Indian Patents. 250289:"USE OF PHOTOSENSITISATION"

Title of Invention	"USE OF PHOTOSENSITISATION"
Abstract	A composition comprising a conjugate of a photosensitiser and a bacteriophage is provided. The conjugate may be used to kill bacteria, particularly MRS A, EMRSA, VRSA, hetero-VRSA or CA-MRSA in a targeted method of photodynamic therapy.

Full Text	USE OF PHOTOSENSITISATION The present invention relates to a composition comprising a conjugate of a photosensitiser and a bacteriophage, particularly a staphylococcal bacteriophage, known as a staphylophage. The invention also relates to the use of the conjugate in a method of photodynamic therapy for infectious diseases. Background The use of antimicrobial agents to counter bacterial infections is becoming increasingly ineffective, due to the rapid emergence of antibiotic resistance amongst many species of pathogenic bacteria. One such pathogen is Staphylococcus aureus (S. aureus), which characteristically causes skin infections such as boils, carbuncles 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 staphylococci, Staphylococcus aureus, streptococci, Corynebacterium spp., E. coli, Klebsiella aerogenes, Klebsiella pneumoniae, Enterobacter aerogenes, Propionibacterium acnes, Bacteroides spp., Pseudomonas aeruginosa and Peptostreptococcus spp. Increasingly, these bacteria are showing resistance to antibiotic treatment. In particular, resistant strains of 5. aureus have emerged. Methicillin-resistant S. aureus (MRSA) was first reported in 1961 (Jevons, M. (1961) British Medical Journal, 1, 124-5), and these strains are now a major cause of hospital-acquired infection throughout the world, as well as being prevalent in many nursing and residential homes. This poses an alarming challenge to healthcare, causing significant infection and morbidity of hundreds of patients in the UK each year (Ayliffe et al, J Hosp Infect (1988), 39, 253-90). Since the first report of MRS A, these organisms have demonstrated resistance to a wide variety of antimicrobials including erythromycin, aminoglycosides, tetracyclines, trimethoprim, sulphonamides and chloramphenicol. MRSA strains have developed that are only susceptible to a single class of clinically-available antibiotics: the glycopeptides such as vancomycin and teicoplanin. However, resistance is developing even to these, as strains tolerant to vancomycin have now been reported (Hiramatsu, K. (1998) American Journal of Medicine, 104, 7S - 10S). These strains are variously known as VRS A (Vancomycin resistant Staphylococcus aureus) and hetero-VRSA (resistant strains arising from exposure to high levels of vancomycin). At present, the management of patients with MRSA infections usually involves the administration of antimicrobial agents and again, there is evidence of the development of resistance to many of the agents used. Due to the emergence of strains which are resistant to virtually all currentlyavailable antimicrobials, MRSA is now a serious threat to health. The term MRSA itself now more accurately applies to methicillin and multiple antimicrobial-resistant S. aureus. Certain strains of MRSA have been found to spread rapidly not only within hospitals, but also between them. These strains have been termed epidemic MRSA (EMRSA). Since the first EMRSA strain (EMRSA-1) was reported in 1981, 17 distinct EMRSA strains have been identified, all of which are resistant to a number of antimicrobials. Recently, the two most prevalent strains have been EMRSA-15 and -16, which account for 60-70% of the 30000 MRSA isolates reported (Livermore, D (2000) Int. J. Antimicrobial Agents, 16, S3 - S10). Importantly, strains of MRSA, (known as community-acquired MRSA (CA-MRSA)) have also started to spread in the community, ie. amongst non-hospitalised individuals. It is clear from the above that alternative methods of countering bacterial infection, particularly infection with MRSA are urgently required. One approach has been to employ a light-activated agent to achieve lethal photosensitization of the organism. This involves treating the organism with a lightactivatable chemical (photosensitiser) which, upon irradiation with light of a suitable wavelength, generates cytotoxic species, resulting in bacteriolysis. This technique has been used to achieve killing of a wide range of bacteria, including S. aureus and MRSA strains, in vitro using toluidine blue 0 (TBO) and aluminium disulphonated phthalocyanine (ALPcS2) as photosensitisers. Neither photosensitiser nor laser light alone exerted a bacteriocidal effect (Wilson et a/, (1994) J Antimicrob Chemother 33, 619-24). In a subsequent study, 16 strains of EMRSA were found to be susceptible to killing by low doses of red light (674 nm) in the presence of AlPcS2 (Griffiths et al, (1997) J Antimicrob Chemother, 40, 873-6). At higher light doses, 100 % killing was achieved. Photodynamic therapy (PDT) is the application of such an approach to the treatment of disease. It is an established procedure in the treatment of carcinoma and forms the basis of a means of sterilising blood products. It has only been more recently that the application of PDT to the treatment of infectious diseases has been evaluated. For example, haematoporphyrins in conjunction with an argon laser have been used to treat post-neurosurgical infections and brain abscesses (Lombard et al, (1985), Photodynamic Therapy of Tumours and other Diseases, Ed. Jori & Perria). One potential problem associated with PDT of infectious diseases is its lack of specificity. Hence, if the photosensitiser binds to, or is taken up by, a host cell, as well as the target organism, then subsequent irradiation may also lead to the death of the host cell. A way to overcome this is by the use of targeting compounds: that is, any compound that is capable of specifically binding to the surface of the pathogen. Several targeting compounds have previously been shown to be successful in eliminating specific strains of bacteria when they were conjugated to a