ZA200602898B

ZA200602898B - Use of photosensitisation

Info

Publication number: ZA200602898B
Application number: ZA200602898A
Authority: ZA
Inventors: Wilson Michael; Nair Sean
Original assignee: Ucl Biomedica Plc
Priority date: 2006-04-10
Filing date: 2006-04-10
Publication date: 2007-07-25

Description

USE OF PHOT OSENSITISATION

The present invention relates to =a composition comprising a conjugsate of a 5S photosensitiser and a bacteriophage, particularly a staphylococcal bacteriophage, known as a staphylophage. The inventi on also relates to the use of the corjugate in 2 method of photodynamic therapy for infectious diseases.

Background

The use of antimicrobial agents to counter bacterial infections is beecoming increasingly ineffective, due to the rapi d emergence of antibiotic resistanc=c amongst many species of pathogenic bacteria. Owne such pathogen is Staphylococcis aureus (8S. aureus), which characteristically cause s skin infections such as boils, cartouncles and impetigo, as well as infecting acne, bums and wounds. If the infecting organism is a toxic strain, such infections, or colonised tampons, may give rise to a life=-threatening toxaemia known as toxic shock syndrome. The organism may also gain access to the bloodstream from these infections, or from foreign bodies such as intravenous catheters, and so cause infections at other sites, such as endocarditis, osteomyelitis, meningitis and pneumonia.

A number of bacteria are responsible for infection of skin and wounds, for example, coagulase-negative staphylo cocci, Staphylococcus aureus, stregptococci,

Corynebacterium spp., E. coli, Klebsi ella aerogenes, Klebsiella pneumo-niae,

Enterobacter aerogenes, Propionibac=terium acnes, Bacteroides spp., Psseudomonas aeruginosa and Peptostreptococcus spp. Increasingly, these bacteria ar-e showing resistance to antibiotic treatment.

In particular, resistant strains wf S. aureus have emerged. Methiczillin-resistant

S. aureus (MRSA) was first reported in 1961 (Jevons, M. (1961) Britista Medical

Journal, 1, 124-5), and these strains asre now a major cause of hospital-ascquired infection throughout the world, as well as being prevalent in many nurs=ing and residential homes. This poses an alarming challenge to healthcare, caus=ing significant

WE 2005/034997 P=CT/GB2004/004305 infection and morbidity of hundreds of patients in the UK each yea-x (Ayliffe ef al, J

Hosp Infect (1988), 39, 253-90).

Since the first report of MRSA, these organisms have demo- nstrated resistance to a wide variety of antimicrobials including erythromycin, aminogdycosides, tetracyclines, trimethoprim, sulphonammnides and chloramphenicol. NARSA strains have developed that are only susceptil®le to a single class of clinical ly-available antibiotics: the glycopeptides such as —vancomycin and teicoplanin. BHowever, resistance is developing even to these. as strains tolerant to vancom=ycin have now been reported (Hiramatsu, K. (1998) American Journal of Medicine=, 104, 7S - 108).

These strains are variously known as WRSA (Vancomycin resistant Staphylococcus aureus) and hetero-VRSA (resistant strains arising from exposure to high levels of vancomycin). At present, the managemment of patients with MRSA Enfections usually involves the administration of antimicarobial agents and again, there is evidence of the development of resistance to many of ghe agents used.

Due to the emergence of strainss which are resistant to virtual ly all currently- available antimicrobials, MRSA is now a serious threat to health. Tkme term MRSA itself now more accurately applies to methicillin and multiple antimi crobial-resistant

S. aureus.

Certain strains of MRSA have Exeen found to spread rapidly maot only within hospitals, but also between them. Thesee strains have been termed epidemic MRSA (EMRSA). Since the first EMRSA strain (EMRSA-1) was reported im 1981, 17 «distinct EMRSA strains have been idemtified, all of which are resistamnt to a number «of antimicrobials. Recently, the two maowst prevalent strains have beene EMRSA-15 and -16, which account for 60-70% of the 30000 MRSA isolates reported

Livermore, D (2000) Int. J. Antimicrotoial Agents, 16, S3 - S10). Immportantly, strains of MRSA, (known as community-acquired MRSA (CA-MRS..A)) have also sstarted to spread in the community, ie. amongst non-hospitalised indi~viduals. ) It is clear from the above that alt_emative methods of counterirnag bacterial infection, particularly infection with MIRSA, are urgently required.

One approach has been to emplowy a light-activated agent to ac _hieve lethal photosenssitization of the organism. This invol-ves treating the organism with a light- activatabBe chemical (photosensitiser) which, mapon irradiation with light of a_ suitable wavelength, generates cytotoxic species, resulting in bacteriolysis. This techmique has been used to achieve killing of a wide range off bacteria, including S. aureus —and

MRSA strains, in vitro using toluidine blue O (TBO) and aluminium disulphaonated phthalocyanine (AIPcS,) as photosensitisers. ENeither photosensitiser nor laseer light alone exerted a bacteriocidal effect (Wilson er al, (1994) J Antimicrob Chenrmother 33, 619-2 4). In a subsequent study, 16 strains © EMRSA were found to be susceptible to killing by low doses of red light (674 nm) in the presence of A_IPcS, (Griffiths et al, (1997) J Antimicrob Chemotheer, 40, 873-6 ). At higher light doses, 100 % killing was achieved.

Photodynamic therapy (PDT) is the appplication of such an approach t=o the treatment of disease. It is an established proceciure in the treatment of carcincoma and forms the basis of a means of sterilising blood products. It has only been momre recently that the application of PDT to the treatment of infectious diseases has been evaluated... For example, haematoporphyrins irx. conjunction with an argon las - er have been usedl to treat post-neurosurgical infection s and brain abscesses (Lombar—d ef al, (1985), Photodynamic Therapy of Tumours awd other Diseases, Ed. Jori & Pe=erria).

