Quorum- Quenching activity of these molecules is described in the examples and it has been validated against a biosensor strain: Chromobacterium violaceum (C. violaceum). The capacity of HT, HTA and DOPAC to inhibit QS-controlled processes which are known to be related with virulence factors such as production of biofilm formation, bioluminescense, swarming, swimming, motility, antibiotic biosynthesis, pigmet biosynthesis, enzymes biosynthesis, toxin biosynthesis, exopolysacharide biosynthesis and sporulation of specific bacterial strains makes them useful in the prevention and treatment of bacterial infection associated with QS, particularly as preservative in food preservation and as excipient in pharmaceutical formulations such as ophthalmic and dental formulations.

Therefore, one aspect of the present invention relates to the use of a compound of formula (I) or (II):

(I) (Π) wherein

R-i , R₂ and R₃ are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, ORa, SR_a, SOR_a, S0₂R_a, OS0₂R_a, OS0₃R_a, N0₂, NHR_a, N(R_a)₂, =N-R_a, N(R_a)COR_a, N(COR_a)₂, N(R_a)S0₂R', N(R_a)C(=NR_a)N(R_a)R_a, CN, halogen, COR_a, COOR_a, OCOR_a, OCOOR_a, OCONHR_a, OCON(R_a)₂, CONHR_a, CON(R_a)₂, CON(R_a)OR_a, CON(R_a)S0₂R_a, PO(OR_a)₂, PO(OR_a)R_a, PO(OR_a)(N(R_a)R_a) and aminoacid ester; each of the R_a groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclyl, or a salt, solvate or isomer thereof, for the inhibition of Quorum Sensing (QS) in multicellular community bacteria, wherein the inhibition of QS implies inhibiting physiological processes of multicellular community bacteria. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w.

The compound of formula (I) and (II) as defined herein can be synthetic or extracted from its natural source.

Another aspect of this invention refers to a pharmaceutical composition comprising a compound of formula (I) or (II) as defined above or a pharmaceutically acceptable salt, solvate or isomer thereof. In a particular embodiment, said compound is present in an effective amount between about 0.005% and about 0.04% w/w of the pharmaceutical composition. In an embodiment, the compound of formula (I) or (II) as defined above or a pharmaceutically acceptable salt, solvate or isomer thereof is present in the composition as an excipient. Another aspect of this invention refers to the use of a compound of formula (I) or (II) as defined above or a pharmaceutically acceptable salt, solvate or isomer thereof, as an excipient for the preparation of pharmaceutical compositions. Preferably, said compound is present in an effective amount between about 0.005% and about 0.04% w/w. Another aspect of this invention refers to a foodstuff comprising a compound, of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof as defined above as a preservative. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w of the foodstuff.

Another aspect of this invention refers to the use of a compound of formula (I) or (II) as defined above or a pharmaceutically acceptable salt, solvate or isomer thereof, as a preservative for the preparation of foodstuff.

Another aspect of this invention refers a pharmaceutical composition selected from an ophthalmic composition and a dental composition comprising a compound of formula (I) or (II) as defined above or a pharmaceutically acceptable salt, solvate or isomer thereof. Another aspect of this invention refers to a pharmaceutical composition comprising a compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof as defined in the present invention, wherein the pharmaceutical composition is selected from an ophthalmic composition and a dental composition.

Another aspect of this invention refers to the use of a compound of formula (I) or (II) as defined above or a pharmaceutically acceptable salt, solvate or isomer thereof, for the preparation of a pharmaceutical composition selected from an ophthalmic composition and a dental composition.

In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w of the ophthalmic or dental composition. A medical device or foodstuff package comprising a compound of formula (I) or (II) as defined above or a salt, solvate or isomer thereof. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w of the medical device or the foodstuff package.

Another aspect of this invention refers to the use of a compound of formula (I) or (II) as defined above or a salt, solvate or isomer thereof, for manufacturing medical devices or foodstuff packages.

Another aspect of this invention refers to a compound of formula (I) or (II) as defined above or a pharmaceutically acceptable salt, solvate or isomer thereof, for use in the treatment of a bacterial infection in a subject. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w. Another aspect of this invention refers to a compound of formula (I) or (II) as defined above or a pharmaceutically acceptable salt, solvate or isomer thereof, for use in the treatment of a disorder associated with biofilm formation in a subject. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w.

Another aspect of this invention refers to a method of disrupting or inhibiting biofilm formation on a surface, the method comprising contacting or manufacturing the surface with a compound of formula (I) or (II) or a salt, solvate or isomer thereof, or a composition comprising the compound, in an amount effective for disrupting or inhibiting biofilm formation on the surface. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w in the composition to be applied to said surface.

Another aspect of this invention refers to a medicament or pharmaceutical composition comprising at least one compound of formula (I) or (II) as defined herein, or a pharmaceutically acceptable salt, solvate or isomer thereof as an excipient, for use in the treatment of a bacterial infection or a disorder associated with biofilm formation by inhibition of quorum sensing. Preferably, said compound is present in an effective amount between about 0.005% and about 0.04% w/w of said medicament or pharmaceutical composition.

Another aspect of this invention refers to a foodstuff comprising at least one compound of formula (I) or (II) as defined herein, or a pharmaceutically acceptable salt, solvate or isomer thereof, as preservative for use in the prevention of food spoilage or foodborne illness. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w of said foodstuff.

Another further aspect refers to a foodstuff package comprising at least one compound of formula (I) or (II) as defined herein, or a salt, solvate or isomer thereof, for use in the prevention of food spoilage or foodborne illness. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w of said foodstuff package.

Another aspect of this invention refers to the use of a compound of formula (I) or (II) as defined above, or a pharmaceutically acceptable salt, solvate or isomer thereof, in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection or a disorder associated with biofilm formation by inhibition of the quorum sensing (QS) of the bacteria. In a preferred embodiment the compound is present in an effective amount between about 0.005% and about 0.04% w/w.

Another aspect of the present invention refers to a method for the treatment and/or prophylaxis of bacterial infections or a disorder associated with biofilm formation said method comprising administering to the subject in need of such a treatment or prophylaxis a therapeutically effective amount of a compound of formula (I) or (II) as defined above, or a pharmaceutically acceptable salt, solvate or isomer thereof to inhibit quorum sensing (QS) in bacteria. Preferably, said compound is present in an effective amount between about 0.005% and about 0.04% w/w.

In another aspect, the present invention provides a method for the treatment and/or prophylaxis of a disorder associated with biofilm formation in a subject, the method comprising administering the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof to a surface or use the compound in the manufacture of said surface in an amount effective to inhibit biofilm formation associated with bacterial quorum sensing on said surface.

Preferably, the compound of formula (I) or (II) as defined herein is present in an effective amount for inhibiting the Quorum Sensing but in a sub-bacterial-growth inhibiting amount, i.e. an amount that does not kill or reduce the growth of the bacteria population.

In a particular embodiment, the compound of formula (I) or (II), or a pharmaceutically acceptable salt, solvate or isomer thereof, as defined herein is administered in a composition comprising said in an effective amount between about 0.005% and about 0.04% w/w, preferably between about 0.005% and about 0.03% w/w, preferably between about 0.005% and about 0.02% w/w, preferably between about 0.005% and about 0.015% w/w, more preferably between about 0.005% and about 0.01 % w/w.

These aspects and preferred embodiments thereof are additionally also defined in the claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. QS circuit. Two regulatory genes (luxR homologue and luxl homologue) are required for QS controlled gene expression. When the cell population is low, insufficient AHL signals are present to activate the LuxR protein (R protein). As the cell population increases, the concentration of the AHL signals increases both intra- and extracellularly. At a critical concentration, the R protein is activated through binding to the AHL signal. The activated R protein acts as a transcription activator or repressor and therefore regulates the quorum-sensing-controlled gene expression (adapted from Cui and Harling, 2005).

Figure 2. Chromatogrham at 280 nm of extract 12%.

Figure 3. Bacterial growth in Muller-Hilton plates supplemented with HT (6 g/ml). Figure 4. Bacterial growth in Muller-Hilton plates supplemented with HT (10 g/ml). Figure 5. Agar-well diffusion test.

Figure 6. Bacterial growth in Muller-Hilton plates supplemented with HT.

Figure 7. Production of violacein by C. violaceum grow in culture media supplemented with HT, HTA and DOPAC.

Figure 8. Production of violacein by C. violaceum grow in culture media supplemented with olive extract 12% and 6%.

Figure 9. Production of violacein by C. violaceum grow in culture media supplemented with olive extract II.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present invention, the following terms have the meaning detailed below.

The compound of formula (I) or (II) of the present invention is a synthetic compound, or it has been extracted and purified from its natural source.

The term "synthetic" means produced by synthesis i.e. is not of natural origin. Preferably, the synthetic compound is purified after the synthetic process. In one embodiment, the compound is more than 80% pure, preferably is more than 90% pure, more preferably is more than 95% pure. In a particular embodiment the purity is between the 98 % and the 100%.