photosensitiser. For example, immunoglobulin G (IgG) has been used to target S. aureus Protein A (Gross et al (1997), Photochemistry and Photobiology, 66, 872-8), monoclonal antibody against Porphyromonas gingivalis lipopolysaccharide (Bhatti et al (2000), Antimicrobial Agents and Chemotherapy, 44, 2615-8) and poIy-L-lysine peptides against P. gingivalis and Actinomyces viscosus (Soukos et al (1998), Antimicrobial Agents and Chemotherapy, 42, 2595-2601). A monoclonal antibody conjugated via dextran chains to the photosensitiser 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 Acad Sci, 618, 383-393). The present inventors have used IgG conjugated to SnCe6 to target EMRSA strains 1, 3, 15 arid 16 (Embleton et al (2002), J Antimicrob Chemother, 50, 857- 864), achieving higher levels of killing than the photosensitiser alone, and selectively killing the EMRSA 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. Hence alternative targeting agents that can target any S. aureus strain are desirable. Bacteriophage are viruses that infect certain bacteria, often causing them to lyse and hence effecting cell death. They have been proposed as antibacterial agents in their own right. However, one of the problems with using staphylococcal bacteriophage (termed staphylophage) in the treatment of S. aureus disease is their restricted host range. Although there are polyvalent staphylophage which can lyse many S. aureus strains, other strains are resistant and hence bacteriophages alone could not provide an effective method of killing 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 have now found that some bacteriophage can serve as an effective, targeted delivery system for photosensitisers. The present inventors have found that when a bacteriophage is linked to a photosensitiser, the photosensitiser-bacteriophage conjugate formed is highly effective in killing bacteria when irradiated with light of a suitable wavelength. Bacteriophage-photosensitiser conjugates could be used to treat or prevent a broad range of bacterial skin and wound infections. The most frequently isolated organisms from skin and wound infections are: coagulase-negative staphylococci, S. aureus, streptococci, e.g. Streptoccocus pyogenes, Corynebacterium spp., E coli, Klebsiella aerogenes, Klebsiella pneumoniae, Enterobacter aerogenes, Propionibacterium acnes, Bacteroides spp., Pseudomonas aeruginosa and Peptostreptococcus spp.. In particular, conjugates of photosensitiser and staphylophage can be used in a method of photodynamic therapy against strains ofStaphylococci spp, particularly against MRSA, EMRSA, VRSA, hetero-VRSA and CA-MRSA. The invention provides a composition comprising a photosensitizing compound (photosensitiser) linked to a bacteriophage to form a photosensitiserbacteriophage conjugate. The bacteriophage may be a staphylococcal phage, and is preferably a staphylophage that can bind to Staphylococcus 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 covalent linkage. The photosensitiser and/or the bacteriophage contain or may be modified to contain groups which can be covalently crosslinked using chemical or photoreactive reagents, to produce crosslinked bonds, for example thiol-thiol crosslinking, amineamine crosslinking, amine-thiol crosslinking, amine-carboxylic acid crosslinking, thiol-carboxylic acid crosslinking, hydroxyl-carboxylic acid crosslinking, hydroxylthiol crosslinking and combinations thereof. The photosensitiser is suitably chosen from porphyrins (e.g. haematoporphyrin derivatives, deuteroporphyrin), phthalocyanines (e.g. zinc, silicon and aluminium phthalocyanines), chlorins (e.g. tin chlorin e6, poly-lysine derivatives of tin chlorin e6, m-tetrahydroxyphenyl chlorin, benzoporphyrin derivatives, tin etiopurpurin), bacteriochlorins, phenothiaziniums (e.g. toluidine blue, methylene blue, dimethylmethylene blue), phenazines (e.g. neutral red), acridines (e.g. acriflavine, proflavin, acridine orange, aminacrine), texaphyrins, cyanines (e.g. merocyanine 540), anthracyclins (e.g. adriamycin and epirubicin), pheophorbides, sapphyrins, fullerene, halogenated xanthenes (e.g. rose bengal), perylenequinonoid pigments (e.g. hypericin, hypocrellin), gilvocarcins, terthiophenes, benzophenanthridines, psoralens and riboflavin. The invention is directed to killing bacteria using the above-described conjugates. The bacteriophage used in the conjugate may 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 embodiment, the infecting bacterium is MRSA, EMRSA, VRSA, hetero-VRSA or CA-MRSA and the conjugate includes the staphylococcal phage 75 or phageΦl 1. Table 1 below shows some examples of bacteria-bacteriophage pairs, although many more examples exist. Further novel bacteriophages can be isolated and/or adapted to the target bacteria. The specificity of the treatment can be modified as required by using monovalent bacteriophages, polyvalent bacteriophages or combinations of monovalent bacteriophages or combinations of monovalent and polyvalent bacteriophages. (Table Removed) The composition of the invention suitably comprises at least 0.010 g/ml, of the photosensitiser, preferably at least 0.02μg/ml, more preferably at least 0.05μg/ml upto 200 μg/ml, preferably up to 100μg/ml, more preferably up to 50 μg/ml. The amount of the bacteriophage in the composition is suitably from 1x105 to lxI010pfu, preferably from 1x1O6 to 1Ix1O9 pfu, more preferably from 1x106 to 1x108 pfu. The composition of the invention may further comprise a source of divalent ions, e.g. Ca+ or Mg2*, preferably Ca2+. Examples include calcium chloride, calcium carbonate and magnesium chloride. The ions are suitably present in an amount of from 5 to 200mM, preferably from 5 to 15 mM, more preferably about 1 OmM. The composition may further comprise one or more ingredients chosen from buffers, salts for adjusting the tonicity, antioxidants, preservatives, gelling agents 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 invention such that any bacteria in the