Ome potential problem associated with PDT of infectious diseases is Mts lack of specificity. Hence, if the photosensitiser bids to, or is taken up by, a host cell, as well as th e target organism, then subsequent irradiation may also lead to the edeath of the host c ell. A way to overcome this is by the use of targeting compounds: that is, any comp ound that is capable of specifically binding to the surface of the pat-hogen.

Several targeting compounds have previously been shown to be successful in eliminatirag specific strains of bacteria when timey were conjugated to a photosenssitiser. For example, immunoglobulim G (IgG) has been used to target S. aureus Protein A (Gross et al (1997), Photochemistry and Photobiology, 66, 872-8), monoclomal antibody against Porphyromonas gingivalis lipopolysaccharide (CBhatti et al (2000), Antimicrobial Agents and Chemothesrapy, 44, 2615-8) and poly-L—lysine peptides against P. gingivalis and Actinomyces~ viscosus (Soukos et al (1998) ,

Antimicrobizal Agents and Chemotherapy, 42, 2=595-2601). A monoclonal antmbody conjugated v—ia dextran chains to the photosensitciser tin (IV) chlorin e6 (SnCe6) was selective for killing P. aeruginosa when exposed to light at 630nm, leaving S. aureus unaffected (Friedberg et al (1991), Ann N'Y Ac ad Sci, 618, 383-393).

S The present inventors have used IgG corajugated to SnCe6 to target EMIRSA strains 1, 3, B 5 and 16 (Embleton et a/ (2002), J Antimicrob Chemother, 50, 8 57- 864), achieving higher levels of killing than the photosensitiser alone, and selectively killing the EPMRSA strains in a mixture with Streptococcus sanguis. However a limitation of IgG is that only strains of S. aureus expressing Protein A can be targeted. Herce alternative targeting agents that can target any S. aureus strainm are desirable. :

Bacteriophage are viruses that infect certain bacteria, often causing the=m to lyse and hence effecting cell death. They have b een proposed as antibacterial &agents in their own right. However, one of the problem s with using staphylococcal bacteriophagze (termed staphylophage) in the tre=atment of S. aureus disease is ~their restricted hosst range. Although there are polyvamlent staphylophage which can lyse many S. aureus strains, other strains are resistarmt and hence bacteriophages alone could not provide an effective method of killings all strains of S. aureus.

It is known that although some bacteriophage will only kill a limited range of bacteria, they will bind to a broader range of bacteria. The present inventors hmave now found that some bacteriophage can serve ass an effective, targeted delivery system for pFotosensitisers.

The present inventors have found that when a bacteriophage is linked toa photosensitisser, the photosensitiser-bacteriophagge conjugate formed is highly effective in k=illing bacteria when irradiated withm light of a suitable wavelengtim.

Bactesriophage-photosensitiser conjugate=s could be used to treat or prewvent a broad range of bacterial skin and wound infections. The most frequently isolated organisms freom skin and wound infections are: «coagulase-negative staphylocoscci, S. aureus, strep-tococci, e.g. Streptoccocus pyogenezs, Corynebacterium spp., E coli,

Klebsiella aczrogenes, Klebsiella pneumoniae, Enterobacter aerogenes,

"

Propoionibacterium acnes, Bacteroides spp., Pseudomonas aeruginosa and

Pepe ostreptococcus spp..

In particular, conjugates of photosensitiser and staphylophage can be used in a me=thod of photodynamic therapy against strains of Staphylococci spp, particulamrly agaimst MRSA, EMRSA, VRSA, hetero-VRS_A and CA-MRSA.

The invention provides a composition «<omprising a photosensitizing compound (photosensitiser) linked to a bacterii ophage to form a photosensitiser- bactesriophage conjugate. The bacteriophage ray be a staphylococcal phage, and is preferably a staphylophage that can bind to Stcaphylococcus aureus, particularly

MRSA, EMRSA, VRSA, hetero-VRSA or CA.-MRSA. The composition may be used in a method of photodynamic therapy.

The bacteriophage is preferably linked to the photosensitiser using a coval ent linkamge. The photosensitiser and/or the bacteriophage contain or may be modified. to contain groups which can be covalently crosslinked using chemical or photoreacti ve reagents, to produce crosslinked bonds, for example thiol-thiol crosslinking, amin_e- amimme crosslinking, amine-thiol crosslinking, aamine-carboxylic acid crosslinking, thiol—carboxylic acid crosslinking, hydroxyl-carboxylic acid crosslinking, hydroxwi- thiol crosslinking and combinations thereof.

The photosensitiser is suitably chosen From porphyrins (e.g. haermatoporphyrin derivatives, deuteroporphyrin), phthalocyanines (e.g. zine, silicon and am luminium phthalocyanines), chlorins (e.g_ tin chlorin e6, poly-lysine derivati—ves of tirm chlorin e6, m-tetrahydroxypheny! chlorira, benzoporphyrin derivatives, tin etiop-urpurin), bacteriochlorins, phenothiaziniumns (e.g. toluidine blue, methylene blue, dimethylmethylene blue), phenazines (e.g. neutral red), acridines (e.g. acrifl avine, proflavin, acridine orange, aminacr-ine), texaphyrins, cyanines (e.g. mero <yanine 540), anthracyclins (e.g. adriamycin and epirubicin), pheophorbides, sapplyrins, fullerene, halogenated xanthenes (e.g. rose bengal), perylenequinonoic pigments (e.g. hypericin, hypocrellin), gilvocarcins, terthiophenes, benzophenanthridines, psoralens and riboflavin.

The invention is directed to killing bacteria using the above-described conjugates. The bactemriophage used in the conjugate ma=y be selected according to the particular organism to be killed, in order to arrive at the conjugate most effective against the particular infecting bacteria. In a preferred emmbodiment, the infecting bacterium is MRSA, FEMRSA, VRSA, hetero-VRSA or «CA-MRSA and the conjugate includes the= staphylococcal phage 75 or phage= ¢11.

