WO2024117979A1

WO2024117979A1 - Photodynamic therapy using organic photosensitizers with high ros- generation capability to combat drug resistance

Info

Publication number: WO2024117979A1
Application number: PCT/SG2023/050799
Authority: WO
Inventors: Cheng WENG; Ren Wei TOH; Jie Wu; Wee Han Ang
Original assignee: National University Of Singapore
Priority date: 2022-12-02
Filing date: 2023-12-01
Publication date: 2024-06-06

Abstract

The disclosure concerns a photodynamic method of killing a microbe, said method comprising a step of contacting a sample comprising the microbe with an effective amount of compound of Formula (I), and irradiating the sample with light for a time and under conditions to kill the microbe. The disclosure also concerns a photodynamic method of treating a bacterial disease or infection.

Description

Photodynamic Therapy Using Organic Photosensitizers with High ROS- Generation Capability to Combat Drug Resistance

Technical Field

The present invention relates, in general terms, to methods of photodynamic therapy.

Background

Photodynamic therapy (PDT) involves the use of light to excite a photosensitizing chemical substance (referred to as a photosensitizer) to produce singlet oxygen (Type II) and or free radicals (Type I) to elicit cell death (phototoxicity). The mechanism of action is a light-induced excitation of the photosensitiser to a singlet state and its conversion to a triplet state, from the triplet state the energy is transferred either to oxygen generating ROS, such as singlet oxygen and free radicals, or to organic molecules; both these reactions results in the generation of radicals. In the context of PDT, two main pathways are characterized by different photochemical mechanisms labelled Type I (the excited photosensitiser reacts directly with the substrate) and Type II (the excited photosensitiser reacts directly with oxygen). ROS are responsible for cytotoxic effects upon cells due to damage and destruction of cell walls, plasma membranes and DNA, hence leading to the death of the cells.

PDT can be used for the treatment of various ocular conditions, such as corneal and scleral infections such as infectious keratitis, keratoconus age-related macular degeneration, ocular squamous neoplasia (OSSN) or ocular surface malignancies (ophthalmologists), and outside of the eye has been used for treatment of skin, bladder, esophageal, breast, lung, gastrointestinal, prostate, and head and neck cancers, for autoimmune diseases such as psoriasis, as well as in skin infections and blood plasma sterilization. Photodynamic therapy (PDT) has been approved for the clinical treatment of a wide range of cancer and non-oncology diseases with superior performance.

PDT may also be used to kill microbial cells including bacteria, fungi and viruses. Skin and soft-tissue infections (SSTIs) are the most frequent forms of methicillin-resistant Staphylococcus aureus (MRSA) infections and their rate has increased about 20 times in the last decade. Additionally, other pathogen species have also developed resistance,

i.e. Vancomycin-Resistant Enterococci (VRE). SSTI incidence is 24.6 per 1000 person- years while among hospitalized patients the incidence is 7% to 10%. Non-antibiotic based antimicrobial therapies are therefore urgently needed in the management of infections in view of the rising number of microorganisms exhibiting resistance to one of more antibiotics.

One of the key features of antimicrobial PDT is its dual selectivity; (i) the antimicrobial effect is limited to the area that is treated afforded by PS localization, and (ii) light to bring about activation is anatomically confined, providing general low toxicity to the host and prevents disorder of the wider commensal microbial community. Antimicrobial PDT lethality is also not prone to induce resistance as bacteria are unlikely to be capable of simultaneously developing mechanisms to counteract all the possible damages caused.

PDT also provides advantages that lessen the need for delicate surgery and lengthy recuperation and minimal formation of scar tissue and disfigurement. However, some forms of PDT apply the photosensitizer systemically, which can cause a side effect is the associated photosensitization of skin tissue.

Current practice involves conjugating photosensitisers to poly-cationic moieties, pathogen specific moieties, nanoparticles, and small molecules in order to improve their targeting ability and hence PDT effect. Such strategies are complicated to execute and these compounds can be expensive to produce.