area bind to the photosensitiser-bacteriophage conjugate; and (b) irradiating the area with light at a wavelength absorbed by the photosensitiser. Suitably the bacteria are as set out above in Table 1, preferably Staphylococcus aureus, more preferably MRSA, EMRSA, VRSA, hetero-VRSA 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 the photosensitiser and to have sufficient energy to activate the photosensitiser. The source of light may be any device or biological system able to generate monochromatic or polychromatic light. Examples include laser, light emitting diode, arc lamp, halogen lamp, incandescent lamp or an emitter of bioluminescence or chemiluminescence. In certain circumstances, 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 laser may have a power of from 1 to lOOmW and a beam diameter of from 1 to 10mm. The light dose for laser irradiation is suitably from 5 to 333 J cm"2, preferably from 5 to 30 J cm"2 for laser light. For white light irradiation, a suitable dose is from 0.01 to 100 kJ/cm2, preferably from 0.1 to 20 kJc/m2, more preferably from 3 to 10 kJ/cm2. 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 (628nm, 10W output) Tunable solid state laser (532-1060nm), including Sd:YAG Light emitting diode (LED) (400-800nm) Diode laser (630-850nm, 25W output), eg. gallium selenium arsenide Tungsten filament lamp Halogen cold light source Fluorescent lamp. In the method of the invention, the composition is suitably in the form of a solution or a suspension in a pharmaceutically acceptable aqueous carrier, but may be in the form of a solid such as a powder or a gel, an ointment or a cream. The composition may be applied to the infected area by painting, spreading, spraying or any other conventional technique. The invention further provides the use of the composition for treatment of the human or animal body. Suitably, the composition is provided for use in the treatment of conditions resulting from bacterial infection, particularly by staphylococci, more particularly by MRSA, EMRSA, VRSA, hetero-VRSA or CA-MRSA. The invention may be used to treat bacterial infection, particularly by staphylococcal bacteria, more particularly by MRSA, EMRSA, VRSA, hetero-VRSA or CA-MRSA to treat or prevent skin infections such as boils, carbuncles, mastitis and impetigo, to treat or prevent infections of acne, bums or wounds, or to treat or prevent endocarditis, osteomyelitis, meningitis and pneumonia, arising as a result of bacterial infection, to treat or prevent infections arising from the use of catheters, implants or other medical devices, or to prevent infection following an operation, such as a Caesarean section. The invention may also be used in the prevention of carriage of the bacteria by carriers who themselves show few, if any, symptoms. Description of the Figures Figure 1 shows the effect of aphage 75-SnCe6 conjugate on different EMRSA strains. Figure 2 shows the effects of conjugate, no conjugate, photosensitiser only or phage only and presence or absence of irradiation on EMRSA-16 and S. epidermidis. Figures 3 to 5 show the effect of the invention on EMRSA-16 and S. aureus 8325-4, varying the light dose. Figure 6 shows the effect of light dose using a fixed concentration of Φl1!-SnCe6 conjugate on EMRSA-16. Figure 7 shows the effect of the invention on strains of VRSA (Mu3), hetero-VRSA (Mu50)and CA-MRSA (MW2). Figure 8 shows the effect of the invention on Streptococcus pyogenes. Figure 9 shows the effect of the invention on Propionibacterium acnes. EXAMPLES Materials and Methods The following 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/1 NaCl) to 1 litre of deionised, distilled water. After mixing, the medium was autoclaved at 121 °C for 15 min. Tryptone Soya Yeast Broth (TSY) One litre of medium was made by adding 39g of Tryptone Soya Broth (Oxoid) (17.0 g/1 pancreatic digest of casein, 3.0 g/1 papaic digest of soybean meal, 2.5 g/l glucose, 2.5 g/1 di-basic potassium phosphate, 5.0 g/1 NaCl) and 0.5% of yeast extract (9.8 g/1 total nitrogen, 5.1 g/1 amino nitrogen, 0.3 g/1 NaCl) to 1 litre of deionised, distilled water. After mixing, the medium was autoclaved at 121 °C for 15 min. Nutrient Broth 2 Top Agar 0.35 % (w/v) of Agar Bacteriological (Agar No. 1, Oxoid) was added to NB2 medium. After mixing, the medium was autoclaved at 121 °C for 15 min. Nutrient Broth 2 Bottom Agar 0.7% (w/v) of Agar Bacteriological was added to NB2 medium. After autoclaving, 10 mM of CaCl2 was added (10ml 1M CaCl2 in 1 litre of NB2). Columbia Blood Agar (CBA) 37.1g of Columbia Agar Base (Oxoid) (23.0 g/1 special peptone, 1.0 g/1 starch, 5.0 g/1 NaCl, 10.0 g/1 agar) was added to 1 litre of deionised, distilled water. After autoclaving, the liquid agar was allowed to cool at room temperature until cool enough to handle. 5% (v/v) defibrinated horse blood (E & O Laboratories, Scotland) was then added. Mannitol Salt Agar (MSA) 111g of Mannitol Salt Agar (Oxoid) (75.0 g/1 NaCl, 10.0 g/1 mannitol, 1.0 g/1 Lab-lemco powder, 10.0 g/1 peptone, 0.025 g/1 phenol red, 15.0 g/1 agar) was added to 1 litre of deionised, distilled water. All mixtures were autoclaved at 121 °C for 15 min. The liquid agar was then poured into plates, covered and allowed to cool overnight. Target organisms The organisms used in the examples were as follows, given as names and NCTC (National Collection of Type Cultures, UK) or ATCC (American Type Culture Collection, USA) numbers: Epidemic methicillin-resistant S. aureus (EMRSA)-l (NCTC 11939) EMRSA-3(NCTC13130) EMRSA-15(NCTC13142) EMRSA-16(NCTC13143) Mu3 (ATCC 700698), is a methicillin-resistant Staphylococcus aureus (MRSA) strain with heterogeneous resistance to vancomycin, designated heterogeneously vancomycin-resistant Staphylococcus aureus (hetero-VRSA) (Hanaki et al (1998). J. Antimicrob. Chemother. 42:199-209) Mu50 is the archetypal VRSA strain (Hiramatsu et al (1997). J. Antimicrob. Chemother. 