Table 1 below shows some examples of bacteria_-bacteriophage pairs, although many more e=xamples exist. Further novel bacteeriophages can be isolated and/or adapted to the target bacteria. The specificity of tie treatment can be modifie d as required by using monovalent bacteriophages, polyvalent bacteriophages or combinations of monovalent bacteriophages or combinations of monovalent and polyvalent bacteriopham ges.

TABLE 1

Bacterium Bacteriophage

Staphylococcus aureus 53,75, 79,80,83, $11, @b12, $13, $147. $ MR11

Staphylococcus epidermidis 48, 71, numerous (182 - different phage)

Staphylococcus spp ¢ 812, SK311, 6131, SEB-I and U16

Streptococcus spp C,, SF370.1, SP24,SFL—, A1 (ATCC 12202-B1) various

Corynebacterium spp $3041 4304S, $15, $1 6, 782

Klebsiella aerogenes and

Klebsiella pneumoniae Plclr100KM

E coli Pl, T1, T3, T4, T7 MS2=

Enterobacter aerogenes Various, P1, M13

Pseudomonas aeruginosa UNL-1, ACQ, UT, tba 1D3, E79, F8 & pf20 B3, F116,

G101, B86, T’M, =Cq, UT], BLB, PP?

Propionibacterium acnes Various, including ATC=C 29399-B1

Bacteroides spp B40-8

Numerous Gram-negative baacteria P1 Various

The compositior of the invention suitably comprisses at least 0.01 pg/ml, of the photosensitiser, pref-erably at least 0.02ug/ml, more pr-eferably at least 0.05ug/ml upto 200 pg/ml, preferalbly up to 100 pg/ml, more preferalbly up to 50 pg/ml. The axmount of the bacteriophage in the composition is suitably from 1x10 * to 1x10 pfu, p referably from 1x106 to 1x10° pfu, more pre=ferably from 1x10 to 1x10® pF.

The composition of the invention ma_y further comprise a source of livalent ions, e.g. Ca’ or Mg”, preferably Ca**. Examples include calcium chloride, calcium carbonate and magnesium chloride. The ioms are suitably present in an amount of firom 5 to 200mM, preferably from 5 to 15 mM, more preferably about 10maM.

The composition may further comprise one or more ingredients cho=sen from buffers, salts for adjusting the tonicity, antiosxidants, preservatives, gelling sagents and remineralisation agents.

The invention further provides a method of killing bacteria, comprising (a) contacting an area to be treated with the composition of the inve=ntion such that any bacteria in the area bind to the photosensitiser-bacteriophagze conjugate; and (b) irradiating the area with light at = wavelength absorbed by the photosensitiser.

Suitably the bacteria are as set out albove in Table 1, preferably aStaphylococcus aureus, more preferably MIRSA, EMRSA, VRSA, hetero-SWRSA or «CA-MRSA.

In the method of the invention, any light source that emits light of &an appropriate wavelength may be used. The wavelength of the light is selected to correspond to the absorption maximum of t"he photosensitiser and to have sufficient energy to activate the photosensitiser. The source of light may be any dev3ce or “biological system able to generate monochromatic or polychromatic light. Examples include laser, light emitting diode, arc lampw, halogen lamp, incandescent lamp or an emifter of bioluminescence or chemilumine=scence. In certain circumstanc es, sunlight may be suitable. Preferably, the wavelength of the light emitted by the light source may be from 200 to 1060nm, preferably from 400 to 750nm. A suitable lamser may have a power of from 1 to 100mW and a be=am diameter of from 1 to 10mrm. The light dose for laser irradiation is suitably freom 5 to 333 J cm, preferably £5om § to . So y

30 J =m? for laser light. For white light irradiation, a suitable dose x's from 0.01 to 100 Jem? preferably from 0.1 to 20 Jem? » more preferably from 3t0 10 J/cm?

The duration of irradiation is suitably from one second to 15 minutes, preferably from 1 to 5 minutes,

The following light sources may be suitable for use in the present invention:

Helium neon (HeNe) gas laser (633nm)

Argon-pumped dye laser (500-700nm, 5W output)

Copper vapour-pumped dye laser (600-800nm)

Excimer-pumped dye laser (400-700nm)

Gold vapour laser (628nam, 10W output) . Tunable solid state laser (532-1060nm), including Sd: WAG

Light emitting diode (LEED) (400-800nm)

Diode laser (630-850nm, 25W output), eg. gallium selenium arsenide

Tungsten filament lamp

Halogen cold light sources

Fluorescent lamp. _In the method of the invention, thae composition is suitably in thae form of a solutiorm or a suspension in a pharmaceutically acceptable aqueous carrier, but may be : in the foo of a solid such as a powder ox a gel, an ointment or a creams . The composition may be applied to the infected area by painting, spreading spraying or any other conventional technique.

The invention further provides thes use of the composition for treatment of the human o r animal body. Suitably, the composition is provided for use in the treatment of conditzions resulting from bacteria) infection, particularly by staphylococci, more particularly by MRSA, EMRSA, VRSA, hetero-VRSA or CA-MRSA.,

The invention may be used to treat bacterial infection, particularl y by staphyloczoccal bacteria, more particularly by MRSA, EMRSA, VRSA, Eaetero-VRSA or CA-MIRSA to treat or prevent skin infections such as boils, carbuncles, mastitis and impetigo, to treat or prevent infections of acne, burns or wounds, or t-o treat or prevent eradocarditis, osteomyelitis, meningitis and pneumonia, ansing as a result of

AMENDED SHEET bacterial infection, to treat or prevent infections arissing from the use of catheters, implants or other medical devices, or to prevent infe=ction following an operation. , such as a Caesarean section.

The iravention may also be used in the preve=ntion of carriage of the bacteria by carriers who themselves show few, if any, symptoms.