Additionally, the irradiation dose and/or exposure times are needed to effect antimicrobial action by PDT are too high for most current clinical applications and presents a substantial limitation.

There remains a need for improved PDT systems and therapeutic products that effectively treat diseases, minimize number of treatments, and minimize harmful effects.

It would be desirable to overcome or ameliorate at least one of the above-described problems.

Summary

The present invention provides a photodynamic method of killing a microbe, said method comprising a step of contacting a sample comprising the microbe with an effective amount of compound of Formula (I), and irradiating the sample with light for a time and under conditions to kill the microbe;

wherein

Ar is aryl; each Ri is independently halo; 2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each R3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when Ar is phenyl and n is 1, R3 is not ortho-substituted carboxyl.

In some embodiments, the compound is a compound of Formula (la):

wherein each Ri is independently halo; 2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each 3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally

substituted acyl; n is an integer selected from 0 to 6; and wherein when n is 1, R3 is not ortho-substituted carboxyl. In some embodiments, R3 is halo or optionally substituted alkyl.

In some embodiments, R3 is halo or C1-C5 alkyl substituted with one or more halo.

In some embodiments, R3 is halo or -CF3.

In some embodiments, the compound is selected from:

In some embodiments, the compound of Formula (I) is provided at a concentration of about 0.1 pM to about 500 pM.

In some embodiments, the compound of Formula (I) is provided at a concentration of about 1 pM to about 5 pM.

In some embodiments, the microbe is a bacterium.

In some embodiments, the microbe is a Gram-positive bacterium.

In some embodiments, the microbe is an antibiotic-sensitive or an antibiotic-resistant bacterium.

In some embodiments, the light is visible light.

In some embodiments, the light is characterised by a wavelength of about 400 nm to about 800 nm.

In some embodiments, the light is characterised by a light intensity of about 5 J/cm² to about 50 J/cm².

In some embodiments, the light is characterised by a light intensity of about 20 J/cm².

In some embodiments, the irradiation is for about 1 min to about 30 min.

In some embodiments, the compound of Formula (I) is non-cytotoxic to mammalian cell.

In some embodiments, the compound of Formula (I) is characterised by a H2O2 photocata lytic efficiency of at least about 10 pM.

The present invention also provides a photodynamic method of disinfecting a surface, the method comprising a step of contacting the surface comprising microbe with a compound of Formula (I), and irradiating the surface with light for a time and under conditions to kill the microbe;

wherein

Ar is aryl; each Ri is independently halo; 2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each 3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when Ar is phenyl and n is 1, R3 is not ortho-substituted carboxyl.

In some embodiments, the surface is a non-biological surface.

The present invention also provides a photodynamic method of treating a bacterial disease or infection, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, solvent or prodrug thereof to a diseased or infected region in a subject in need thereof, and irradiating the diseased or infected region with light.

The present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvent or prodrug thereof for use in photodynamically treating a bacterial disease or infection.

The present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvent or prodrug thereof in the manufacture of a medicament for photodynamically treating a bacterial disease or infection.

In some embodiments, the bacterial disease or infection is characterised by an antibiotics resistance.

In some embodiments, the bacterial disease or infection is selected from gastroenteritis, skin infection, ear infection, sinus infection, pneumonia, urinary tract infection, sexually transmitted infection (such as Gonorrhea), Tuberculosis, Anthrax, Tetanus, Leptospirosis, Cholera, Botulism, Pseudomonas Infection, MRSA Infection, E. coli Infection, Meningitis, and Syphilis.

The present invention also provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically acceptable carrier, excipient or diluent.

Brief description of the drawings

Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

Figure 1. Antibacterial photodynamic therapy using Eosin Y ranging from 0 to 10 pM.

Figure 2. (A) Cell viability assays against mouse epidermal fibroblast cell line L929 using Eosin Y with or without white light; (B) Inhibition graphs of MSSA RN4220, MRSA BAA-1768, and L929 using Eosin Y at 5 pM with or without white light.