40:135-136) MW2 is a Community-acquired MRSA 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 genome sequence of the prototypic CA-MRSA strain, MW2, has revealed the presence of additional virulence factors not commonly present in other S. aureus strains (Baba et al (2002), Lancet. 25;359(9320):1819-27). Staphylococcus epidermidis (NCTC 11047) Streptococcus pyogenes (ATCC 12202) Propionibacterium acnes (ATCC 29399) Staphyloccus aureus 8324-5 (Novick (1967) Virology 33; 156-166). All were maintained by weekly subculture on CB A. Bacteriopbage Phage 75 (Public Health Laboratory Service, UK) is a serogroup F staphylococcal phage, capable of infecting EMRSA-16, EMRSA-3 and weakly infectingEMRSA-15. Bacteriophage 411 (landolo et al, (2002), Gene 289 (1-2); 109-118) is a temperate bacteriophage of serological group B.Φl 1 is a transducing phage with a low lysogenisation frequency. It infects S.aureus lytic group III strains which include many human and animal pathogens. Bacteriophage propagation Mid-exponential EMRSA-16 (300ul) was added to 15ml Falcon tubes. Approximately 10s pfu of phage 75 were added to the tubes and allowed to incubate at room temperature for 30 min to allow the phage to infect the bacteria. 9ml of cooled molten top NB2 agar (with lOmM CaC^), was added to the tubes, and the mixture poured onto undried NB2 base agar plates. The plates were left to incubate at 37 °C overnight. The next morning 1 ml of NB2 with 10 mM CaCl2 was added to each plate, and the top agar with the liquid medium was scraped into a small centrifuge tube. The collected agar was then spun in a centrifuge at 15000 rpm for 15 min at 4°C. The supernatant was collected and passed through a 0.45 μm (Nalgene) filter to remove any bacterial cells. The resulting solution of phage 75 was stored at 4°C. Bacteriophage precipitation Phage precipitation was carried out to purify the phage 75 from the NB2 medium after propagation. To 5ml of phage 75 in NB2, 1.3 ml of 5M NaCl (1M final concentration) and 0.2 ml Ix phosphate buffered saline (PBS) (8.0g/l NaCl, 0.2g/l KC1, 1.15 g/1 Na2HPO4t 0.2g/l KH2PO4) were added, and 20% PEG (polyethylene glycol 8000, Sigma) was added to the solution and stirred slowly overnight until completely dissolved. The solution was then placed on ice overnight and the next morning the solution was centrifuged at 8OOOrpm for 20 min at 4°C. The supernatant was removed and the remaining pellet was resuspended in 2.5ml 1x PBS, and filtered through a 0.45 urn filter. Photosensitiser The photosensitiser used was tin (IV) chlorin e6 (SnCe6) (Frontier Scientific, Lancashire, UK), which is photoactivatable at 633 nm. Preparation of conjugate 2mg of SnCe6 was dissolved with stirring in 800μl of activation buffer (0.1 M MES (2-(N-morpholino(ethanesulphonic acid) (Sigma)), 0.5 M NaCl, pH 5.5). An EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiirnidehydrochloride) (Sigma) solution (4mg in 1 ml activation buffer) and a S-NHS (Nhydroxysulphosuccinimide) (Fluka) solution (2.7 mg in 250 ul activation buffer) were made. To the dissolved SnCe6, 200ul of dissolved EDC and S-NHS were added, and the mixture was left for 1 to 4 hours at room temperature with stirring to provide a stable amine-reactive intermediate. The mixture was covered in aluminium foil as SnCe6 is a light sensitive reagent. The reaction was quenched by adding 1.4μl (3- mercaptoethanol (Sigma). Experiments were carried out using the reagents at a molar ratio of SnCe6: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 M NaOH. 1.5ml of phage 75 was then added to the amine-reactive solution to allow the amino groups on the phage to react with the carboxyl groups of the SnCe6, and then mixed for 4 to 16 hours. The reaction was quenched with 2.5ul ethanolamine (Sigma). The photosensitiser-phage conjugate (PS-phage) was separated from free PS after conjugation by precipitating the PS-phage twice, as described above in Bacteriophage Precipitation. The PS-phage was then dialysed against PBS. In the examples below, the concentration of phage 75 is 7.3x106 pfu/ml and the concentration of SnCe6/bacteriophage-SnCe6 is 1.5 μg/ml. Laser The laser used was a Model 127 Stabilite helium-neon (He/Ne) laser (Spectra Physics, USA) with a power output of 35 mW. The laser emitted radiation in a collimated beam, diameter 1.25 mm, with a wavelength of 633nm. Example 1 A culture of EMRSA-16 in the mid-exponential growth phase was diluted to Ixl07cfu/ml. 20μl samples of the diluted bacteria were then placed into wells of a 96-well plate (Nunc), together with a magnetic stirrer bar. 100 ul of the phage 75-SnCe6 conjugate prepared above and calcium chloride (CaCl2) to a final concentration of 10 mM was added to the bacteria. The contents of the wells were left to incubate at room temperature for 5 min, with stirring. Controls were performed with 100 ul IxPBS added to the bacteria and used as a reference for experimental samples. The experiment was carried out in duplicate. After incubation, the contents of the well were directly exposed to the laser light for 5 min, with stirring, corresponding to an energy density of 21 J/cm2. Aluminium foil was placed in the surrounding wells to allow any escaping laser light to be reflected back into the target well. Controls were performed with no laser irradiation. After exposure to the laser, 100 ul samples were immediately taken from each well and serially diluted, from" 10' to 10"4, in 1 ml TSY in 1.5 ml Eppendorf tubes. Aliquots of 50 ul of each dilution were then placed and spread out on half a CB A plate. The plates were placed in a 37°C incubator overnight. The following morning the number of survivors was counted, the average between the four sets was taken and multiplied by the appropriate dilution factor, and graphically analysed. Phageat7.3xl06pfu/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 EMRSA-1 in place of EMRSA-16. It was found that 99.98% of the bacteria were killed. Example 3 Example 1 was repeated, using EMRSA-3 in place of EMRSA-16. It was found that over 99.99% of the bacteria were killed. Example 4 Example 