Description of the Figures

Figure 1 shows the effect of a phage 75-SnCe6 conj ugate on different EMRSA strains.

Figure 2 shows the effects of conjugate, no conjugaste, photosensitiser only or phaage only and preseence or absence of irradiation on EMR=SA-16 and S. epidermidis.

Figures 3 to 5 show the effect of the invention on EMMRSA-16 and S. aureus 83254, varying the ligsht dose.

Figure 6 shows the effect of light dose using a fixed_ concentration of ®11-SnCe6 conjugate on “TEMRSA-16.

Figure 7 showws the effect of the invention on strainss of VRSA (Mu3), hetero-VR_SA (Mu50) and CA-MRSA (MW2).

Figure 8 shows the effect of the invention on Strepteococeus pyogenes.

Figure 9 shows the effect of the invention on Propiconibacterium acnes.

EXAMPLES

Materials aned Methods

The fosllowing media were prepared:

Nutrient Broth 2 (NB2) medium

One litre of medium was made= by adding 25g of Nutrient Broth 2 (Oxoid) (10.0 g/1 Lab-Lemco powder, 10.0 g/1 peptone, 5.0 g/l NaCl) to 1 litre of eionised, distilled water. After mixing, the medium was autoclaved at 121°C for 1S min.

Ss

Tryptone Soya Yeast Broth (TSY)

One litre of medium was madee by adding 39g of Tryptone Soya Beroth (Oxoid) (17.0 g/l pancreatic digest of casein, 3.0 g/l papaic digest of soytmean meal, 2.5 g/l glucose, 2.5 g/l di-basic potassium phosphate, 5.0 g/l NaCl) and 0 .5% of yeast 190 extract (9.8 &/1 total nitrogen, 5.1 g/l amino nitrogen, 0.3 g/l NaCl) to 1 litre of deionised, distilled water. After mixing, the medium was autoclaved at 21°C for 15 min.

Nutrient Broth 2 Top Agar

LES : 0.35 % (w/v) of Agar Bactera ological (Agar No. 1, Oxoid) was added to NB2 medium. After mixing, the medium was autoclaved at 121°C for 15 mimn.

Nutrient Broth 2 Bottom Agar 0.7% (w/v) of Agar Bacterio logical was added to NB2 medium. After autoclaving, 10 mM of CaCl, was added (10m] 1M CaCl, in 1 litre of NAB2).

Columbia Blood Agar (CBA) 37.1g of Columbia Agar Basse (Oxoid) (23.0 g/l special peptone_, 1.0 g/l starch, 5.0 g/l NaCl, 10.0 g/1 agar) wwas added to 1 litre of deionised, dis=tilled water.

After autoclaving, the liquid agar was allowed to cool at room temperature until cool enough to handle. 5% (v/v) defibrimated horse blood (E & O Laboratories, Scotland) was then added. a

A11-

MR annitol Salt Agar (MSA) 111g of Mannitol Salt Agar (Oxoid) (75.0 g/l NaCl, 10.0 g/l maranitol, 1.0 g/l

Lab-lemco powder, 10.0 g/l peptone, 0_025 g/1 phenol red, 15.0 g/l agar’) was added to- 1 litre of deionised, distilled water.

All mixtures were autoclaved at 121°C for 15 min. The liquid amgar was then poured into plates, covered and allowed to cool ovemight.

Target organisms

The organisms used in the exarmples were as follows, given as mames and

NICTC (National Collection of Type Cultures, UK) or ATCC (Americam Type

Culture Collection, USA) numbers:

Epidemic methicillin-resistant S. aurews (EMRSA)-1 (NCTC 11939)

EMRSA-3 (NCTC 13130)

EMRSA-15 (NCTC 13142)

EMRSA-16 (NCTC 13143)

Mu3 (ATCC 700698), is a methicillin resistant Staphylococcus aureus (MRSA) sstrain with heterogeneous resistance to vancomycin, designated heterogeneously wancomycin-resistant Staphylococcus aureus (hetero-VRSA) (Hanaki «ef a! (1998). J.

Antimicrob. Chemother. 42:199-209)

T™Mu50 is the archetypal VRSA strain (CHiramatsu ef al (1997). J. Antimicrob. «Chemother. 40:135-136)

MMW? is a Community-acquired MRS A strain. Community acquired MRSA strains ®(CA-MRSA) share the presence of staphylococcal cassette chromosome mec #(SCCmec) type IV in their genomes, are frequently virulent, and predominantly cause skin and soft tissue infections. The ge=nome sequence of the prototypic CA-MRSA strain, MW2, has revealed the presen«ce of additional virulence factors not commonly present in other S. aureus strains (Ba¥oa er al (2002), Lancet. 25;359(9 320):1819-27).

Staphylococcus epidermidis (NCTC 11047)

Streptococcus pyogenes (ATCC 12202) Propionibacterium acnes (ATCC 29399)

S-taphyloccus aureus 8324-5 (Novick (1967) Virology 33; 156-166).

All were maintained by weekly seabculture on CBA.

BRacteriophage

Phage 75 (Public Health Laboratory Service, UK) is a serogroup F staphylococcal phage, capable of infecting EMRSA-16, EMRSA-3 and weal<ly irmfecting EMRSA-15.

Bacteriophage ¢11 (Tandolo et a/, (2002), Gene 289 (1-2); 109-118) i sa temperate bacteriophage of serological group B. ¢11 is a transducing phage with a low lysogenisation frequency. It infects SS.aureus lytic group II strains whichh include many human and animal pathogens.

B-acteriophage propagation

Mid-exponential EMRSA-16 (30041) was added to 15ml Falcon tube=s.

A _pproximately 10° pfu of phage 75 were added to the tubes and allowed to iracubate at- room temperature for 30 min to allow the phage to infect the bacteria. 9m of cooled molten top NB2 agar (with 10mMC CaCl,) was added to the tubes, ancq the mixture poured onto undried NB2 base agzar plates. The plates were left to immcubate at 37°C overnight.