Figure 3. (A) General procedures for the synthesis of Eosin Y derivatives; (B) Synthesis route for Eosin Y derivatives.

Figure 4. Semi-quantitative analysis of H2O2 production using Eosin Y and its diverse derivatives at near-physiological conditions under white light irradiation using 18 W white light LED strips.

Figure 5. Antibacterial photodynamic therapy using E5B.

Detailed description

"Alkyl" refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, /so-propyl, n-butyl, /so- butyl, n-hexyl, and the like.

"Alkylene" refers to divalent alkyl groups preferably having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. Examples of such alkylene groups include methylene (-CH2-), ethylene (-CH2CH2-), and the propylene isomers (e.g., -CH2CH2CH2- and -CH(CH3)CH2-), and the like.

"Alkoxy" refers to the group alkyl-O- where the alkyl group is as described above. Examples include, methoxy, ethoxy, n-propoxy, /so-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

"Oxo/hydroxy" refers to groups =0, H0-.

"Aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (eg. phenyl) or multiple condensed rings (eg. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl and the like.

"Acyl" refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(0)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

"Oxyacyl" refers to groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

"Acyloxy" refers to the groups -OC(O)-alkyl, -OC(O)-aryl, -C(O)O-heteroaryl, and -C(O)O-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein.

"Amino" refers to the group -NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

"Heteroaryl" refers to a monovalent aromatic heterocyclic group which fulfils the Huckel criteria for aromaticity (ie. contains 4n + 2 n electrons) and preferably has from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur within the ring (and includes oxides of sulfur, selenium and nitrogen). Such heteroaryl groups can have a single ring (eg. pyridyl, pyrrolyl or N- oxides thereof or furyl) or multiple condensed rings (eg. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl).

Examples of heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiophene, benzo[b]thiophene, triazole, imidazopyridine and the like.

"Heterocyclyl" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring. The most preferred heteroatom is nitrogen. It will be understood that where, for instance, R2 or R' is an optionally substituted heterocyclyl which has one or more ring heteroatoms, the heterocyclyl group can be connected to the core molecule of the compounds of the present invention, through a C-C or C-heteroatom bond, in particular a C-N bond.

Examples of heterocyclyl include, but are not limited to, quinolizine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1, 2,3,4- tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazolidine, morpholino, pyrrolidine, tetrahydrofuranyl, and the like.

In this specification "optionally substituted" is taken to mean that a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino,

phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono-and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclyl amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclyl, and the like, and may also include a bond to a solid support material, (for example, substituted onto a polymer resin). For instance, an "optionally substituted amino" group may include amino acid and peptide residues.

The inventors believe that the promise of PDT in antibacterial fields has not yet been fully fulfilled particularly using conventional organic Type I photosensitiers (PSs). The inventors have found that Type I photosensitizers, such as Eosin Y and its derivatives thereof, possessed high intracellular ROS-generation properties to induce bactericidal effects under low light-dose white illumination within Gram-positive bacteria, and in particular Methicillin-resistant Staphyloccocus aureus (MRSA). This provides a PDT platform to combat multidrug resistance in the fundamental antibacterial domains without suffering photobleaching of traditional PSs for subsequent extension and translation of clinical trials.

The inventors have derivatised Eosin Y, a hybrid photosensitiser (both Type I and II) to synthesize new analogues which exhibited stronger Type I characteristics. This was ascertained by determining semi-quantitatively the hydrogen peroxide generating abilities of the new analogues (Figure 4), since hydrogen peroxide is formed only by Type I processes. This is believed to be advantageous as Type I PS are less oxygendependent and bacterial pathogens are more sensitive to hydroxyl radical damage formed from Type I processes. In this regard, Type I PS which are also small molecules may be advantageous for use in antimicrobial photodynamic therapy.