The expression "extracted from its natural source" means that is obtained by extraction from its natural source, separated and further purified. Means, techniques and methods of extraction and purification are know in the art and include, among others, evaporation, liquid-liquid extraction, decantation, chromatography, centrifuging, crystallization, filtration and precipitation. The compound of the present invention, extracted from its natural source, is at least more than 80% pure, preferably is more than 90% pure, more preferably is more than 95% pure. In a particular embodiment the purity is between the 98 % and the 100%. Natural oils that contain a compound of the invention are excluded when the compound is present in the oil in an amount less than 70%.

"Alkyl" refers to a straight or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, containing no unsaturation, and which is attached to the rest of the molecule by a single bond. Alkyl groups preferably have from 1 to about 22 carbon atoms. One more preferred class of alkyl groups has from 1 to about 12 carbon atoms; and even more preferably from 1 to about 6 carbon atoms. Alkyl groups having 1 , 2, 3, 4 or 5 carbon atoms are particularly preferred. Methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, tert-butyl, sec-butyl and iso-butyl are particularly preferred alkyl groups. As used herein, the term alkyl, unless otherwise stated, refers to both cyclic and noncyclic groups, although cyclic groups will comprise at least three carbon ring members, such as cyclopropyl or cyclohexyl. Alkyl radicals may be optionally substituted by one or more substituents, such as an aryl group, like in benzyl or phenethyl.

"Alkenyl" and "Alkynyl" refer to a straight or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, containing at least one unsaturation (one carbon-carbon double or triple bond respectively) and which is attached to the rest of the molecule by a single bond. Alkenyl and alkynyl groups preferably have from 2 to about 22 carbon atoms. One more preferred class of alkenyl and alkynyl groups has from 2 to about 12 carbon atoms; and even more preferably from 2 to about 6 carbon atoms. Alkenyl and alkynyl groups having 2, 3, 4 or 5 carbon atoms are particularly preferred. The terms alkenyl and alkynyl as used herein refer to both cyclic and noncyclic groups, although cyclic groups will comprise at least three carbon ring members. Alkenyl and alkenyl radicals may be optionally substituted by one or more substituents.

"Aryl" refers to a radical derived from an aromatic hydrocarbon by removal of a hydrogen atom from a ring carbon atom. Suitable aryl groups in the present invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups. Typical aryl groups contain from 1 to 3 separated and/or fused rings and from 6 to about 22 carbon ring atoms. Preferably aryl groups contain from 6 to about 10 carbon ring atoms. Aryl radicals may be optionally substituted by one or more substituents. Specially preferred aryl groups include substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl, substituted or unsubstituted phenanthryl and substituted or unsubstituted anthryl.

"Heterocyclyl" refers to a cyclic radical having as ring members atoms of at least two different elements. Suitable heterocyclyl radicals include heteroaromatic and heteroalicyclic groups containing from 1 to 3 separated and/or fused rings and from 5 to about 18 ring atoms. Preferably heteroaromatic and heteroalicyclic groups contain from 5 to about 10 ring atoms. Heterocycles are described in: Katritzky, Alan R., Rees, C. W., and Scriven, E. Comprehensive Heterocyclic Chemistry (1996) Pergamon Press; Paquette, Leo A.; Principles of Modern Heterocyclic Chemistry W.A. Benjamin, New York, (1968), particularly Chapters 1 , 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28. Suitable heteroaromatic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., coumarinyl including 8- coumarinyl, quinolyl including 8-quinolyl, isoquinolyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Suitable heteroalicyclic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., pyrrolidinyl, tetrahydrofuryl, dihydrofuryl, tetrahydrothienyl, tetrahydrothiopyranyl, piperidyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1 ,2,3,6- tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1 ,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1 .0]hexyl, 3- azabicyclo[4.1 .0]heptyl, 3H-indolyl, and quinolizinyl. Heterocylic radicals may be optionally substituted by one or more substituents.

The organic groups above defined may be substituted at one or more available positions by one or more suitable groups such as OR', =0, SR', SOR', S0₂R', OS0₂R', OS0₃R', N0₂, NHR\ N(R')₂, =N-R\ N(R')COR\ N(COR')₂, N(R')S0₂R\ N(R')C(=NR')N(R')R', CN, halogen, COR', COOR', OCOR', OCOOR', OCONHR', OCON(R')₂, CONHR', CON(R')₂, CON(R')OR\ CON(R')S0₂R', PO(OR')₂, PO(OR')R', PO(OR')(N(R')R'), substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C₂-Ci₂ alkenyl, substituted or unsubstituted C₂-Ci₂ alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclyl, wherein each of the R' groups is independently selected from the group consisting of hydrogen, OH, N0₂, NH₂, SH, CN, halogen, COH, COalkyl, COOH, substituted or unsubstituted Ci-C₁₂ alkyl, substituted or unsubstituted C₂-Ci₂ alkenyl, substituted or unsubstituted C₂-Ci₂ alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list.

"Halogen" substituents in the present invention include F, CI, Br, and I.

In one preferred embodiment of the present invention, the compounds of formula (I) or (II) are selected from the compounds wherein R-i , R₂ and R₃ are independently selected from the group consisting of hydrogen, substituted or unsubstituted C C₂₂ alkyl, substituted or unsubstituted C₂-C₂₂ alkenyl, substituted or unsubstituted C₂-C₂₂ alkynyl, C₆-C₂₂ substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclyl having from 5 to 18 ring atoms, OR_a, SR_a, SOR_a, S0₂R_a, OS0₂R_a, OS0₃R_a, N0_2, NHR_a, N(R_a)₂, =N-R_a, N(R_a)COR_a, N(COR_a)₂, N(R_a)S0₂R', N(R_a)C(=NR_a)N(R_a)R_a, CN, halogen, COR_a, COOR_a, OCOR_a, OCOOR_a, OCONHR_a, OCON(R_a)₂, CONHR_a, CON(R_a)₂, CON(R_a)OR_a, CON(R_a)S0₂R_a, PO(OR_a)₂, PO(OR_a)R_a, PO(OR_a)(N(R_a)R_a) and aminoacid ester;

And each of the R_a groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C C₂₂ alkyl, substituted or unsubstituted C₂-C₂₂ alkenyl, substituted or unsubstituted C₂-C₂₂ alkynyl, substituted or unsubstituted C₆-C₂₂ aryl, and substituted or unsubstituted heterocyclyl having from 5 to 18 ring atoms.

Preferred compounds of formula (I) are hydroxytyrosol (R-i , R₂, R₃ are H) and the following hydroxytyrosol derivatives:

(1 ) carboxylic acid esters, such as acetate (Ri, R₂, are H and R₃ is -COCH₃),

(2) sulphonate esters, such as alkyl- or aralkylsulphonyl (for example, methanesulphonyl);

(3) phosphate esters;

(4) phosphonate esters;

(5) phosphoramidate esters; (6) amino acid esters (for example, alanine, L-valyl or L-isoleucyl).

Thus, in one more preferred embodiment of the present invention, the compound is selected from a compound of formula (I) wherein R-i , R₂ and R₃ are independently selected from the group consisting of hydrogen, S0₂R_a, COR_a, PO(OR_a)2, PO(ORa)Ra, PO(OR_a)(N(R_a)R_a) and aminoacid ester;

and each of the Ra groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C22 alkyl, substituted or unsubstituted C2-C22 alkenyl, substituted or unsubstituted C2-C22 alkynyl, substituted or unsubstituted C₆-C₂2 aryl, and substituted or unsubstituted heterocyclic group having from 5 to 18 ring atoms.

Some specific most preferred compounds of formula (I) in the present invention are for example the following:

Hydroxytyrosol (Ri, R₂, R3 are H),

Hydroxytyrosol acetate (Ri, R₂, are H and R₃ is -COCH₃),

A specific most preferred compound of formula (II) in the present invention is for example the following:

3,4-dihydroxyphenylacetic acid (Ri, R₂, R3 are H) or its salts.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio. In some embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency or listed in the European or U.S. Pharmacopeia, or other generally recognized international pharmacopeia for use particularly in humans.

Non-limiting examples of salts in the present invention are sulphates; hydrohalide salts; phosphates; lower alkane sulphonates; arylsulphonates; salts of C1 -C20 aliphatic mono-, di- or tribasic acids which may contain one or more double bonds, an aryl nucleus or other functional groups such as hydroxy, amino, or keto; salts of aromatic acids in which the aromatic nuclei may or may not be substituted with groups such as hydroxyl, lower alkoxyl, amino, mono- or di- lower alkylamino sulphonamido. Also included within the scope of the invention are quaternary salts of the tertiary nitrogen atom with lower alkyl halides or sulphates, and oxygenated derivatives of the tertiary nitrogen atom, such as the N-oxides. In preparing dosage formulations, those skilled in the art will select the pharmaceutically acceptable salts. Therefore, in some particular embodiment, the salts of the present invention are pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to any salt which, upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. The preparation of salts can be carried out by methods known in the art.