The next morning 1 ml of NB2 with 10 mM CaCl, was added to each_ plate, ard the top agar with the liquid medium wvas scraped into a small centrifuge t-ube.

Thhe collected agar was then spun in a centrifuge at 15000 rpm for 15 min at 23°C.

The supernatant was collected and passed. through a 0.45um (Nalgene) filter ato remove any bacterial cells. The resulting solution of phage 75 was stored at <3°C.

Bacteriophage precipitation

Phage precipitation was carried owt to purify the phage 75 from the NIB2 medium after propagation. To 5ml of pha ge 75 in NB2, 1.3 m! of SM NaCl ( IM firaal concentration) and 0.2 ml 1x phosph ate buffered saline (PBS) (8.0g/! NaCl, 0.2g1KCi, 1.15 g/l Na,HPO,, 0.2g/1 KH,P0,) were added, and 20% PEG

(polyexhylene glycol 8000, Sigma) was added to the solution and stirred slowly overnizght until completely dissolved. The solution was then placed on ice overni ght and thee next morning the solution was centrifuged at 3000rpm for 20 min at 4°C.

The suapernatant was removed and the remaining pellet was resuspended in 2.5m} 1x

PBS, mand filtered through a 0.45 pm filter.

PhotoOsensitiser

The photosensitiser used was tin ('W) chiorin 6 (SnCe6) (Frontier Scien tific,

Lancashire, UK), which is photoactivatable at 633 nm. :

Prepmaration of conjugate 2mg of SnCe6 was dissolved with stirring in 800 pul of activation buffer (0.1

M MRES (2-(N-morpholino(ethanesulphonuic acid) (Sigma)), 0.5 M NaCl, pH 5.5). An

EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (Sigma) solution (dmg in 1 m) activation buffer) and a S-NHS ( N- hydmroxysulphosuccinimide) (Fluka) solution (2.7 mg in 250 ul activation buffer) weree made.

To the dissolved SnCe6, 200pl off dissolved EDC and S-NHS were addled, and_ the mixture was left for 1 to 4 hours at room temperature with stirring to perovide a stable amine-reactive intermediate. The mixture was covered in aluminium foil as

SnCCe6 is a light sensitive reagent. The reaction was quenched by adding 1.41 §- me=rcaptoethanol (Sigma).

Experiments were carried out using the reagents at a molar ratio of

Sn Ce6:EDC:S-NHS of 1:1:2.5.

The pH of the reactive SnCe6 mixture was neutralised to 7.0 by adding 0.7ml 1 BM NaOH. 1.5mi of phage 75 was them added to the amine-reactive solutiora to allow the amino groups on the phage to react with the carboxyl groups of the SnCe®6, armd then mixed for 4 to 16 hours. The weaction was quenched with 2.5u} ethanolamine (Sigma).

The photosensitiser-phage conjugate (PSS-phage) was separated from free PS after conjugaticsn by precipitating the PS-phage twice, as described above in

Bacteriophage WPrecipitation. The PS-phage wass then dialysed against PBS.

In the examples below, the concentratiomn of phage 75 is 7.3x10° pfu/mm] and the concentration of SnCe6/bacteriophage-SnCe=6 is 1.5 pg/ml.

Laser

The laser used was a Model 127 Stabilit-e helium-neon (He/Ne) laser (Spectra

Physics, USA) ~with a power output of 35 mW. ~The laser emitted radiation irm a collimated bearm, diameter 1.25 mm, with a wawelength of 633nm.

Example 1 "A culture of EMRSA-16 in the mid-exp=onential growth phase was di_luted to 1x10’cf/ml. 2 O'ul samples of the diluted bacte=ria were then placed into weHls of a 96-well plate (I™unc), together with a magnetic sstirrer bar. 100 pl ofthe phage 75-SnCe6 conjugate= prepared above and calcium_ chloride (CaCl) to a final concentration of 10 mM was added to the bacteria. The cortents of the wells were eft to incubate at room temperature for 5 min, with stirring. Controls were performed with 100 ul 1xPBS added to thee bacteria and used as a refereence for experimental samples. The experiment was carmried out in duplicate.

After inecubation, the contents of the wel 1 were directly exposed to thes laser light for 5 min, with stirring, corresponding to a-n energy density of 21 J/cm’.

Aluminium foil was placed in the surrounding v=wells to allow any escaping lamser light to be reflected boack into the target well. Control _s were performed with no lasser irradiation.

After ex posure to the laser, 100 pu] samples were immediately taken fiirom each well and serially diluted, from 10! to 10%, in 1 ron! TSY in 1.5 mi Eppendorf wtubes.

Aliquots of 50 p 1] of each dilution were then plasced and spread out on half a CCBA plate. The platess were placed in a 37°C incubator overnight. The following nmorning the number of survivors was counted, the average between the four sets w-as taken and multiplied by the appropriate dilution factor, and graphically analysed. .

Phage at 7.3x10°pfu/ml!

SnCe6/phage at 1.5 ug/ml

It was found that over 99.9% of the EMRSA-16 were killed.

Example 2 .

Example 1 was repeated, using EMIRSA-1 in place of EMRSA-16. It was fourad that 99.98% of the bacteria were killexd.

Example 3

Example 1 was repeated, using EMER SA-3 in place of EMRSA-16. It was fournd that over 99.99% of the bacteria were killed.

Exampled

Example 1 was repeated, using EMIRSA-15 in place of EMRSA-16. It was fourad that over 99.99% of the bacteria were= killed.

Example 5

Example 1 was repeated, using S. epidermidis in place of EMRSA—16. It was fourad that over 99.99% of the bacteria were killed.

Result for Examples 1 to 5 are presented in Figure 1,

Exammpleé6

Example 1 was repeated, using 10pl each EMRSA-16 and S. epide=rmidis in place of the 20ul samples of EMRSA-16. S amples were plated on MBA p lates for enurmeration.