1 was repeated, using EMRSA-15 in place of EMRSA-16. It was found 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 found that over 99.99% of the bacteria were killed. Result for Examples 1 to 5 are presented in Figure 1. Example 6 Example 1 was repeated, using lOul each EMRSA-16 and S. epidermidis in place of the 20^1 samples of EMRSA-16. Samples were plated on MBA plates for enumeration. Phage at 7.3xl06pfu/ml SnCe6/phage at 1.5 μg/ml 21 J/cm2 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 strains 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 Φll-SnCe6 Conjugates against Staphvlococcus aureus and a laser light source Bacteriophage 011 was propagated and precipitated as described above for phage 75, except.that S aureus strain 8325-4 was used as the propagating strain. Tin chlorin e6 (SnCe6) was conjugated onto Staphylococcus phage Φ1l using the method described above, achieving bound concentrations of 2.3 and 3.5 ug ml"1 SnCe6 with the phage 011 at 4.7 x 107 pfu.ml"1. These Φ11-SnCe6 conjugates were then incubated with various strains of Staphylococcus aureus and exposed to laser light at 633nm from a 35mW HeNe laser (21 J/cm2) for 5 minutes. The final concentration of conjugated SnCe6 was 1.15 μg ml"1. The results show that Φll-SnCe6 conjugates achieved 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 concentration (1.15μg ml"1) did not achieve any kill. The results are presented in Figure 3. We have also shown that this Φ11-SnCe6 conjugate is effective against a methicillin-resistant strain of the organism (EMRSA-16), achieving 88.11% kill, even though Φ11 only infects this strain under stringent optimal conditions. A range of control experiments such as; light without photosensitiser (L+S-), photosensitiser without light (L-S+), and unconjugated phage at 1 x 107 pfu ml"1 (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 (lOmM) we are now achieving 99.88% kills against S. aureus 8325-4 using Φ1 1-SnCe6 conjugates (1.75μg ml"1). The results are presented in Figure 5. For Figures 3 to 5 the photosensitiser (either SnCe6 or Φ11-SnCe6) was added to give a final concentration of 1.15 ug ml"1 (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 minutes in the case of Figure 5. The effect of varying the light dose on the kills obtained with the SnCe6- phage Φ1l conjugate was investigated. The experiments were carried out as described above except that the bacterial suspensions were exposed to light from the Helium/Neon laser for different periods of time - these were 1, 5, 10, 20 and 30 minutes. In each case, the concentration of the Φ11-SnCe6 conjugate (final concentration equivalent to 3.5 ug ml"1 of SnCe6) was the same. Incubation of the organism with the Φ11-SnCe6 conjugate for upto 60 minutes in the dark had no significant effect on the viable count. However, significant reductions in the viable count were obtained when the suspensions were exposed to laser light in the presence of theΦ11-SnCe6 conjugate - greater kills were obtained with the longer exposure times. Using an exposure time of 30 minutes, a reduction in the viable count of approximately 99.9999% was obtained. Φ11-SnCe6 was used to give a final concentration of 3.5 μg ml"1 (with respect to SnCe6). The light source was a 35 mW Helium/Neon laser and irradiation (when used) was for 1, 5,10,20 or 30 minutes. The results are presented in Figure 6. In Figures 3 to 6 SnCe6 = tin chlorin e6 Φ11-SnCe6 = tin chlorin e6 conjugated to bacteriophage Φ11 PBS = Phosphate buffered saline L+S+ = bacteria irradiated in the presence of conjugate L+S- = bacteria irradiated in the absence of conjugate L-S+ = bacteria exposed to conjugate in the absence of light L-S- = bacteria exposed neither to light nor conjugate Example 8 Lethal Photosensitisation of Staphylcoccus aureus using a phage 75-tin (IV) chlorin e6 conjugate and a white light source Bacterial strains: S. aureus 8325-4 EMRSA-16 Light 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 directed onto a 96 well plate at a distance of 5 cm. A square of 4-wells is placed at the centre of the light source. Approx light intensity = 44,000 lux or 470^W/nm Phage 75 was conjugated to SnCe6 as described above. Phages were used at a concentration of 1 x 107 pfu/ml. Ovemight cultures of S. aureus grown in nutrient broth were centrifuged, resuspended in PBS and adjusted to an OD of 0.05 at 600nm (approximately 4 x 107 cfu/ml) 50ul of bacterial culture was aliquoted into a 96-well plate and 50μl of the one of the following solutions added to the wells: 1) 3.5μg/ml SnCe6-phage 75 (final concentration 1.75μg/ml, 1 xl06pfu/well) in PBS 2) 1.75μg/ml SnCe6-phage 75 (final concentration 0.875ug/ml, 5 xlO5 pfu/well) in PBS 3) 3.5μg/ml SnCe6 in PBS (final concentration 1.75μg/ml) 4) 1.75μg/ml SnCe6 in PBS (final concentration 0.875μg/ml) 5) PBS 6) Phage 75 at a concentration of 5 x 10s or 1 xlO6 pfu/well in PBS Wells were either exposed 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 each well, serially diluted and spread onto Columbia blood agar. Agar plates were incubated overnight at 37°C and counted the next day. Results (Table Removed) this is calculated compared to bacteria incubated with PBS and kept in the dark All results are the average of replicate experiments. Controls included bacteria incubated with SnCe6, phage 75-SnCe6 and phage 75 without exposure to white light. Phage 75 was also exposed to white light. All controls had bacterial counts which were not significantly different to the control suspension which had no photosensitiser added and was not irradiated. Example 9 Further tests were carried out on S. aureus strains Mu3, Mu50 and MW2. To suspensions of vancomycin-resistant strains of Staphylococcus aureus (Mu3 and Mu50) or a community-acquired strain of MRSA (MW2), saline, phage 75, SnCe6 or phage 75-SnCe6 was added and samples exposed to light from a 35 mW Helium/Neon laser. The concentration of SnCe6 used was 1.5 ug/ml, the phage concentration was 5.1 x 107 plaque-forming