In Type I photosensitized reactions, the PS is excited by a light source into an excited triplet state. Typically, PS, like most other molecules, are in a singlet state in their normal ground state. When the PS absorbs light it is converted to a different singlet state with a greater energy content. Because the excited singlet state has a very short

life-time, there is little opportunity for it to react with another molecule via either electron or energy transfer. The excited singlet state rapidly undergoes a process known as intersystem crossing, in which there is a transition to a slightly lower energy level as the spin of an electron flips. The spin flip puts the molecule into a triplet energy state. Because transitions between triplet and singlet energy states are relatively forbidden, the excited triplet state tends to have a longer lifetime than an excited singlet state, and hence is more likely to enter into a reaction with oxygen. The excited, triplet state PS then either directly transfer electrons to molecular oxygen or transfer electrons of substrates to molecular oxygen to form reactive oxygen species that include superoxide anions, hydrogen peroxide and hydroxyl radical. Type I PDT is more effective in hypoxic conditions compared to Type II PDT, because the oxygen can be regenerated intracellularly such as via disproportionation reactions catalysed by superoxide dismutase, hence less oxygen dependence.

In a Type II PS, the transfer of energy from the PS to oxygen produces an excited singlet state of oxygen (singlet oxygen). Singlet oxygen is a highly reactive oxygen species that has an excited state lifetime of a few microseconds in most biological environments, e.g., 3 to 4.5 ps in water.

These PSs are xanthene-based organic dyes that are distinct from the current reported organic Type I PSs including naturally occurring PSs and those with backbones, phenothiazine, porphyrin, fullerene, bodipy, naphthalimide, triphenylamine, and tetraphenylethene. In addition, these xanthene-based PSs is easy to access for affordable and scalable production with low manufacturing costs.

Compared to recently reported Type I aggregate-induced emission luminogens (AIEgens) for antimicrobial photodynamic therapy (APDT) as a means to circumvent aggregate-induced quenching, these organic Type I PSs does not suffer from aggregate- induced photobleaching and may bypass the multidrug resistance of MRSA at a highly diluted concentration (1 pM) under a low light-dose white irradiation (20 J/cm²) that is competitive to the state of the art APDT.

The high photo-toxicity and negligible dark-toxicity at a wide range of administration concentrations allows for highly efficient photo therapeutic efficacies against both drug resistant and susceptible Gram-positive pathogens under low light-dose white

illumination for spatiotemporally controlled APDT with great potential for clinical translation.

As a low light dose is required for APDT, the compounds provide high quantum yields with marginal photobleaching and low photon energy consumed, and are hence an eco- and economic friendly APDT option.

The compounds may show superior bacterial targeting and extraordinary biosafety, and may selectively exert photo-bactericidal effects on Gram-positive bacteria, in particular MRSA, to combat drug resistance without impacting on Gram-negative pathogens as well as normal mammalian cells for clinical translation.

As Eosin Y is commercially available and its various derivatives are readily accessible to commercialize with affordable and scalable production, there may be easy access to the production of Eosin Y and its various derivatives for APDT.

The compounds are easy to functionalize and tune the backbone of Eosin Y to satisfy specific or diverse requirements of customers. As the compounds are generally nontoxic, they are easy to transport, store, and handle. They are thus affordable and scalable for industry production for commercialized APDT.

Accordingly, the present invention provides a photodynamic method of killing a microbe, said method comprising a step of contacting a sample comprising the microbe with an effective amount of compound of Formula (I), and irradiating the sample with light for a time and under conditions to kill the microbe;

wherein

Ar is aryl; each Ri is independently halo;

R2 is oxo, optionally substituted alkoxy, optionally substituted amino, optionally

substituted N-heterocyclyl or optionally substituted N-heteroaryl; each R3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when Ar is phenyl and n is 1, R3 is not ortho-substituted carboxyl.

wherein each Ri is independently halo;

R2 is oxo or optionally substituted alkoxy; each R3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 1 to 6; and wherein when Ar is phenyl and n is 1, R3 is not ortho-substituted carboxyl.