Phage at 7.3x10°pfu/ml

SnCe6/phage at 1.5 pg/ml

21 J/cm? laser light

It was found that over 99.99% of both bacterial strains were killed in the mixed culture.

Comparative Example

Example 6 was repeated, firstly in the absence of conjugate, and without exposing to laser light, secondly with SnCe6 photosensitiser and exposure to laser light, and thirdly with phage 75 and without exposure to laser light.

The results for Example 6 and for the Comparative Example are presented in

Figure 2.

The Examples show that the conjugate is highly effective at killing all of the

EMRSA strains tested. Since phage 75 is only capable of infecting EMRSA. -15 and

EMRSA-16, this indicates that the phage is able to successfully bind to straims it is incapable of infecting, thus acting as an effective targetting agent. The attached photosensitisers then effected the killing upon laser irradiation.

Significant kills were also obtained with S. epidermidis, both alone and in a mixture with MRSA, indicating that the phage also bound to non-related staphylococcal strains. The phage 75-SnCe6 conjugate is useful for a variety” of staphylococcal infections.

Example 7

Targeted Photodynamic Therapy using ®11-SnCe6 Conjugates against

Staphylococcus aureus and a laser light source

Bacteriophage ®11 was propagated and precipitated as described above for phage 75, except that S qureus strain 8325-4 was used as the propagating strain. Tin chlorin 6 (SnCe6) was conjugated onto Staphylococcus phage ®11 using thme method described above, achieving bound concentrations of 2.3 and 3.5 pg mi”! SnCe6 with the phage ®©11 at 4.7 x 10” pfu.ml’. These ®11-SnCe6 conjugates were ther incubated with various strains of Staphylococcus aureus and exposed to laser light at

633nm from a 35mW HeNe laser (21 J/cm?) for 5 minute=s. The final concentration of conjugated SnCe6 was 1.15 pg ml. ~~ Theresults show that ®11-SnCe6 conjugates achmieved a 92.33% kill of S. aureus 8325-4 (compared to control counts in phosphate= buffered saline) after 5 minutes exposure, whilst SnCe6 at a corresponding conc=entration (1.15 ug ml) did not achieve any kill. The results are presented in Figure 3.

We have also shown that this ®11-SnCe6 conjugzate is effective against a methicillin-resistant strain of the organism (EMRSA-16)», achieving 88.11% kill, even though ®1 1 only infects this strain under stringent «optimal conditions. A range of control experiments such as; light without photosensittiser (L+S-), photosensitiser without light (I.-S+), and unconjugated phage at 1 x 107 —pfu ml" (L-S-); did not result in significant kills. The results are presented in Figure 4 .

By increasing the light dose to 10 minutes in the presence of calcium (10mM) we are now achieving 99.88% kills against S. aureus 8325-4 using ®11-SnCe6 conjugates (1.75 pg ml). The results are presented in Figure 5.

For Figures 3 to 5 the photosensitiser (either SnCe6 or ®11-SnCe6) was added to give a final concentration of 1.15 pg ml’ (with respect to SnCe6). The light source was a 35 mW Helium/Neon laser and irradiation &when used) was for 5 minutes in the case of Figures 3 and 4, and for 10 minute=s in the case of Figure 5.

The effect of varying the light dose on the kills obtained with the SnCe6- phage ®11 conjugate was investigated. The experiments were carried out as described above except that the bacterial suspensions we=re exposed to light from the

Helium/Neon laser for different periods of time - these wwere 1, 5, 10, 20 and 30 minutes. In each case, the concentration of the ®11-SnCee6 conjugate (final concentration equivalent to 3.5 pg ml” of SnCe6) was th_e same.

Incubati on of the organism with the ®11-SnCe6 conjugate for upto 60 minutes in the dlark had no significant effect on the viablee count. However, significant reductions in the viable count were obtained vvhen the suspensions were exposed to laser light in the presence of the ®11-SnCe6 eonjugate - greater kills were ombtained with the longer exposure timnes. Using an exposure time of ~ 30 minutes, a reeduction in the viable count of approximately 99.9999% was obtairaed. aD11-SnCe6 was used to give a final concentration of 3.5 ppg m1! (with respect to

SnCe6). The light source was a 35 mW Heliurm/Neon laser and irraciliation (when used) was for 1, 5, 10, 20 or 30 minutes. The results are presented ir Figure 6.

In Figures 3 to 6

SnCeb = tin chlorin e6

D11-SnCe6 = tin chlorin e6 conjugamted to bacteriophage ©11

PBS = Phosphate buffered saline

L_+S+ = bacteria irradiated in the presence of conjugate

LL_+S- = bacteria irradiated in the absence of conjugate

XL_-S+ = bacteria exposed to conjugate in the absence of light

A.-S- = bacteria exposed neither to 1x ght nor conjugate

EExample 8

Lethal Photosensitisation of Stapfeylcoccus eureus using a phage 75-tin (IV) chlorin e6 conjimgate and a white light source

Bacterial strains: S. aureus 8325-4

EMRSA-16

Wight source: KL200 (Schott). This is a 20-watt halogen cold light =source. The light guide attached to it is a flexible optic fibre bundle which is directedll onto a 96 well plate at a distance of 5 cm. A squares of 4-wells is placed at the centre of the light source.

Approx light intensity = 44,000 lux or 470pW/nm

Phage 75 was conjugated to SnCe6 as described above. Phages we=re used at 2 «concentration of 1 x 10” pfu/ml.