units/ml and the light energy dose was 21 J/cm2. The numbers above the bars represent the % kill of the organism relative to the sample to which saline only was added. The results are presented in Figure 7. Example 10 Lethal photosensitization of Streptococcus pvogenes using tin cblorin e6 (SnCe6). Streptococcus pyogenes ATCC 12202 was grown in Brain Heart Infusion broth at 37°C in an atmosphere consisting of 5%C02 in air. The cells were harvested by centrifugation and re-suspended in phosphate buffered saline (PBS) and diluted to Ix 107cfu/ml in PBS. 20μl samples of the diluted bacterial suspension were then placed into wells of a 96-well plate, together with a magnetic stirrer bar. 100μl of different concentrations (1-50μg/ml) of the SnCe6 in PBS was added to the bacterial suspensions. Controls were performed with 100 μ1 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 Helium/Neon laser emitting light with a wavelength of 633nm for 10 min, with stirring, corresponding to an energy density of 42 J/cm2. Aluminium foil was placed in the surrounding wells to allow any escaping laser light to be reflected back into the target well. Control wells were not irradiated with laser light. After exposure to the laser light, 100 nl samples were immediately taken from each well and serially diluted, from 10'1 to 10"5, in 1 ml TSY in 1.5 ml Eppendorf tubes. Duplicate 50 μl 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 and 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 irradiation of the organism with laser light in the absence of the photosensitiser. However, irradiation of the organism in the presence of SnCe6 resulted in a concentration-dependent decrease in the viable count. A 99.9997% kill of the organism was obtained using a photosensitiser concentration of 50 μ/ml. The results are presented in Figure 8. In Figure 8 L+ (open bars) = cultures irradiated with laser light in the absence of SnCe6 as well as in the presence of various concentrations of the photosensitiser; L - (shaded bars) = cultures incubated in the dark in the absence of SnCe6 as well as in the presence of various concentrations of the photosensitiser. Example 11 Lethal photosensitization of Propionibacterium acnes using tin chlorin e6 (SnCefl. Propionibacterium acnes ATCC 29399 was grown in pre-reduced Brain Heart Infusion broth at 37°C in an anaerobic atmosphere. The cells were harvested by centrifugation and re-suspended in phosphate buffered saline (PBS) and diluted to 1xl08cfu/ml in PBS. 20 \i\ samples of the diluted bacterial suspension were then placed into wells of a 96-well plate, together with a magnetic stirrer bar. 100 ul of different concentrations (1 - 50μg/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 Helium/Neon laser emitting light with a wavelength of 633nm for 10 min, with stirring, corresponding to an energy density of 42 J/cm2. Aluminium foil was placed in the surrounding wells to allow any escaping laser light to be reflected back into the target well. Control wells were not irradiated with laser light. After exposure to the laser light, 100 μl samples were immediately taken from each well and serially diluted, from 10'1 to 10'5, in 1 ml of pre-reduced TSY in 1.5 ml Eppendorf tubes. Duplicate 50 ul aliquots of each dilution were then spread out on half a CBA plate. The plates were incubated anaerobically at 37°C and 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 irradiation of the organism with laser light in the absence of the photosensitiser. However, irradiation of the organism in the presence of SnCe6 resulted in a concentration-dependent decrease in the viable count. A 100% kill of the organism was obtained using a photosensitiser concentration of 50 μg/ml. The results 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 the photosensitiser; L - (shaded bars) = cultures incubated in the dark in the absence of SnCe6 as well as in the presence of various concentrations of the photosensitiser. Example 12 Preparation of conjugate of TBO and bacteriophage 1mg of toluidine blue 0 (TBO) was dissolved in 800 ul of activation buffer (0.1M MES, 0.5M NaCl pH5.5) together with 0.4mg EDC and 0.6mg of S-NHS and 200 ul of phage (5 x 107pfu/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 proceed for a further 2 to 4 hours after which time the reaction was quenched by adding hydroxylamine to a final concentration of lOmM. The TBO-phage conjugate was separated from free TBO by two rounds of phage precipitation followed by dialysis against PBS. I/We claim: 1. A composition comprising a conjugate of a photosensitiser selected from a group consisting of porphyrins, phthalocyanines, chlorins, bacteriochlorins, phenothiaziniums, phenazines, acridines, texaphyrins, anthracyclins, pheophorbides, sapphyrins, fullerene, halogenated xanthenes, perylenequinonoid pigments, gilvocarcins, terthiophenes, benzophenanthridines, psoralens and riboflavin and a staphylococcal bacteriophage, wherein the photosensitiser is covalently linked to the bacteriophage, the concentration of the photosensitiser is from 0.01 to 200 µg/ml, and the concentration of the bacteriophage is from 1x105 to 1xl010 pfu/ml. 2. The composition as claimed in claim 1, wherein the photosensitiser is tin (IV) chlorin e6 (SnCe6). 3. The composition as claimed in claim 1 or claim 2, wherein the bacteriophage is chosen from phage 53,75, 79,80, 83, Φ11, Φ 12, Φ13, Φ147, ΦMR11, 48,71, Φ 812, SK311,Φ 131, SB-land U16. 4. The composition as claimed in claim 3, wherein the bacteriophage is phage 75 or phage Φ 11. 5. The composition as claimed in any of the preceding claims, which optionally comprises a source of Ca2+ ions, preferably calcium chloride. 6. The composition as claimed in any of claims 1 to 5, in the form of a solution in a pharmaceutically acceptable carrier. 7. The composition as claimed in any of claims 1 to 6, wherein the composition optionally comprises one or more of a buffer, salt, antioxidant, preservative, gelling agent or remineralisation agent.