For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stochiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. Generally, nonaqueous media like ether, ethyl acetate, ethanol, 2-propanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, Ν,Ν-dialkylenethanolamine, triethanolamine and basic aminoacids salts. Since hydroxytyrosol has three hydroxyl groups, alkali addition salts are particularly preferred such as Na⁺ and NX₄ ⁺ (wherein X is independently selected from H or a C C₄ alkyl group).

The compounds of the invention may be in crystalline form either as free compounds or as solvates (e.g. hydrates, alcoholates, particularly methanolates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art. The compounds of the invention may present different polymorphic forms, and it is intended that the invention encompasses all such forms.

Any compound referred to herein is intended to represent such specific compound as well as certain variations, forms or isomers thereof. In particular, compounds referred to herein may have asymmetric centres and therefore exist in different enantiomeric or diastereomeric forms. Thus, any given compound referred to herein is intended to represent any one of a racemate, one or more enantiomeric forms, one or more diastereomeric forms, and mixtures thereof. Likewise, stereoisomerism or geometric isomerism about the double bond is also possible, therefore in some cases the molecule could exist as (E)-isomer or (Z)-isomer (trans and cis isomers). If the molecule contains several double bonds, each double bond will have its own stereoisomerism, that could be the same as, or different to, the stereoisomerism of the other double bonds of the molecule. Furthermore, compounds referred to herein may exist as atropisomers. All the stereoisomers including enantiomers, diastereoisomers, geometric isomers and atropisomers of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.

Unless otherwise stated, the compounds comprised in the pharmaceutical compositions of the invention are also meant to include isotopically-labelled forms i.e. compounds which differ only in the presence of one or more isotopically-enriched atoms. For example, compounds having the present structures except for the replacement of at least one hydrogen atom by a deuterium or tritium, or the replacement of at least one carbon by ¹³C- or ¹⁴C-enriched carbon, or the replacement of at least one nitrogen by ¹⁵N-enriched nitrogen are within the scope of this invention.

The term "quorum sensing" as used herein refers to the process by which bacteria produce and detect signaling molecules with which they coordinate gene expression and regulate processes beneficial to the microbial community. Quorum sensing can occur within a single bacterial species as well as between diverse species, and can regulate a host of different processes, in essence, serving as a simple communication network.

The term "inhibit (or inhibition of) quorum sensing" or "quench (or quenching of) quorum sensing" or "quorum-quenching" as used herein means altering the quorum sensing process, i.e. altering the physiological process of bacteria, by blocking the key steps of quorum sensing, such as signal generation, signal accumulation or signal reception, such that coordination of gene expression and process regulation in microbial communities is impaired or prevented. Therefore, the compounds described herein are named "quorum quenchers" (QQ) or "quorum sensing inhibitors" (QSI).

The term "multicellular community bacteria" refers to bacteria at high cell densities, i.e. when they switch from a nomadic existence, each bacterium lives on its own, to community behaviour, i.e. as a group of bacterium living and interacting with one another due to the quorum sensing. The "physiological processes of bacteria" are a wide variety of physiological processes unique to the life-cycle of microbes, preferably bacteria. These processes include but are not limited to production of N-acyl homoserine lactones, biofilm formation, bioluminescense, swarming, swimming, motility, antibiotic biosynthesis, pigmet biosynthesis, enzymes biosynthesis, toxin biosynthesis, exopolysacharide biosynthesis and sporulation.

The term "viability of the bacteria" means the capacity of normal growth and development of each bacterium, i.e. the capacity of normal growth and development when the bacteria do not behave as a bacteria multicellular community. The compounds of the present invention do not affect the viability of the bacteria since they are not antimicrobial agents.

By an "effective amount" or a "therapeutically effective amount" of a compound, drug or pharmacologically active agent is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, the particular active agent or agents, and the like.

In a preferred embodiment of this invention, the effective amount does not affect the viability of bacteria. Preferably, the compound of formula (I) or (II) as defined herein is present in a sub-bacterial-growth inhibiting amount, i.e. an amount that, when contacting with a population of bacteria, does not kill or reduce the growth of the bacteria population.

The sub-bacterial-growth inhibiting amount in each case can be readily determined by the skilled person using conventional methods to measure the minimum amount of the compound resulting in inhibition of bacterial growth. For example, following the exemplary assays described herein in Examples 3 and 4.

In a particular embodiment, the compound of formula (I) or (II) as defined in the present invention or a pharmaceutically acceptable salt, solvate or isomer thereof may be present in a pharmaceutical formulation, food formulation or cosmetic formulation.

Thus, it is not always possible to specify an exact "effective amount". However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

As described above, in this invention the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is present in an effective amount for the inhibition of QS which is not sufficient to behave as antimicrobial agent. As it is shown in the examples, the amount of compounds of formula (I) or (II) needed to behave as antimicrobial agent is quite superior to the amount needed to behave as a Quorum-Quencher (QQ).

In a preferred embodiment the effective amount is between about between about 0.005 % and about 0.04 % weight, between about 0.0075 % weight and about 0.0375 % weight, between about 0.001 % weight and about 0.035 % weight, between about 0.00125 % weight and about 0.0325 % weight, between about 0.0015 % weight and about 0.0325 % weight, between about 0.00175 % weight and about 0.03 % weight, and more preferably between about 0.0018 % weight and about 0.032 % weight. In a particular embodiment, the effective amount is between about 0.005% and about 0.02% weight, preferably between about 0.005% weight and about 0.015% weight, more preferably between about 0.005% weight and about 0.01 % weight. In some embodiments the effective amount is about 0.001 % weight, about 0.002 % weight, about 0.003 % weight or about 0.004 % weight. The percentages (% w/w) are expressed as weight of the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof by the total weight of the composition comprising the compound or by weight of the foodstuff, foodstuff package, medical device or surface.

In another embodiment the effective amount is expressed in μg mL or μg g ^g of the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof by mL or g of the composition comprising the compound), therefore effective amount is about 75 and about 375 μg mL (or μg g), between about 100 and about 350 μg mL (or μg g), between about 125 and about 325 μg mL (or μg g), between about 150 and about 325 μg mL (or μg g), between about 175 and about 300 μg mL (or μg g), and more preferably between about 180 and about 320 μg mL (or μg g). In a particular embodiment, the effective amount is between about 50 and about 200 μg mL (or μg g), preferably between 50 and about 150 μg mL (or μg g), more preferably between about 50 and about 100 μg mL (or μg g). In some embodiments the effective amount is about 100 μg/mL (or μg/g), about 200 μg/mL (or μg/g), about 300 μg/mL (or μg g) or about 400 μg/mL (or μg/g).

When the compound of formula (I) or (II) or a salt, solvate or isomer thereof as defined herein is present on a surface, it is preferably in an effective amount of between about 1 and about 200 μς/οηι², preferably between about 1 and about 100 μς/οηι², preferably between about 1 and about 50 μς/οηη², more preferably between about 5 and about 300 g/cm².

The terms "microbial", "antimicrobial", "microbe" and the like in this text are related to microorganisms, particularly to bacteria or fungi, and preferably in this text to bacteria. Particularly, "antimicrobial" refers either to compounds that kill microbes (bacteria or fungi), i.e. microbiocidal compounds, or to compounds that prevent the growth of microbes, i.e. microbiostatic compounds.

As used herein, the terms "pathogenic bacterium", "pathogenic bacteria", "bacterium" or "bacteria" refer to both gram-negative and gram-positive bacterial cells capable of infecting and causing disease in a mammalian host, as well as producing infection- related symptoms in the infected host, such as fever or other signs of inflammation, intestinal symptoms, respiratory symptoms, dehydration, and the like. In one embodiment the bacteria are gram-negative bacteria. In another embodiment the bacteria are gram-positive bacteria. In another further embodiment the bacteria are gram-positive bacteria together with gram-negative bacteria. In another embodiment there is only one bacteria specie or different bacteria species; one bacteria genus or different bacteria genus, infecting or causing disease.