Overnight cultures of S. aureus grown in nutrient broth were cen trifuged, resuspended in PBS and adjusted to an OD of 0.05 at 600nm (apgproximately 4 x 10’ cfu/ml) 50ul of bacterial culture was al iquoted into a 96-well plate and 5#0ul of the one of the following solutions added to time wells: 1) 3.5pug/ml SnCe6-phage 75 (final concentration 1.75pug/ml, 1 x 10° pfu/well) in PBS 2) 1.75pg/ml SnCe6-phage 75 «final concentration 0.875ug/ml, 5= x10° pfu/well) in

PBS

3) 3.5ug/ml SnCe6 in PBS (final concentration 1.75ug/mi) 4) 1.75pg/ml SnCe6 in PBS (fimal concentration 0.875ug/ml) 5) PBS 6) Phage 75 at a concentration of 5 x 10° or 1 x10° pfw/well in PES

Wells were either expos ed to white light (4 wells at a time=) or wrapped in tin foil and stored in the dark.

After various exposure times an aliquot was taken from ea ch well, serially diluted and spread onto Columbwia blood agar. Agar plates were in. cubated overnight at 37°C and counted the next dazy.

Results

Table 2

EEMRSA-16

I EE A

+r +r = Kill ~ this is calculated compared to bacteria incubated with PBS and kept in the ark

All results are the average of replicate experiments.

Controls included bacteria incubated wi th SnCe6, phage 75-SnCe6 and phag € 75 wvithout exposure to white light. Phage “75 was also exposed to white light.

AAll controls had bacterial counts which were not significantly different to the control ssuspension which had no photosensitiser added and was not irradiated.

Example9

Further tests were carried out or §. aureus strains Mu3, Mu50 and M™W2. To suspensions of vancomycin-resistant strains of Staphylococcus aureus (Mu3 and

Mu50) or 2 community-acquired strain ©f MRSA (MW?2), saline, phage 75, SnCe6 or pehage 75-SnCe6 was added and samples exposed to light from a 35 mW

FHelium/Neon laser.

The concentration of SnCe6 usec was 1.5 pg/ml, the phage concentra tion was 5.1 x 10” plaque-forming units/ml and the light energy dose was 21 J/cm®. Thae mumbers above the bars represent the % kill of the organism relative to the sample to wwhich saline only was added. The results are presented in Figure 7.

Example 10

Lethal photosensitization of Streptococcus pyogenes using tim _chlorip e6 (SnCe6).

Streptococcus pyogenes ATCC 12202 was grown in Braain Heart Infusion broth at 37°C in an atmosphere consisting of 5%CO, in air. The «ells were harvested by centrifugation and re-suspended in phosphate buffered saline (PBS) and diluted to 1x10’cfu/ml in PBS. 20 pl samples of the diluted bacterial suspension were then placed into wells of a 96-well plate, together with a magnetic stimrer bar. 100 pl of different concentrations (1 — 50 ug/ml) of the SnCe6 in PBS was added to the bacterial suspensions. Controls were performed with 100 ul PBS added to the bacteria and either irradiated (L+S-) or kept in the dark (L-S-). The experiment was carried out in duplicate, © After incubation, the contents of some of the wells were «exposed to light from the 35 mW Heliurn/Neon laser emitting light with a wavelength of 633nm for 10 min, with stirring, corresponding to an energy density of 42 J/chm?. Aluminium foil was placed in the surrounding wells to allow any escaping laser Right to be reflected back into the target well. Control wells were not irradiated with Raser light.

After exposure to the laser light, 100 pul samples were im-mediately taken from each well and serially diluted, from 10" to 10%, in 1 m! TSY in 1 .5 m! Eppendorf tubes. Duplicate SO pl aliquots of each dilution were then spread. out on half a CBA plate. The plates were placed in a 37°C incubator for up to 48 h znd the resulting colonies were counted to determine the number of surviving organisms.

Incubation of the organism in the dark with increasing concentrations of

SnCe6 had no significant effect on the viable count. Neither did Arradiation of the organism with laser light in the absence of the photosensitiser. H_ owever, irradiation ofthe organism in the presence of SnCe6 resulted in a concentragtion-dependent decrease in the viable count. A 99.9997% kill of the organism waas obtained using a photosensitiser concentration of 50 pg/ml. The results are presented in Figure 8. In

Figure 8 :

L+ (open bars) = cultures irradiated with laser light in thes absence of SnCe6 as well as in the presence of various concentrations of the photos. ensitiser;

L - (shaded bars) = cultures incubate=d in the dark in the absence of SnCe6 as well ass in the presence of various concentrations of the photosensitiser.

Exampple1l

Lethal photosensitization of Propionibacte-rium acnes using tin chlorim ¢6 (SnCe-6),

Propionibacterium acnes ATCC 29899 was grown in pre-reducecd Brain

Heart Mnfusion broth at 37°C in an anaerobic atmosphere. The cells were Imarvested by centrifugation and re-suspended in phosphates buffered saline (PBS) and d_iluted to ” 1x10%=fw/m] in PBS. 20 ul samples of the di_ luted bacterial suspension were then placed into wells of a 96-well plate, together with a magnetic stirrer bar. 1 00 pl of different concentrations (1 — 50 ug/ml) of thes SnCe6 in PBS was added tc» the bacteri_al suspensions. Controls were performed with 100 pl PBS added tos the bacteria and either irradiated (L+S-) or kept iin the dark (L-S-). The exper—iment was carried out in duplicate.

After incubation, the contents of som e of the wells were exposed to light from the 35 mW Helium/Neon laser emitting light- with a wavelength of 633nmm for 10 min, with stirring, corresponding to an energ_y density of 42 J/cm? Alumirium foil was placed in the surrounding wells to allow any escaping laser light to be reflected back irato the target well, Control wells were not irradiated with laser light.

After exposure to the laser light, 100 pl samples were immediately taken from each well and serially diluted, from 10" to 10, in 1 ml of pre-reduced TS™Y in 1.5 ml

Eppenclorf tubes. Duplicate 50 pl aliquots of ~ each dilution were then spread out on half a CCBA plate. The plates were incubated anaerobically at 37°C and th_e resulting colonies were counted to determine the number of surviving organisms.