Accordingly Eosin Y is excluded from this scope. Eosin B is also excluded from this scope.

In some embodiments, when Ar is phenyl and n is 1, R3 is not ortho-substituted carboxyl and para-substituted carboxyl.

The compound of Formula (I) may be used in antibacterial photodynamic therapy or as potent photodynamic therapy (PDT) agents. The molecules may also be used as fluorophores for imaging, and for H2O2 photoproduction.

In general, PDT is a multi-stage process. First a photosensitiser, ideally with negligible

toxicity other than its phototoxicity, is administered in the absence of light, either systemically or topically. When a sufficient amount of photosensitiser accumulates in the diseased tissue, the photosensitiser is locally activated by exposure to light for a specified period. The light dose supplies sufficient energy to stimulate the photosensitiser, but not enough to damage neighbouring healthy tissue. The reactive oxygen and/or free radicals kills the target cells.

The compound of Formula (I) were found to be effective in killing microbes while not being adverse to mammalian cells. In particular, variations at R3 was found to not adversely impact its cytotoxicity to mammalian cells and may further improve its bactericidal properties. Additionally, low photosensitiser dosage and/or low light intensities may be used and is thus less harmful for the subject. In contrast, the prior art compounds focuses conjugation techniques to improve the photosensitiser's targeting ability. Additionally, modifications commonly centres around the xanthene structure. Further, Eosin Y has thus far been only employed as a fluorescent dye for staining and imaging and as a photocatalyst in synthetic organic reactions.

In some embodiments, Ar is CG-CM aryl. The aryl may be a single ring or multiple condensed rings such as naphthalene. In other embodiments, Ar is Ce aryl.

In some embodiments, the compound is a compound of Formula (la):

wherein each Ri is independently halo;

R2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each R3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when n is 1, R3 is not ortho-substituted carboxyl.

In some embodiments, the compound is a compound of Formula (la):

wherein each Ri is independently halo;

R2 is oxo or optionally substituted alkoxy; each R3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 1 to 6; and wherein when n is 1, R3 is not ortho-substituted carboxyl.

In some embodiments, each Ri is independently selected from F, Br, Cl or I. In other embodiments, each Ri is independently F, Br or Cl. In other embodiments, each Ri is independently F or Br. In other embodiments, each Ri is independently Br.

In some embodiments, 2 is -OH or =0. In some embodiments, 2 is -OH. In some embodiments, 2 is optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl. The N-heterocyclyl and N-heteraryl of 2 may be linked to the Markush structure via the nitrogen atom. In some embodiments, 2 is optionally substituted amino. The optionally substituent may be C1-C5 alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl.

In some embodiments, each 3 is independently halo, optionally substituted alkyl, optionally substituted oxyacyl, optionally substituted acyloxy, or optionally substituted acyl. In some embodiments, each 3 is independently halo, optionally substituted alkyl, optionally substituted oxyacyl, or optionally substituted acyl.

In some embodiments, the optional substituent is selected from halo, alkyl, or alkenyl. In some embodiments, the optional substituent is selected from halo or alkyl. In some embodiments, the optional substituent is selected from F, Cl, Br, I or C1-C5 alkyl. In

some embodiments, the optional substituent is selected from F, Br, methyl, ethyl, or propyl.

In some embodiments, R3 is halo or optionally substituted alkyl. In some embodiments, R3 is halo or C1-C5 alkyl substituted with one or more halo. In some embodiments, R3 is halo or -CF3.

In some embodiments, the compound is selected from:

In some embodiments, the compound of Formula (I) is provided at a concentration of about 0.1 pM to about 500 pM. In some embodiments, the compound of Formula (I) is provided at a concentration of about 1 pM to about 5 pM. In some embodiments, the microbe is a bacterium. In some embodiments, the microbe is Gram-positive bacteria and/or Gram-negative bacteria. In some embodiments, the microbe is Gram-positive bacteria. In some embodiments, the microbe is an antibioticresistant bacterium. In some embodiments, the microbe is an antibiotic- resistant Gram-

positive bacteria.