In some embodiments, and without limitation, the bacteria is of a genus selected from the group consisting of Acinetobacter, Actinobacillus, Aeromonas, Aggregatibacter, Agrobacterium, Bacillus, Bordetella, Brucella, Burkholderia, Campylobacter, Chromobacterium, Cyanobacteria, Enterobacter, Erwinia, Escherichia, Franciscella, Fusobacterium, Haemophilus, Helicobacter, Hemophilus, Klebsiella, Lactobacillus, Legionella, Listeria, Micrococcus, Moraxella, Mycobacterium, Neisseria, Nitrosomas, Obesumbacterium, Pantoea, Pasteurella, Pediococcus, Porphyromonas, Prevotella, Proteus, Pseudomonas, Ralstonia, Rhisobium, Rhodobacter, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Tannerella, Treponema, Vibrio, Xenorhabdus, Yersinia and mixtures thereof. For example, in some embodiments and without limitation, the bacteria is of a species selected from the group consisting of Aeromonas hydrophila, Aeromonas salmonicida, Aggregatibacter actinomycetemcomitans, Agrobacterium tumefaciens, Bacillus Cereus, Bacillus Subtilis, Burkholderia cepacia, Campylobacter jejuni, Chromobacterium violaceum, Enterobacter agglomeran, Erwinia carotovora, Erwinia chrysanthemi, Escherichia coli, Fusobacterium nucleatum, Haemophilus injiuenzae, Helicobacter pylori, Lactobacillus Plantarum, Listeria Monocytogenes, Klebsiella Pneuumoniae, Micrococcus Luteus, Mycobacterium tuberculosis, Neisseria meningitidis, Neisseria gonorrhoeae, Nitrosomas europaea, Obesumbacterium proteus, Pantoea stewartii, Pediococcus acidilactici, Prevotella intermedia, Porphyromonas gingivalis, Pseudomonas aureofaciens, Pseudomonas aeruginosa, Pseudomonas Phosphoreum, Pseudomonas syringae, Ralstonia solanacearum, Rhisobium etli, Rhisobium leguminosarum, Rhodobacter sphaeroides, Salmonella typhimurium, Serratia liguefaciens, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus enteritis, Tannerella forsythensis, Treponema denticola, Vibrio anguillarum, Vibrio fischeri, Vibrio cholerae, Vibrio harveyi, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Xenorhabdus nematophilus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia medievalis, Yersinia ruckeri and mixtures thereof.

The particular bacteria that cause food spoilage, or foodborne illness, or respiratory tract infections, or ocular infections, or periodontal diseases, or urinary tract infections, or other quorum sensing related infections, diseases or biofilm formation are known in the art.

In one embodiment the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is used as an excipient for the preparation of pharmaceutical compositions. In one embodiment, the pharmaceutical composition is an ophthalmologic formulation. In another embodiment the pharmaceutical composition is a dental formulation.

The term "excipient" as referred herein is an ingredient included in a pharmaceutical preparation for the purpose of improving its physical qualities, for example, the stability. A compound of formula (I) or (II) as defined above, or a pharmaceutically acceptable salt, solvate or isomer thereof, as described in this document may be used as an excipient for the preparation of any medicament or pharmaceutical composition.

In one embodiment, the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof for the inhibition of quorum sensing (QS) is for use in the treatment in a subject in vivo. In such embodiments, the use comprises administering the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof to a subject, wherein the subject is for example, an animal, such as a mammal, such as a primate, such as a human, or a plant. Preferably the subject is a mammalian subject and more preferably the mammalian subject is human. In one embodiment, the subject is afflicted with a bacterial infection associated with bacterial QS and the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is administered in an amount effective to treat the bacterial infection. In another embodiment, the subject is afflicted with a disorder associated with biofilm formation and the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is administered in an amount effective to treat that disorder.

In another embodiment, the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof for the inhibition of quorum sensing (QS) is for use in the treatment in a subject ex vivo. In such an embodiment, for example, the use comprises administering the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof to a surface or use the compound in the manufacture of said surface in an amount effective to inhibit biofilm formation associated with bacterial quorum sensing on surface (including without limitation, a medical device or a foodpackage).

As used herein, to treat a bacterial infection in a subject means to reduce the virulence of the bacteria in the subject. The term "bacterial infection" shall mean any deleterious presence of bacteria in the subject.

"Bacterial infection" refers herein to bacterial infections associated with quorum sensing, wherein bacterial infections are caused by pathogenic bacteria. These bacterial infections include, but are not limited to, bacteremia, septicemia, endo- and pericarditis, sinusitis, upper respiratory tract infection, urinary tract infections, chronic bronchitis, pneumonia, cerebral and pulmonary lesions, meningitis, dermatitis or folliculitis, necrotizing fascitis, cellulitis, osteomylitis, enterocolitis, bacterial dental infections, contact lens-associated kerititis and conjunctivitis.

"Biofilm formation" or "bacterial biofilm formation" refers to the formation of drug- impervious communities of bacteria. Biofilms are dense extracellular polymeric matrices in which the bacteria embed themselves. Biofilms allow bacteria to create a microenvironment that attaches the bacteria to the host surface and which contains excreted enzymes and other factors allowing the bacteria to evade host immune responses including antibodies and cellular immune responses. Such biofilms can also exclude antibiotics. Further, biofilms can be extremely resistant to removal and disinfection. Biofilms are inherent in dental plaques, and are found on surgical instruments, food processing and agriculture equipment and water treatment and power generating machinery and equipment. Therefore, as used herein, the term "biofilm" refers to a thin layer of microorganisms, preferably bacteria, adhering to the surface of a structure, which may be organic or inorganic, together with the polymers that they secrete.

A "disorder associated with biofilm formation" in a subject is selected from the group consisting of cystic fibrosis, dental caries, periodonitis, otitis media, muscular skeletal infections, necrotizing fasciitis, biliaty tract infection, osteomyelitis, bacterial prostatitis, endocarditis, native valve endocarditis, cystic fibrosis pneumonia, meloidosis, or skin lesions associated with bullous impetigo, atopic dermatitis and pemphigus foliaceus or implanted (medical)device-related infections. In some embodiments, the condition is a nosocomial infection, including but not limited to, pneumonia or an infection associated with sutures, exit sites, arteriovenous sites, sclera buckles, contact lenses, urinary catheter cystitis, peritoneal dialysis (CAPD) peritonitis, lUDs, endotracheal tubes, Hickman catheters, central venous catheters, mechanical heart valves, vascular grafts, biliary stent blockage, and orthopedic devices.

Also provided is a method of modulating biofilm formation on a surface, the method comprising contacting or manufacturing the surface with a compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof, in an amount effective for disrupt or inhibit biofilm formation on the surface.

In another embodiment the invention relates to the use of the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof for the preparation of a cream, gel, powder, suspension, emulsion or the like to contact the compound with a surface. The surface may be inanimate or animate.

In one embodiment, the surface is an inanimate surface. Exemplary inanimate surfaces include, but are not limited to, metal, glass, plastic, wood and stone surfaces. Said materials are used for manufacturing medical devices or foodstuff packages.

In an alternative embodiment, when the surface is inanimate, the composition may be coated or applied to the surface of medical apparatus that comes into contact with potential sites in the body where multi-species infections may reside. By way of example, the compositions disclosed herein may be applied to catheters for preventing catheter induced infection. As catheters generally have a gelatinous film applied to them to ease insertion, the compounds of the present invention can conveniently be added to such compositions.

Percutaneous devices (such as catheters) and implanted medical devices (including, but not limited to, pacemakers, vascular grafts, stents, and heart valves) commonly serve as foci for bacterial infection. The tendency of some microorganisms to adhere to and colonize the surface of the device promotes such infections, which increase the morbidity and mortality associated with use of the devices.

For example, a compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is used to inhibit biofilm formation on substrates used to manufacture medical devices associated with non-invasive and invasive medical procedures. Such substrates include, without limitation, tubular, sheet, rod and articles of proper shape for use in a number of medical devices such as vascular grafts, aortic grafts, arterial, venous, or vascular tubing, vascular stents, dialysis membranes, tubing or connectors, blood oxygenator tubing or membranes, surgical instruments, ultrafiltration membranes, intra-aortic balloons, stents, blood bags, catheters, sutures, soft or hard tissue prostheses, synthetic prostheses, prosthetic heart valves, tissue adhesives, cardiac pacemaker leads, artificial organs, endotracheal tubes, lenses for the eye such as contact or intraocular lenses, blood handling equipment, apheresis equipment, diagnostic and monitoring catheters and sensors, biosensors, dental devices, drug delivery systems, or bodily implants of any kind. For example, arthroscopic surgery is routinely performed with use of medical devices that minimize the invasiveness of the procedure. Such devices include, for example and without limitation, ultrathin microfiberoptic endoscopes that offer the laryngologist unique access to the limited spaces of the temporal bone and skull base. In another example, a stent supplemented with one or more compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof can be constructed. Stents are used to maintain an open lumen in tissues including the tracheo-bronchial system, the biliary hepatic system, the esophageal bowel system, and the urinary tract system. In another embodiment the surface is an animate surface. Exemplary animate surfaces include, but are not limited to, mammalian tissues, mammalian membranes, mammalian skin, plants. Preferably the surface is mammalian skin and in a particular embodiment the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is used for the preparation of a cosmetic composition. In another embodiment the invention relates to the use of the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof for the preparation of an ophthalmic composition. Preferably, the ophthalmic composition comprises from 0.1 g to 300 mg, more preferably comprises from 1 g to 100 mg, of a compound of formula (I) or (II).

In one embodiment, the ophthalmic composition is administered in form of eye drops, injection or a cream.

Periodontal diseases contribute greatly to the incidence of tooth loss in adults, and are often the result of bacterial infection and biofilm formation. Dental plaque consists of tens, possibly hundreds of species, including both Gram negative and Gram positive bacteria. Known periodontal pathogens include Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythensis, Prevotella intermedia, Treponema denticola, and Fusobacterium nucleatum. Dental decay is caused by very few bacterial species, mainly Streptococcus mutans and in the later stages by Lactobacilli species.