Incubation of the organism in the dark< with increasing concentrations of

SnCe6 had no significant effect on the viable= count. Neither did irradiatiomn of the organissm with laser light in the absence of th-e photosensitiser. However, i_tradiation of the Organism in the presence of SnCe6 resmilted in a concentration-depe-ndent decrease in the viable count. A 100% kill of the orgammism was obtained using a photosensitiseer concentration of 50 pg/ml. The result.s are presented in Figure 9. In

Figure 9

L+ (open bars) = cultures irradiated with laser light in the absence of SnCe6 as well as in the presence of various concentrations of thee photosensitiser;

L - (shaded bars) = cultures incubated in the ark in the absence of SnCe6 as well as in the presence of various concentrations of thes photosensitiser.

Example 12 .

Preparation ef conjugate of TBO and bacteriophag=e 1mg o f toluidine blue O (TBO) was dissolved in 800 ul of activation buffer (0.IMMES, 0 .5M NaCl pH5.5) together with 0.4mg EIDC and 0.6mg of S-NHS and 200 pl of phage (5 x 10” pfu/ml). The reaction was allowed to proceed for 15 to 30 minutes with stirring after which time the EDC was neutralised by adding 1.4 ul of 2- mercaptoethanol. The reaction was allowed to proceedl for a further 2 to 4 hours after which time the= reaction was quenched by adding hydrox ylamine to a final concentration of 10mM.

The TEB3O-phage conjugate was separated from fimee TBO by two rounds of phage precipitation followed by dialysis against PBS.

Claims

WwNO 2005/034997 PCT/GB=2004/004305 CLAIMS

1. A composition compri sing a conjugate of a photosensitiser and a bacteriophage.

2. A composition according to claim 1, wherein the bacterioplaage is a staphylococcal bacteriophage.

3. A composition according to claim 1 or 2, wherein the photossensitiser is covalently linked to the bacteriophage.

4, A composition according to any of claims 1 to 3, wherein the photosensitiser is chosen from Porphy/rins, phthalocyanines, chlorins, bacteriochlorins, phenothiaziniums, phenazines, acridines, texaphyrins, cy=zanines, anthracyclins, pheophorbides, sapphyxrins, fullerene, halogenated xantheness, perylenequinonoid pigments, gilvocarcins, terthiophenes, benzophenanthri dines, psoralens and riboflavin.

5. A composition according to claim 4, wherein the photosensditiser is tin (IV) chlorin e6 (SnCeS6).

6. A composition according to any of the preceding claims, wherein the bacteriophage is chosen from phage 53, 75, 79, 80, 83, ¢11, $12, $13, ¢1477, ¢ MR11, 48, 71, ¢ 812, SK311, $131, SB-1, U16, C,, SF370.1, SP24, SFL, _Al, ATCC 12202-B1, f304L, $3048, $15, $16, 7 82, Plclrl00KM, P1, Ti, T3, T4, TZ MS2, P1, M13, UNL-1, ACQ, UT], tbalD3, E79, F8, pf20 B3, F116, G101, B86, TJM, ACq, UT], BLB, PP7, ATCC 29399-B1 and B40-8.

8. A composition accordimg to any of the preceding claims, wimerein the concentration of the photosensitiser is from 0.01 to 200 pg/ml.

9. A composition according to any of the preceding claims, whaerein the concentration of the bacteriophage is from 1x10’ to 1x10" pfu/ml.

10. A composition according to any of the preceding claims, whaich further comprises a source of Ca?* ionss, preferably calcium chloride.

J).

11. A composition according to any of claims 1 to 10, in the form of a solution in a pharmaceutically acceptable camrmier.

12. A composition according to any of claims 1 to 11, wherein the composition further comprises one or more «fa buffer, salt, antioxidart, preservative, S gelling agemt or remineralisation agent,

13. A method of killing bacteria, comprising (a) contactin g an area to be treate=d with a composition accomrding to any of thme preceding claims, such that any~ bacteria present bind to te photeosensitiser-bacteriophage conjugaate; and (b) irradiating the area with Ii ght &at a wavelength absorbed by the photosensitiser.

14. A method according to claim 1_3, wherein the bacteria are staphylococcus, particularly MRSA, EMRSA VRSA, hetero-VRSA or CA-MRSA.

15. A method according to any of claims 13 or 14, wherein the light is laser light or white light.

16. A method according to claim 155, wherein the laser light iss from a helium neon gas laser.

17. A method according to any of claims 15 or 16, wherein the laser light has a wavelen gth of from 200 to 1060nm.

18. A method according to any of cl aims 15 to 17, wherein the laser has a power of from. 1 to 100mW and a beam diameter of from 1 to 10mm.

19. A ‘method according to claim | 8. wherein the light dose of laser irradiation is fr-om 5 to 333 Jom.

20. A method according to claim 15, wherein the light dose of ~white light 3s from 0.01 to 100 J/cm?

21. ~A method according to any of clazims 15 to 20, wherein the «duration of 1 radiation is form one second to % § minutes. 22, AA method according to any of clai ms 13 to 21, wherein the composition is present in or on the area to be trea_ted at a concentration of fiom

0.00001 to 1% wiv. AMENDED SHEET

23. Use of a composition according to any of claims 1 to 12, for treatment off the human or animal body.

24. Use of a composition according to any of claims 1 to 12, in tie manufacture of a medicament for treatmxaent of bacterial infection.

25. Use according to claim 24, wherein the bacterial infection is aS. amureus, particularly MRSA, EMRSA, VRSA, hetero-VRSA or CAMRSA.

26. Use of a bacteriophage aus a targeting agent in photodynamic ~therapy (PDT).

27. Use according to claim 226, wherein the bacteriophage is a staphylococcal phage.

28. A composition accordin gto any of claims 1 to 12, substantia lly as d_escribed in the Examples.

29. A method according to any of claims 13 to 22, substantially =as dlescribe in the Examples.