Full Text

USE OF PHOTOSENSITISATION
The present invention relates to a composition comprising a conjugate of a
photosensitiser and a bacteriophage, particularly a staphylococcal bacteriophage,
known as a staphylophage. The invention also relates to the use of the conjugate in a
method of photodynamic therapy for infectious diseases.
Background
The use of antimicrobial agents to counter bacterial infections is becoming
increasingly ineffective, due to the rapid emergence of antibiotic resistance amongst
many species of pathogenic bacteria. One such pathogen is Staphylococcus aureus (S.
aureus), which characteristically causes skin infections such as boils, carbuncles 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 staphylococci, Staphylococcus aureus, streptococci,
Corynebacterium spp., E. coli, Klebsiella aerogenes, Klebsiella pneumoniae,
Enterobacter aerogenes, Propionibacterium acnes, Bacteroides spp., Pseudomonas
aeruginosa and Peptostreptococcus spp. Increasingly, these bacteria are showing
resistance to antibiotic treatment.
In particular, resistant strains of 5. aureus have emerged. Methicillin-resistant
S. aureus (MRSA) was first reported in 1961 (Jevons, M. (1961) British Medical
Journal, 1, 124-5), and these strains are now a major cause of hospital-acquired
infection throughout the world, as well as being prevalent in many nursing and
residential homes. This poses an alarming challenge to healthcare, causing significant
infection and morbidity of hundreds of patients in the UK each year (Ayliffe et al, J
Hosp Infect (1988), 39, 253-90).
Since the first report of MRS A, these organisms have demonstrated resistance
to a wide variety of antimicrobials including erythromycin, aminoglycosides,
tetracyclines, trimethoprim, sulphonamides and chloramphenicol. MRSA strains
have developed that are only susceptible to a single class of clinically-available
antibiotics: the glycopeptides such as vancomycin and teicoplanin. However,
resistance is developing even to these, as strains tolerant to vancomycin have now
been reported (Hiramatsu, K. (1998) American Journal of Medicine, 104, 7S - 10S).
These strains are variously known as VRS A (Vancomycin resistant Staphylococcus
aureus) and hetero-VRSA (resistant strains arising from exposure to high levels of
vancomycin). At present, the management of patients with MRSA infections usually
involves the administration of antimicrobial agents and again, there is evidence of the
development of resistance to many of the agents used.
Due to the emergence of strains which are resistant to virtually all currentlyavailable
antimicrobials, MRSA is now a serious threat to health. The term MRSA
itself now more accurately applies to methicillin and multiple antimicrobial-resistant
S. aureus.
Certain strains of MRSA have been found to spread rapidly not only within
hospitals, but also between them. These strains have been termed epidemic MRSA
(EMRSA). Since the first EMRSA strain (EMRSA-1) was reported in 1981, 17
distinct EMRSA strains have been identified, all of which are resistant to a number
of antimicrobials. Recently, the two most prevalent strains have been EMRSA-15
and -16, which account for 60-70% of the 30000 MRSA isolates reported
(Livermore, D (2000) Int. J. Antimicrobial Agents, 16, S3 - S10). Importantly,
strains of MRSA, (known as community-acquired MRSA (CA-MRSA)) have also
started to spread in the community, ie. amongst non-hospitalised individuals.
It is clear from the above that alternative methods of countering bacterial
infection, particularly infection with MRSA are urgently required.
One approach has been to employ a light-activated agent to achieve lethal
photosensitization of the organism. This involves treating the organism with a lightactivatable
chemical (photosensitiser) which, upon irradiation with light of a suitable
wavelength, generates cytotoxic species, resulting in bacteriolysis. This technique has
been used to achieve killing of a wide range of bacteria, including S. aureus and
MRSA strains, in vitro using toluidine blue 0 (TBO) and aluminium disulphonated
phthalocyanine (ALPcS2) as photosensitisers. Neither photosensitiser nor laser light
alone exerted a bacteriocidal effect (Wilson et a/, (1994) J Antimicrob Chemother
33, 619-24). In a subsequent study, 16 strains of EMRSA were found to be
susceptible to killing by low doses of red light (674 nm) in the presence of AlPcS2
(Griffiths et al, (1997) J Antimicrob Chemother, 40, 873-6). At higher light doses,
100 % killing was achieved.
Photodynamic therapy (PDT) is the application of such an approach to the
treatment of disease. It is an established procedure in the treatment of carcinoma and
forms the basis of a means of sterilising blood products. It has only been more
recently that the application of PDT to the treatment of infectious diseases has been
evaluated. For example, haematoporphyrins in conjunction with an argon laser have
been used to treat post-neurosurgical infections and brain abscesses (Lombard et al,
(1985), Photodynamic Therapy of Tumours and other Diseases, Ed. Jori & Perria).
One potential problem associated with PDT of infectious diseases is its lack
of specificity. Hence, if the photosensitiser binds to, or is taken up by, a host cell, as
well as the target organism, then subsequent irradiation may also lead to the death of
the host cell. A way to overcome this is by the use of targeting compounds: that is,
any compound that is capable of specifically binding to the surface of the pathogen.
Several targeting compounds have previously been shown to be successful in
eliminating specific strains of bacteria when they were conjugated to a
photosensitiser. For example, immunoglobulin G (IgG) has been used to target S.
aureus Protein A (Gross et al (1997), Photochemistry and Photobiology, 66, 872-8),
monoclonal antibody against Porphyromonas gingivalis lipopolysaccharide (Bhatti et
al (2000), Antimicrobial Agents and Chemotherapy, 44, 2615-8) and poIy-L-lysine
peptides against P. gingivalis and Actinomyces viscosus (Soukos et al (1998),
Antimicrobial Agents and Chemotherapy, 42, 2595-2601). A monoclonal antibody
conjugated via dextran chains to the photosensitiser 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 Acad Sci, 618, 383-393).