In some embodiments, the light is visible light. In some embodiments, the light is characterised by a wavelength of about 400 nm to about 800 nm. This wavelength range is used to simulate sunlight. In some embodiments, the wavelength is about 400 nm to about 700 nm, about 400 nm to about 600 nm, about 400 nm to about 550 nm, or about 400 nm to about 500 nm.

In some embodiments, the light is characterised by a light intensity of about 5 J/cm² to about 50 J/cm². In some embodiments, the light is characterised by a light intensity of about 20 J/cm².

In some embodiments, the irradiation is for about 1 min to about 30 min. In some embodiments, the irradiation is for about 1 min to about 10 min.

In some embodiments, the compound of Formula (I) is characterised by a H2O2 photocata lytic efficiency of at least about 10 pM. The compound of Formula (I) may have a concentration of about 10 pM.

The method may be an in vivo method, and/or an in vitro method. For example, the method may be used on adherent cells.

wherein

Ar is aryl; each Ri is independently halo;

R2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each 3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when Ar is phenyl and n is 1, 3 is not ortho-substituted carboxyl.

In some embodiments, the surface is a non-biological surface.

The present invention also provides a compound of Formula (I) for use in photodynamically treating a bacterial disease or infection.

The bacterial disease or infection may be treated by contacting a diseased or infected region with the compound of Formula (I) or a pharmaceutically acceptable salt, solvent or prodrug thereof, and subsequently irradiating the diseased or infected region with light. For example, the compounds may be topically applied to the diseased or infected region, or systematically administered. Alternatively, the compound of Formula (I) may be injected into the diseased or infected tissue or region. The compound of Formula (I) may penetrate the diseased or infected tissue or region. The diseased or infected area may be a superficial region or within the body. Thereafter the diseased or infected area may be subjected to photoirradiation (light can be delivered over entire wound or at

specific spots using an endoscope) for a specific period of time. Depending on the type of light, the treatment may penetrate the diseased or infected tissue or region, for example to a depth of about 1 cm to about 5 cm. After irradiation, the diseased or infected area is allowed to heal on its own.

In some embodiments, the bacterial disease or infection is characterised by an antibiotic resistance.

In some embodiments, the diseased or infected tissue or region is selected from a surface of a skin, an epidermis layer, a dermis layer, a lining of an organ (mucosa), or a combination thereof.

The compound of the invention can be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and

alkylammonium. In particular, the present invention includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the phosphate group.

Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

It will be appreciated that any compound that is a prodrug of the compound of formula (I) is also within the scope and spirit of the invention. Thus the compound of the invention can be administered to a subject in the form of a pharmaceutically acceptable pro-drug. The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compound of the invention. Such derivatives would readily occur to those skilled in the art. Other texts which generally describe prodrugs (and the preparation thereof) include: Design of Prodrugs, 1985, H. Bundgaard (Elsevier); The Practice of Medicinal Chemistry, 1996, Camille G. Wermuth et al., Chapter 31 (Academic Press); and A Textbook of Drug Design and Development, 1991, Bundgaard et al., Chapter 5, (Harwood Academic Publishers). For example, if R2 is hydroxyl, it may be esterified.

The compound of the invention may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) 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 compound of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered to the patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of macular degeneration.

As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight

per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.

Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.

The compound of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Modes of administration including injections, topical or intravenous administration may also be possible. For example, solutions or suspensions of the compound or composition of the invention may be formulated as eye drops, or as a membranous ocular patch, which is applied directly to the surface of the eye. Topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.

The compound or composition of the invention may also be suitable for intravenous administration. For example, a compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered intravenously at a dose of up to 16 mg/m².