Therefore, in another embodiment the invention relates to the use of the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof for the preparation of dental formulation. Preferably, the dental composition comprises from 0.1 μg to 300 mg, more preferably comprises from 1 μg to 100 mg, of a compound of formula (I) or (II). In preferred embodiments the composition is a paste. In other embodiments, the composition may be toothpaste, a mouthwash, or a chewing gum.

By dental, it is taken to mean any part of the oral cavity, not simply the teeth. So, infections of the roots, gums and so on are taken to be covered by this term. More particularly, the bacterial dental infection may be periodontal disease. Periodontal disease is a type of disease that affects one or more of the periodontal tissues: alveolar bone, periodontal ligament, cementum or gingival. While many different diseases affect the tooth-supporting structures, plaque-induced inflammatory lesions make up the vast majority of periodontal diseases and have traditionally been divided into two categories: gingivitis or periodontitis.

The dose of compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof administered to a mammalian subject range from about 0.1 μg to about 400 mg/day. In some embodiments, the dose is about 0.1 Mg/day, about 0.5 Mg/day, about 1 g/day, about 5 Mg/day, about 10 Mg/day, about 25 Mg/day, about 50 Mg/day, about 75 Mg/day, about 100 Mg/day, about 125 Mg/day, about 150 Mg/day, about 175 Mg/day, about 200 Mg/day, about 225 Mg/day, about 250 Mg/day, about 275 μg day, about 300 Mg/day, about 325 Mg/day, about 350 Mg/day, about 375 Mg/day, about 400 Mg/day, about 425 Mg/day, about 450 Mg/day, about 475 Mg/day, about 500 μg day, about 750 Mg/day, about 1 mg/day, about 5 mg/day, about 10 mg/day, about 25 mg/day, about 30 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 60 mg/day, about 70 mg/day, about 80 mg/day, about 90 mg/day, about 100 mg/day, about 150 mg/day, about 200 mg/day , about 250 mg/day, about 300 mg/day, about 350 mg/day or about 400 mg/day. In some embodiments, the maximum dosage is about 200 mg/day. In some embodiments, the maximum dosage is about 300 mg/day. If desired, the effective daily dose is divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. The dosage regimen of a compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof composition alone or in combination to be used in treatment of bacterial infections (or biofilm formation) associated with QS will be determined by the attending physician considering various factors which modify the action of the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof, e.g., the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.

In another embodiment the invention relates to the use of the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof for the fabrication of a foodstuff package.

In another embodiment, one or more compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is used to inhibit biofilm formation associated with bacterial QS on a foodstuff package by contacting or manufacture the package with a compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof in an amount effective to inhibit biofilm formation.

For example, one or more compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is used to inhibit biofilm formation on substrates used to manufacture foodpackage. Materials suitable for the manufacture of the foodpackage include polymeric materials, glass, ceramics, metals, and the like. Food packaging types are tins, cans, packets, films, bottles, bags, trays, boxes, cartons and the like. The term "disrupt" in the scope of this invention refers to interrupt or impede the progress.

In a particular embodiment, the compound of formula (I) or (II) as defined in the present invention or a pharmaceutically acceptable salt, solvate or isomer thereof may be present in the pharmaceutical formulation, food formulation or cosmetic formulation together with or adsorbed in dextrins, such as maltodextrins or cyclodextrins, or starch.

In another embodiment the compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof is used as a preservative for the preparation of foodstuff.

The term "preservative" means that, added to foods, preserves or tends to preserve the food, preventing food spoilage or foodborne illness.

The term "food" or "foodstuff" refers to a substance that can be used or prepared for use as food. Herein, foodstuff includes solid, semisolid and liquid food. Solid food includes, for example and without limitation, meat, fish, seafood, groceries, pasta, bread, rice, dairy products as cheese, snacks, flour, bread, pastry products, cereals and the like. Semisolid food includes, for example and without limitation, jams, yoghurts, jelly and the like. Liquid food includes, for example and without limitation, milk, juices, sauces, wine, beer, vinegar and the like.

The expression "food spoilage" in this invention refers to the process of food becoming damaged irreparably by bacteria. The deterioration is produced to the point in which the food is not edible to humans or its quality of edibility becomes reduced.

The expression "foodborne illness", also called "foodborne disease" or "food poisoning" is any illness resulting from the consumption of contaminated food. There are two types of food poisoning: infectious agent and toxic agent. The present invention refers to food poisoning by infectious agent, wherein the infectious agent refers to the presence of bacteria or other microbes which infect the body after consumption. The bacteria that cause foodborne illness are known in the art.

To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term "about". It is understood that, whether the term "about" is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value.

The following examples are merely illustrative of certain embodiments of the invention and cannot be considered as restricting it in any way.

EXAMPLES

Materials and methods

Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. In this study we will determine the MIC of the five selected compounds against a selection of bacteria (Table 1 ) using the microdilution technique.

The microdilution test was performed in sterile microdilution plates with 96 wells U- shaped. Different concentrations of the selected compounds were dispensed in the wells of rows 1 -10 of microdilution plates in 100μΙ_, with a multichannel pipette. Each well of the microdilution plate was then inoculated with 100μΙ_ of concentrated suspension of the corresponding inoculum. The growth control wells had 100μΙ_ of sterile, free of compounds culture media, and were inoculated with 100μΙ_ of concentrated suspensions of inoculum. The row 12 of the microdilution plate was used as a control of sterility with only medium. The microdilution plates were incubated at the recommended temperature for each bacterial strain for 72 hours. The MIC was determined using a plate reader at 600 nm (Infinite® M200 micro plate reader, Tecan, Grodig, Austria).

Antipathogenic properties against selected bacterial strains

The ability of test compounds to inhibit bacterial communication systems or Quorum Sensing was determined by using the biosensor bacterium Chromobacterium violaceum. It was evaluated based on the inhibition of violacein production by the bacterium C. violaceum. In these tests violacein produced by C. violaceum CECT 494 bacteria was quantified when grew in broth with different concentrations of test compounds. The culture medium (1 mL) was centrifuged at 13000 g (13000 rpm) for 10 minutes and after removing the supernatant the violacein was extracted with dimethylsulfoxide (DMSO). The pigment production in culture medium supplemented with compounds / extracts was quantified using a UV spectrophotometer (Hewlett Packard 8453) at OD₅₈₅. Bacterial Strains Causing:

1 Erwinia spp. (fruits and vegetables)

2 Pseudomonas spp. (fruits and vegetables, milk and meat)

3 Leuconostoc spp. (meat)

Food spoilage

4 Aerobacter spp. (meat)

5 Lactobacillus spp. (meat and milk)

6 Micrococcus luteus (milk)

7 Bacillus subtilis (milk)

8 Escherichia coli 0157:1-17 CECT 4267

9 Escherichia coli 0157:1-17 CECT 4076

10 Yersinia enterocolitica CECT 4315

1 1 Salmonella typhimurium

Foodborne illness

12 Aeromonas hydrophila CECT 389

13 Shigella sonnei CECT 457

14 Listeria monocytogenes CECT 91 1

15 Listeria monocytogenes CECT 940

16 Staphylococcus aureus

Respiratory tract

17 Pseudomonas aeruginosa

infections

18 Streptococcus pneumoniae

Table 1 . Selected bacterial strains.

Example 1. Determination of the % of degradation of HT, HTA and DOPAC.

The aim of this study was to determine the % of degradation of different compounds when incubated in the culture media used to determine the antimicrobial capacity. The selected compounds were (1 ) Hydroxytyrosol (3,4-dihydroxyphenylethanol; DOPET), (2) Hydroxytyrosol acetate (2-(3,4-dihydroxyphenyl)-ethyl acetate or 4-(acetoxyethyl)- 1 ,2-dihydroxybenzene) and (3) 3,4-Dihydroxyphenylacetic acid (DOPAC). The percentage of degradation of the selected compounds was analysed by HPLC (Agilent 1 100 Series, Agilent Technologies, Waldbronn, Germany) equipped with a binary pump (G1312 A), a degasser (G1322 A), a photodiode array diode array detector (G1315 B) and a mass detector in series (Agilent Technologies). The samples were injected by a model L-7200 autosampler. The mass detector was an ion trap spectrometer (G2445A) equipped with an electrospray ionization (ESI) system.

As Table 2 shows, there was not a significant degradation of the different compounds when incubated in the culture media Mueller-Hinton so positive results if any must not be related with production of significant amounts of pro-oxidants (quinones/semiquinones) or hydrogen peroxide (Halliwell, B., Arch. Biochem. Biophys., 2008, 476, 107-1 12.). This culture media was the same used in previous studies to determine the antimicrobial activity of HT (Bisignano, G.et al., J. Pharm. Pharmac, 1999, 51 , 971 -974).

Table 2. Percentage of compound (HT, HTA and DOPAC) remaining in the culture media after 24 hours of incubation.

Example 2. Determination of the phenolic composition (content of Hydroxytyrosol, Hydroxytyrosol acetate and DOPAC) of the olive extracts.

The aim of this study is determine the phenolic composition (content of Hydroxytyrosol, Hydroxytyrosol acetate and DOPAC) of the olive extracts included in this study: (1 ) Olive extract 12%, (2) Olive extract 6% and (3) Olive extract II.

Chromatographic separations of these olive extracts were carried out a C18 Mediterranea Sea column (Teknokroma, Barcelona, Spain) (RP-18, 250mmx4mm; 5 μηη particle size), with 1 % formic acid (A) and acetonitrile (B) as solvents (99.9%, HPLC grade; Merck, Darmstadt, Germany). The flow rate of 1 mL-min^~1 and all chromatograms were recorded at 280 nm. The HPLC system was equipped with an Agilent 1 100 Series diode array and a mass detector in series (Agilent Technologies, Waldbronn, Germany). The HPLC system consisted of a binary pump (G1312 A), an auto sampler (G1313 A) a degasser (G1322 A), and photodiode-array detector (G1315 B) controlled by software (v. A08.03). The mass detector was an ion trap spectrometer (G2445A) equipped with an electrospray ionization (ESI) system and controlled by software (v. 4.1 ). The nebulizer gas was nitrogen; the pressure and the flow rate of the dryer gas were set at 65 psi and 1 1 L rmin^"1, respectively. The full scan mass covered the range from m/z 100-1000 collision-induced fragmentation experiments were performed in the ion trap using helium as collision gas, with voltage ramping cycles from 0.3 to 2V. The heated capillary and voltage were maintained at 350 °C and 4 kV, respectively.

The Chromatogram analysis at 280 nm showed different peaks corresponding to Hydroxytyrosol, Hydroxytyrosol acetate and DOPAC (Figure 2). These phenolic compounds were identified according to their UV spectra and retention times by chromatographic comparisons with authentic standards. Hydroxytyrosol was quantified as Hydroxytyrosol (3,4- dihydroxyphenylethanol; DOPET) at 280 nm, Hydroxytyrosol acetate was quantified as Hydroxytyrosol acetate (2-(3,4-dihydroxyphenyl)-ethyl acetate or 4-(acetoxyethyl)-1 ,2-dihydroxybenzene) and DOPAC as 3,4- Dihydroxyphenylacetic acid at 280 nm. Table 3 indicates the concentration of these phenolic compounds in each extract. As it could be observed, the obtained concentrations were very low (always lower than 0.5 mg/g).

Table 3. Concentration (mg/g) of phenolic compounds detected in the selected extracts

Example 3. Evaluation of the antimicrobial capacity of (1 ) Hydroxytyrosol (3,4- dihydroxyphenylethanol or DOPET), (2) Hydroxytyrosol acetate (2-(3,4- dihydroxyphenvD-ethyl acetate or 4-(acetoxyethyl)-1 ,2-dihvdroxybenzene), (3) 3,4-Dihvdroxyphenylacetic acid (DOPAC), (4) Olive extract 12%, (5) Olive extract 6% and (6) Olive extract II.

The antimicrobial activity has been defined by determining the minimum inhibitory concentration (MIC) of the different compounds. The MIC is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. The MIC of the six selected compounds has been determined against a selection of bacteria using the microdilution technique.

Minimum Inhibitory Concentration (MIC) of the selected compounds. The MIC of microorganisms involved in causing food spoilage is shown in Table 4. In general, all the tested bacterial strains were able to grow in culture media supplemented with the selected compounds at concentrations lower than 1000 μg mL.

Table 4. MIC of bacterial strains involve in causing food spoilage.

An exception was observed in the case of the bacterium Lactobacillus plantarum when treated with HT and HTA, which showed a MIC of 400 μg mL. Thus, the most effective compounds were HT and HTA; although their antimicrobial capacity is much reduced. In fact, Erwinia carotovora, Leuconostoc, and Klebsiella pneumoniae subsp. Pneumoniae where inhibited when supplemented with HT and HTA at around 1000 μg mL meanwhile, concentrations of 2000 or higher where needed when supplemented with the rest of the tested compounds (DOPAC, extract 12%, extract 6% and olive extract II). Therefore, although higher antimicrobial activity against bacterial strains involve in causing food spoilage was observed for HT and HTA, these compounds are not very effective inhibiting bacterial growth.

Table 5 HT(1 ) HTA (2) DOPAC(3) 12% (4) 6% (5) Olive (6)

E.coli 0157:H7 >2000 >2000 2000 >2000 >2000 >2000

Yersinia

>2000 >2000 2000 >2000 >2000 >2000 enterocolitica

Salmonella

>2000 2000 2000 >2000 >2000 >2000 typhymurium

Aeromonas

>2000 >2000 >2000 >2000 >2000 >2000 hydrophyla

Shigella sonnei 1000 >2000 >2000 >2000 >2000 >2000

Listera 2000 >2000 >2000 >2000 >2000 >2000 monocytogenes

Table 5. MIC of bacterial strains involve in causing foodborne illness

The MIC of microorganisms involved in causing foodborne illness is shown in Table 5. It was observed that these bacteria are less susceptible to the tested compounds than microorganisms that cause food spoilage. In most of the cases, the MICs were around 2000 μg mL or higher. Only Shigella sonnei exhibited a MIC of 1000 μg mL when treated with HT. DOPAC also seemed to be effective inhibiting growth of Escherichia coli 0157:H7, Yersinia enterocolitica and Salmonella typhymurium, which showed MIC of 2000 μg mL. Therefore, the higher antimicrobial activity against bacteria involved in causing bacterial food-borne illness was observed for HT, being S. sonnei the most susceptible bacterium. However, as previously reported none of the tested compounds or extracts showed a high antimicrobial activity as very high concentrations (around 1000 g/mL) are needed.

Table 6. MIC of bacterial strains involve in causing respiratory tract infections

The MIC of bacterial strains involve in causing respiratory tract infections is shown in Table 6. Both tested strains, Staphylococcus aureus and Pseudomonas aeruginosa, were very resistant to the tested compounds with MICs higher than >1000 μg mL for all the tested compounds.

Based on the obtained results it could be concluded that among the tested compounds HT, HTA and DOPAC showed the highest antimicrobial activity, being HT and HTA the most effective ones. On the other hand, the olive extracts 6% and 12% as well as the olive extract II presented a very limited antimicrobial activity, showing MICs of 2000 μg mL or higher. However, none of the tested compounds can be considered as effective antimicrobial agents as very high concentrations are needed in almost all the cases.

Example 4. Corroboration of the data presented in a previous published research paper (Bisignano et al., 1999).

The aim of this study is to corroborate the data presented in a previous published research paper (Bisignano, G. et al., J. Pharm. Pharmac, 1999, 51 , 971 -974.), which evaluated the in-vitro susceptibility of several bacterial strains to hydroxytyrosol. The following experiments were carried following the same materials and methods described in this publication. Two bacterial strains were selected (Salmonella typhi and Staphylococcus aureus). However, since the materials and methods section is sometimes confusing this study has been approached in four different ways to validate the obtained results.

4.1 Antimicrobial activity of HT using the agar diffusion test

The inhibitory activity of HT was assayed by the agar-well diffusion test. Plates were made by adding approximately 10⁶ cfu/ml of an overnight culture of Salmonella spp. y Staphylococcus aureus to the Mueller-Hilton agar (12 %). The culture media was supplemented with the selected concentrations of HT. Table 7 shows the concentration selected for this study. The experiment was carried out three times and there were three replicates per sample.

Table 7. Volumes of stock solutions and culture media used in the agar diffusion test.

Stock solutions of HT (10 mg/mL) in dimethylsulphoxide (DMSO) were diluted with DMSO (1 :10, to reach 1 mg/mL)) and subsequently diluted in sterile buffer up to reach the desired concentration (500 μg/mL), following the method described in the above mentioned paper (Bisignano et al., 1999). Additionally, the same method was carried out using stock solutions made in water to compare the obtained results by using DMSO.

Results Example 4.1

The tested concentrations (selected following the paper materials and methods) did not report any antimicrobial effect by the addition of this HT extract in any of the tested bacterial strains (Figures 3 and 4). Therefore the addition of DMSO did not enhance the antimicrobial activity reported for HT.

4.2 Antimicrobial activity of HT using the agar-well diffusion test The inhibitory activity of HT was assayed by the agar-well diffusion test (Figure 5). Plates were made by adding approximately 10⁶ cfu/ml of an overnight culture of Salmonella spp. and Staphylococcus aureus to the Muller- Hilton agar (12 %). Wells were filled with 20 μΙ_ of different concentrations of HT. Plates were incubated for 24 h at 37 °C prior to the determination of inhibition zone sizes by contrast camera imaging (Synoptics, Cambridge, United Kingdom). The experiment was carried out three times and there were three replicates per honey sample.

Stock solutions of HT (10 mg/mL) in DMSO were diluted with DMSO (1 :10 to reach 1 mg/mL). Subsequently, dilution series were done in sterile buffer:DMSO (1 :1 ) up to reach the desired concentration (5, 20, 50, 100, 200, 500 μg mL), following the method described in the above mentioned paper (Bisignano et al., 1999). Additionally, the same method was carried out using stock solutions made in water to compare the obtained results by using DMSO.