The compound or composition of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug is orally administerable.

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

The compound or composition of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The compound or composition of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the compounds of the invention.

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

Preferred unit dosage composition are those containing a daily dose or unit, daily subdose, as herein above described, or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the active ingredients particularly mentioned above, the composition of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters,

waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Examples

To probe the therapeutic performance of Eosin Y on pathogenic microorganisms, in particular multidrug-resistant species, we selected a wound-isolated methicillin- resistant Staphyloccocus aureus (MRSA) strain, S. aureus ATCC BAA-1768, to evaluate the capability of Eosin Y in photo-induced intracellular H2O2 production to exert bactericidal effects. Using plate colony counting methods, Eosin Y showed negligible dark-toxicity ranging from 0 to 10 pM and demonstrated quantitative bactericidal effects on MRSA BAA-1768 even at a low concentration of 5 pM when exposed to white illumination. Likewise, Eosin Y inflicted significant cell growth inhibition on methicillin- susceptible Staphyloccocus aureus (MSSA) RN4220 pathogens via photo-induced intracellular oxidative stress (Figures 1 and 2B). The dark and photo-cytotoxicity of Eosin Y towards the mouse epidermal fibroblast cell line, L929, as the normal mammalian cell model was determined. At the concentration required to achieve bactericidal effects, Eosin Y was essentially non-cytotoxic to L929 cells under dark or white illumination, indicating that there was a great potential to translate Eosin Y- enabled APDT to preclinical trial (Figure 2).

Eosin Y was subsequently derivatised in terms of the substituents on the acceptors of the backbone to tune the photophysical properties for further enhancement in APDT efficacies. A strategy for the affordable and scalable synthesis of Eosin Y derivatives with moderate to excellent yields was developed (Figure 3).

Next semi-quantitative peroxides test strips were utilised to measure the photoproduction of H2O2 from dioxygen using Eosin Y and its derivatives (10 pM). Gratifyingly, E3B and E5B demonstrated 2 and 4-fold photo-catalytic efficacies in the H2O2 production, respectively, in contrast to Eosin Y whereas other PSs manifested worse or similar performance at identical conditions (Figure 4). Thereafter, the dark and phototoxicity of E5B against MRSA pathogens was determined. E5B demonstrated no dark-

toxicity and achieved >99.9% bactericidal potency at only 1 pM under a low light dose at merely 20 J/cm² (Figure 5).

In summary, Eosin Y and its derivatives thereof are efficacious organic PDT agent for the selective targeting of Gram-positive pathogens, in particular MRSA, to combat multidrug resistance. Based on this discovery, a panel of new Eosin Y analogues to further improve its antibacterial photodynamic therapeutic effects was developed, from which Eosin Y analogue(s) E3B and E5B, which is efficacious at therapeutically-relevant doses of irradiation. Upon a low light-dose irradiation from a normal household white lamp, the Eosin Y analogue E5B can overwhelm the anti-ROS defense system of bacteria without drug deactivation. It can induce bactericidal effects on both MSSA and MRSA pathogens, without harming normal mammalian cells, through reacting with molecular oxygen to produce cytotoxic ROS intracellularly. Thus, it provided a new platform for the extension and translation of PDT in the preclinical antimicrobial fields. An efficient method for production of Eosin Y analogues was developed with moderate to high yields for the affordable and scalable production. Superior photo-bactericidal PS from the panel of Eosin Y derivatives and its therapeutic efficacies on pathogenic bacteria were demonstrated.

It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is/are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention

but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

1. A photodynamic method of killing a microbe, said method comprising a step of contacting a sample comprising the microbe with an effective amount of compound of Formula (I), and irradiating the sample with light for a time and under conditions to kill the microbe;

wherein

2. The method according to claim 1, wherein the compound is a compound of

wherein each Ri is independently halo; 2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each 3 is independently halo, optionally substituted alkyl, optionally substituted

alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when n is 1, R3 is not ortho-substituted carboxyl.