Results Example 4.2

The tested concentrations did not report any antimicrobial effect by the addition of this HT extract in any of the tested bacterial strains (Figure 6). Therefore the addition of DMSO did not enhance the antimicrobial activity reported for HT.

4.3 Antimicrobial activity of HT using the flask-incubation assays

Flask-incubation assays were carried out to quantify the inhibitory activity of HT. Salmonella spp. and Staphylococcus aureus bacteria were incubated for 24 h and inoculated to OD₆oonm= 0.1 in 5 mL tubes containing ISO-SENS broth (Scharlau Chemie, S.A.) and ISO-SENS broth supplemented with different HT concentrations (5, 20, 50, 100, 200, 500 Mg/mL). The flasks were incubated at 37 °C in a shaking incubator.

Stock solutions of HT (10 mg/mL) in DMSO were used to reach the desired concentrations (5, 20, 50, 100, 200, 500 Mg/mL) (Table 8), following the method described in the above mentioned paper (Bisignano et al., 1999). Additionally, the same method was carried out using stock solutions made in water to compare the obtained results by using DMSO.

Final HT [ ] Stock solution (μί)

Culture media (μί) Final volumen (mL) (Mg/mL) (10 mg/ml )

0 0 5000 5

10 5 4995 5 50 25 4975 5

100 50 4950 5

200 100 4900 5

500 250 4750 5

Volumes of stock solutions and culture media used in the flask-incubation

Results Example 4.3

No growth inhibition was observed in any of the tested tubes including those prepared with DMSO and those prepared with water.

4.4 Antimicrobial activity of HT using the flask-incubation assays

The materials and methods used in this assay are the same as described for the section 4.3. In this case, a stock solutions of HT (10 mg/mL) in DMSO were diluted with DMSO (1 :10 to reach 1 mg/mL), following the method described in the above mentioned paper (Bisignano et al., 1999). Additionally, the same method was carried out using stock solutions made in water to compare the obtained results by using DMSO. Also, DMSO was added without the addition of HT to determine its inhibitory effect. As indicated in Table 9, the final concentration of HT was reached using two different stock solutions. The final concentration defined as 1000 (A) refers to the use of a stock solution of 1000 μg/mL, while the final concentration defined as 1000 (B) refers to the use of a stock solution of 10000 μg/mL. The tested concentrations are showed in Table 9.

Final HT [ ] Stock solution (μί)

Culture media (μί) Final volumen (mL) ( g/mL) (1000 μg//ml )

0 0 5000 5

10 50 4950 5

50 250 4750 5

100 500 4500 5

200 1000 4000 5

500 2500 2500 5

1000 (A) 5000 0 5

Final HT [ ] Stock solution (μί)

Culture media (μί) Final volumen (mL) ( g/mL) (10000 μg//ml )

1000 (B) 500 4500 5 Table 9. Volumes of stock solutions and culture media used in the flask-incubation assays.

Results Example 4.4

The inhibition observed in this study is showed in Table 10. As it can be observed only the use of a concentration of 1000 μς of HT diluted in DMSO inhibited growth of Salmonella spp. On the other hand, the addition of 500 μς of HT diluted in DMSO inhibited growth of Staphylococcus aureus. No growth inhibition was observed in those tubes supplemented with HT diluted in water instead of DMSO. Therefore, the observed inhibition in those tubes supplemented with HT diluted in DMSO was probably due to the addition of DMSO.

Table 10. Inhibitory activity of HT diluted in DMSO and water. Additionally, DMSO was added in the same volume to determine its inhibitory effect.

Results Examples 4

The obtained results showed that HT cannot be considered as an antimicrobial agent. In fact, the previous described antimicrobial activity was not confirmed, which opens great doubts of previously published results. Therefore, if a decrease in the concentration of AHLs produced by the biosensor strain C. violaceum is observed, it cannot be attributed to any bactericidal or bacteriostatic effect. Example 5. Evaluation of the anti-Quorum Sensing (anti-QS) capacity of HT, HTA, DOPAC and three olive extracts.

The aim of this study was the evaluation of the anti-Quorum Sensing (anti-QS) capacity of (1 ) Hydroxytyrosol (3,4-dihydroxyphenylethanol; DOPET), (2) Hydroxytyrosol acetate (2-(3,4-dihydroxyphenyl)-ethyl acetate or 4-(acetoxyethyl)-1 ,2-dihydroxybenzene), (3) 3,4-Dihydroxyphenylacetic acid (DOPAC), (4) Olive extract 12%, (5) Olive extract 6% and (6) Olive extract II.

Screening of the QS inhibitory activity of selected compounds was carried out based on their ability to inhibit the production of a purple pigment violacein in Chromobacterium violaceum. QS in this wild type strain is known to control production of violacein in response to autoinducer molecules such as N-acyl-homoserine lactones (AHLs). The production of violacein by C. violaceum grow in culture media supplemented with the six selected compounds was quantified at OD₅₈₅ using a UV-Vis spectrophotometer (Hewlet Packard 8453).

Results Example 5. Production of violacein by C. violaceum grow in culture media supplemented with the selected compounds:

Figures 7, 8 and 9 show the production of violacein by C. violaceum grow in culture media supplemented with the selected compounds at different concentrations: 50, 100, 200, 400, 600, 800 and 1000 g/mL. The addition of HT, HTA and DOPAC (Figure 7) significantly affected the production of violacein. Culture media supplemented with these compounds at the minimum tested concentration (50 g/mL) produced an inhibition of the violacein production between 20 and 50 % when compared to the control (untreated strain). In general, increasing concentrations, up to 200 μg mL, did not significantly increase the obtained inhibition of violacein production. However, the addition of a concentration of 400 μg mL of HTA or higher almost completely inhibited the violacein production. The use of 800 and 1000 μg mL of HT and DOPAC also showed a significant drop in violacein production by almost 70% of the total production.

The effect of the addition of olive extract at 12% on the violacein production by C. violaceum showed variable results. In general, addition of any of the tested concentrations reduced violacein production by at least 20%, which means that the obtained inhibition was not concentration dependent. In any case, the obtained violacein inhibition was not higher than 30%. Additionally, the addition of olive extract at 6% did not showed a clear tendency. The violacein produced by C. violaceum grow in culture media supplemented with the olive extract at 6% was very variable, showing significant deviation between the different replicates. What is more, the use of increasing concentrations did not show higher anti-QS activity. Therefore, no clear conclusion can be obtained; although the expected anti-QS activity of this compound would be very low.

The addition of olive extract II did not showed any anti-QS activity as production of violacein was not significantly reduced by any of the tested concentrations.

The obtained results showed that among the tested compounds, HT, HTA and DOPAC showed the highest anti-QS activity even at the lowest tested concentration (50 μg mL). HTA was the most efficient compound reducing almost completely the production of violacein by C. violaceum when applied at the highest tested concentrations (600, 800 and 1000 Mg/mL).

In conclusion, the present study evidenced the capacity of HT, HTA and DOPAC as QS inhibitors in the biosensor strain C. violaceum.

Conclusions.

Based on the obtained results it could be concluded that none of the tested compounds and olive extracts can be considered as effective antimicrobial agents against the tested bacterial strains.

However, the present study evidenced the capacity of HT, HTA and DOPAC as QS inhibitors in the biosensor strain C. violaceum. Many natural extracts (fruits, herbs, spices) have been suggested to inhibit QS by a combination of mechanisms; however, the anti-QS capacity of HT, HTA and DOPAC has not been previously described. Considering that QS inhibitors may help to attenuate virulence and reduce biofilm formation, they are of interest in the development of novel natural microbial intervention strategies.

Claims

A compound of formula (I)

(I) (II) wherein R-i , R₂ and R₃ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, OR_a, SR_a, SOR_a, S0₂R_a, OS0₂R_a, OS0₃R_a, N0₂, NHR_a, N(R_a)₂, =N-R_a, N(R_a)COR_a, N(COR_a)₂, N(R_a)S0₂R', N(R_a)C(=NR_a)N(R_a)R_a, CN, halogen, COR_a, COOR_a, OCOR_a, OCOOR_a, OCONHR_a, OCON(R_a)₂, CONHR_a, CON(R_a)₂, CON(R_a)OR_a, CON(R_a)S0₂R_a, PO(OR_a)₂, PO(OR_a)R_a, PO(OR_a)(N(R_a)R_a) and aminoacid ester; each of the R_a groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclyl, or a salt, solvate or isomer thereof, for use in the inhibition of quorum sensing (QS) in multicellular community bacteria in a subject, wherein the inhibition of QS implies inhibiting physiological processes of multicellular community bacteria.

2. Use of a compound of formula (I) or (II), or a salt, solvate or isomer thereof, as defined in claim 1 , for the in vitro or ex vivo inhibition of quorum sensing (QS) in multicellular community bacteria, wherein the inhibition of QS implies inhibiting physiological processes of multicellular community bacteria.

3. The compound for use or the use according to claim 1 or 2, wherein the compound of formula (I) or (II), or a salt, solvate or isomer thereof, is used in a sub-bacterial- growth inhibiting amount.

4. The compound for use or the use according to any previous claim, wherein said compound is present in an effective amount between about 0.005 % and about 0.04 % w/w.

5. The compound for use or the use according to claim 4, wherein said compound is present in an effective amount between about 0.005 % and about 0.015 % w/w.

6. The compound for use or the use according to any previous claim wherein: R-i , R₂ and R₃ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C₁-C₂₂ alkyl, substituted or unsubstituted C₂-C₂₂ alkenyl, substituted or unsubstituted C₂-C₂₂ alkynyl, substituted or unsubstituted C₆-C₂₂ aryl, substituted or unsubstituted heterocyclyl having from 5 to 18 ring atoms, OR_a, SR_a,

SORa, S0₂R_a, OS0₂R_a, OS0₃Ra, N0₂, NHR_a, N(R_a)₂, =N-R_a, N(R_a)CORa, N(COR_a)₂, N(R_a)S0₂R', N(Ra)C(=NRa)N(R_a)Ra, CN, halogen, COR_a, COOR_a, OCOR_a, OCOOR_a, OCONHRa, OCON(R_a)₂, CONHR_a, CON(R_a)₂, CON(R_a)OR_a, CON(R_a)S0₂Ra, PO(OR_a)₂, PO(OR_a)Ra, PO(OR_a)(N(R_a)Ra) and aminoacid ester; and each of the R_a groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C₁-C₂₂ alkyl, substituted or unsubstituted C₂-C₂₂ alkenyl, substituted or unsubstituted C₂-C₂₂ alkynyl, substituted or unsubstituted C₆-C₂₂ aryl, and substituted or unsubstituted heterocyclyl having from 5 to 18 ring atoms.

7. The compound for use or the use according to any of claims 1 to 5 wherein:

R-i , R₂ and R₃ are each independently selected from the group consisting of hydrogen, S0₂R_a, CORa, PO(OR_a)₂, PO(OR_a)Ra, PO(OR_a)(N(R_a)Ra) and aminoacid ester.

8. The compound for use or the use according to any of claims 1 to 5 wherein:

R-i , R₂ and R₃ are each independently selected from the group consisting of hydrogen

9. The compound for use or the use according to claim 8 wherein the compound of formula (I) is hydroxytyrosol.

10. The compound for use or the use according to claim 8 wherein the compound of formula (I) is hydroxytyrosol acetate.

1 1 . The compound for use or the use according to claim 8 wherein the compound of formula (II) is 3,4-dihydroxyphenylacetic acid.

12. The compound for use or the use according to any previous claim wherein the inhibited physiological process of the multicellular community bacteria is selected from the group consisting of production of N-acyl homoserine lactones, biofilm formation, bioluminescense, swarming, swimming, motility, antibiotic biosynthesis, pigmet biosynthesis, enzymes biosynthesis, toxin biosynthesis, exopolysacharide biosynthesis and sporulation.

13. The compound for use or the use according to any previous claim wherein the bacteria is of a genus selected from the group consisting of Acinetobacter, Actinobacillus, Aeromonas, Aggregatibacter, Agrobacterium, Bacillus, Bordetella, Brucella, Burkholderia, Campylobacter, Chromobacterium, Cyanobacteria, Enterobacter, Erwinia, Escherichia, Franciscella, Fusobacterium, Haemophilus, Helicobacter, Hemophilus, Klebsiella, Lactobacillus, Legionella, Listeria, Micrococcus, Moraxella, Mycobacterium, Neisseria, Nitrosomas, Obesumbacterium, Pantoea, Pasteurella, Pediococcus, Porphyromonas, Prevotella, Proteus, Pseudomonas, Ralstonia, Rhisobium, Rhodobacter, Salmonella, Serratia, Shigella, Staphyllococcus, Streptococcus, Tannerella, Treponema, Vibrio, Xenorhabdus, Yersinia and mixtures thereof.

14. A compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof as defined in any of claims 1 to 1 1 , for use in the treatment of a bacterial infection or a disorder or condition associated with bacterial biofilm formation in a subject.

15. The compound for use according to claim 14, wherein said compound is used in a sub-bacterial-growth inhibiting amount.

16. The compound for use according to claim 14 or 15, wherein said compound is present in an effective amount between about 0.005 % and about 0.04 % w/w.

17. The compound for use according to claim 16, wherein the compound is present in an effective amount between about 0.005 % and about 0.015 % w/w.

18. The compound according to any of claims14 to 17, wherein the bacterial infection is selected from the group consisting of bacteremia, septicemia, endo- and pericarditis, sinusitis, upper respiratory tract infection, urinary tract infections, chronic bronchitis, pneumonia, cerebral and pulmonary lesions, meningitis, dermatitis or folliculitis, necrotizing fascitis, cellulitis, urinary tract infections, osteomylitis, enterocolitis, bacterial dental infections, contact lens-associated kerititis and conjunctivitis.

19. The compound according to any of claims 14 to 18, wherein the disorder associated with bacterial biofilm formation is selected from the group consisting of cystic fibrosis, dental caries, periodonitis, otitis media, muscular skeletal infections, necrotizing fasciitis, biliaty tract infection, osteomyelitis, bacterial prostatitis, endocarditis, native valve endocarditis, cystic fibrosis pneumonia, meloidosis, or skin lesions associated with bullous impetigo, atopic dermatitis and pemphigus foliaceus or implanted device-related infections or wherein the condition associated with biofilm formation is a nosocomial infection or an infection associated with sutures, exit sites, arteriovenous sites, sclera buckles, contact lenses, urinary catheter cystitis, peritoneal dialysis (CAPD) peritonitis, lUDs, endotracheal tubes, Hickman catheters, central venous catheters, mechanical heart valves, vascular grafts, biliary stent blockage, and orthopedic devices.

20. A pharmaceutical composition comprising a compound of formula (I) or (II) or a salt, solvate or isomer thereof as defined in any of claims 1 to 1 1 , in a sub-bacterial- growth inhibiting amount.

21 . A pharmaceutical composition according to claim 20, wherein the compound is present in an effective amount between about 0.005% and about 0.04% w/w.

22. A pharmaceutical composition according to claim 21 , wherein the compound is present in an effective amount between about 0.005% and about 0.015% w/w.

23. A pharmaceutical composition according to any of claims 20 to 22 comprising a compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof as defined in any of claims 1 to 1 1 as an excipient.

24. A pharmaceutical composition according to any of claims 20 to 23, wherein the pharmaceutical composition is selected from an ophthalmic composition and a dental composition.

25. A foodstuff comprising a compound of formula (I) or (II) or a pharmaceutically acceptable salt, solvate or isomer thereof as defined in any of claims 1 to 1 1 as a preservative.

26. The foodstuff according to claim 25, wherein said compound is present in a sub- bacterial-growth inhibiting amount.

27. The foodstuff according to claim 25 or 26, wherein said compound is present in an effective amount between about 0.005 % and about 0.04 % w/w of the foodstuff.

28. A medical device or a foodstuff package comprising a compound of formula (I) or (II) or a salt, solvate or isomer thereof as defined in any of claims 1 to 1 1.

29. A method of disrupting bacterial biofilm formation on a surface, the method comprising contacting the surface with a composition comprising a compound of formula (I) or (II) as defined in any of claims 1 to 1 1 or a pharmaceutically acceptable salt, solvate or isomer thereof, in an amount effective for disrupt or inhibit biofilm formation on the surface.

30. A method according to claim 29, wherein said compound is present in a sub- bacterial-growth inhibiting amount.

31 . The method according to claim 129 or 30 wherein said compound is present in a effective amount between about 0.005 % and about 0.04 % w/w in the composition to be applied to said surface.

32. A method of disrupting bacterial biofilm formation on a surface, the method comprising manufacturing the surface with a compound of formula (I) or (II) as defined in any of claims 1 to 1 1 or a salt, solvate or isomer thereof.

33. A method according to claim 32, wherein said compound is present in a sub- bacterial-growth inhibiting amount.

34. The method according to claim 32 or 33, wherein said compound is present in a effective amount between about 0.005 % and about 0.04 % w/w.

35. The method according to any of claims 29 to 34 wherein the surface is selected from the group consisting of a surface of a medical device and a surface of a foodstuff package.

36. A medicament or pharmaceutical composition comprising at least one compound of formula (I) or (II) as defined in any of claims 1 to 1 1 , or a pharmaceutically acceptable salt, solvate or isomer thereof as an excipient, for use in the treatment of a bacterial infection or a disorder associated with bacterial biofilm formation by inhibition of quorum sensing.

37. A medicament or pharmaceutical composition for use according to claim 36, wherein said compound is used in a sub-bacterial-growth inhibiting amount.

38. A medicament or pharmaceutical composition for use according to claim 36 or 37, wherein said compound is present in an effective amount between about 0.005% and about 0.04% w/w.