3. The method according to claim 1 or 2, wherein R3 is halo or optionally substituted alkyl.

4. The method according to any one of claims 1 to 3, wherein R3 is halo or C1-C5 alkyl substituted with one or more halo.

5. The method according to any one of claims 1 to 4, wherein R3 is halo or -CF3.

6. A photodynamic method of killing a microbe, said method comprising a step of contacting a sample comprising the microbe with an effective amount of compound of Formula (I), and irradiating the sample with light for a time and under conditions to kill the microbe; wherein the compound of Formula (I) is selected from:

7. The method according to any one of claims 1 to 6, wherein the compound of Formula (I) is provided at a concentration of about 0.1 pM to about 500 pM, or preferably about 1 pM to about 5 pM.

8. The method according to any one of claims 1 to 7, wherein the microbe is a bacterium.

9. The method according to any one of claims 1 to 8, wherein the microbe is a Gram-positive bacterium.

10. The method according to any one of claims 1 to 9, wherein the microbe is an antibiotic-sensitive or an antibiotic-resistant bacterium.

11. The method according to any one of claims 1 to 10, wherein the light is visible light.

12. The method according to any one of claims 1 to 11, wherein the light is characterised by a wavelength of about 400 nm to about 800 nm.

13. The method according to any one of claims 1 to 12, wherein the light is characterised by a light intensity of about 5 J/cm² to about 50 J/cm², or preferably about 20 J/cm².

14. The method according to any one of claims 1 to 13, wherein the irradiation is for about 1 min to about 30 min.

15. The method according to any one of claims 1 to 14, wherein the compound of Formula (I) is non-cytotoxic to mammalian cell.

16. The method according to any one of claims 1 to 15, wherein the compound of Formula (I) is characterised by a H2O2 photocata lytic efficiency of about 10 pM.

17. The method according to any one of claims 1 to 16, wherein the method is an in vitro method.

18. A photodynamic method of disinfecting a surface, the method comprising a step of contacting the surface comprising microbe with a compound of Formula (I), and irradiating the surface with light for a time and under conditions to kill the microbe;

wherein

19. The method according to claim 18, wherein the surface is a non-biological surface.

20. A photodynamic method of treating a bacterial disease or infection, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, solvent or prodrug thereof to a diseased or infected region in a subject in need thereof, and irradiating the diseased or infected region with light;

Ar is aryl;

each Ri is independently halo; 2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each 3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when Ar is phenyl and n is 1, 3 is not ortho-substituted carboxyl.

21. A compound of Formula (I) or a pharmaceutically acceptable salt, solvent or prodrug thereof for use in photodynamically treating a bacterial disease or infection;

wherein

Ar is aryl; each Ri is independently halo; 2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each 3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when Ar is phenyl and n is 1, 3 is not ortho-substituted carboxyl.

22. Use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvent or prodrug thereof in the manufacture of a medicament for photodynamically treating a bacterial disease or infection;

wherein

Ar is aryl; each Ri is independently halo;

R2 is oxo, optionally substituted alkoxy or optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; each 3 is independently halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted acyl; n is an integer selected from 0 to 6; and wherein when Ar is phenyl and n is 1, R3 is not ortho-substituted carboxyl.

23. The method, compound or use according to any one of claims 20 to 22, wherein the bacterial disease or infection is characterised by an antibiotics resistance.

24. The method, compound or use according to any one of claims 20 to 23, wherein the bacterial disease or infection is selected from gastroenteritis, skin infection, ear infection, sinus infection, pneumonia, urinary tract infection, sexually transmitted infection (such as Gonorrhea), Tuberculosis, Anthrax, Tetanus, Leptospirosis, Cholera, Botulism, Pseudomonas Infection, MRSA Infection, E. coli Infection, Meningitis, and Syphilis.

25. A pharmaceutical composition comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically acceptable carrier, excipient or diluent.