The present inventors have used IgG conjugated to SnCe6 to target EMRSA
strains 1, 3, 15 arid 16 (Embleton et al (2002), J Antimicrob Chemother, 50, 857-
864), achieving higher levels of killing than the photosensitiser alone, and selectively
killing the EMRSA 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. Hence alternative targeting agents that can target any S. aureus strain are
desirable.
Bacteriophage are viruses that infect certain bacteria, often causing them to
lyse and hence effecting cell death. They have been proposed as antibacterial agents
in their own right. However, one of the problems with using staphylococcal
bacteriophage (termed staphylophage) in the treatment of S. aureus disease is their
restricted host range. Although there are polyvalent staphylophage which can lyse
many S. aureus strains, other strains are resistant and hence bacteriophages alone
could not provide an effective method of killing 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 have
now found that some bacteriophage can serve as an effective, targeted delivery
system for photosensitisers.
The present inventors have found that when a bacteriophage is linked to a
photosensitiser, the photosensitiser-bacteriophage conjugate formed is highly
effective in killing bacteria when irradiated with light of a suitable wavelength.
Bacteriophage-photosensitiser conjugates could be used to treat or prevent a
broad range of bacterial skin and wound infections. The most frequently isolated
organisms from skin and wound infections are: coagulase-negative staphylococci, S.
aureus, streptococci, e.g. Streptoccocus pyogenes, Corynebacterium spp., E coli,
Klebsiella aerogenes, Klebsiella pneumoniae, Enterobacter aerogenes,
Propionibacterium acnes, Bacteroides spp., Pseudomonas aeruginosa and
Peptostreptococcus spp..
In particular, conjugates of photosensitiser and staphylophage can be used in
a method of photodynamic therapy against strains ofStaphylococci spp, particularly
against MRSA, EMRSA, VRSA, hetero-VRSA and CA-MRSA.
The invention provides a composition comprising a photosensitizing
compound (photosensitiser) linked to a bacteriophage to form a photosensitiserbacteriophage
conjugate. The bacteriophage may be a staphylococcal phage, and is
preferably a staphylophage that can bind to Staphylococcus 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 covalent
linkage. The photosensitiser and/or the bacteriophage contain or may be modified to
contain groups which can be covalently crosslinked using chemical or photoreactive
reagents, to produce crosslinked bonds, for example thiol-thiol crosslinking, amineamine
crosslinking, amine-thiol crosslinking, amine-carboxylic acid crosslinking,
thiol-carboxylic acid crosslinking, hydroxyl-carboxylic acid crosslinking, hydroxylthiol
crosslinking and combinations thereof.
The photosensitiser is suitably chosen from porphyrins (e.g.
haematoporphyrin derivatives, deuteroporphyrin), phthalocyanines (e.g. zinc, silicon
and aluminium phthalocyanines), chlorins (e.g. tin chlorin e6, poly-lysine derivatives
of tin chlorin e6, m-tetrahydroxyphenyl chlorin, benzoporphyrin derivatives, tin
etiopurpurin), bacteriochlorins, phenothiaziniums (e.g. toluidine blue, methylene
blue, dimethylmethylene blue), phenazines (e.g. neutral red), acridines (e.g.
acriflavine, proflavin, acridine orange, aminacrine), texaphyrins, cyanines (e.g.
merocyanine 540), anthracyclins (e.g. adriamycin and epirubicin), pheophorbides,
sapphyrins, fullerene, halogenated xanthenes (e.g. rose bengal), perylenequinonoid
pigments (e.g. hypericin, hypocrellin), gilvocarcins, terthiophenes,
benzophenanthridines, psoralens and riboflavin.
The invention is directed to killing bacteria using the above-described
conjugates. The bacteriophage used in the conjugate may 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 embodiment, the infecting
bacterium is MRSA, EMRSA, VRSA, hetero-VRSA or CA-MRSA and the
conjugate includes the staphylococcal phage 75 or phageΦl 1.
Table 1 below shows some examples of bacteria-bacteriophage pairs,
although many more examples exist. Further novel bacteriophages can be isolated
and/or adapted to the target bacteria. The specificity of the treatment can be modified
as required by using monovalent bacteriophages, polyvalent bacteriophages or
combinations of monovalent bacteriophages or combinations of monovalent and
polyvalent bacteriophages.
(Table Removed)
The composition of the invention suitably comprises at least 0.010 g/ml, of
the photosensitiser, preferably at least 0.02μg/ml, more preferably at least 0.05μg/ml
upto 200 μg/ml, preferably up to 100μg/ml, more preferably up to 50 μg/ml. The
amount of the bacteriophage in the composition is suitably from 1x105 to lxI010pfu,
preferably from 1x1O6 to 1Ix1O9 pfu, more preferably from 1x106 to 1x108 pfu.
The composition of the invention may further comprise a source of divalent
ions, e.g. Ca+ or Mg2*, preferably Ca2+. Examples include calcium chloride, calcium
carbonate and magnesium chloride. The ions are suitably present in an amount of
from 5 to 200mM, preferably from 5 to 15 mM, more preferably about 1 OmM.
The composition may further comprise one or more ingredients chosen from
buffers, salts for adjusting the tonicity, antioxidants, preservatives, gelling agents 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 invention such
that any bacteria in the area bind to the photosensitiser-bacteriophage
conjugate; and
(b) irradiating the area with light at a wavelength absorbed by the
photosensitiser.
Suitably the bacteria are as set out above in Table 1, preferably
Staphylococcus aureus, more preferably MRSA, EMRSA, VRSA, hetero-VRSA 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 the photosensitiser and to have sufficient
energy to activate the photosensitiser. The source of light may be any device or
biological system able to generate monochromatic or polychromatic light. Examples
include laser, light emitting diode, arc lamp, halogen lamp, incandescent lamp or an
emitter of bioluminescence or chemiluminescence. In certain circumstances, 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 laser may
have a power of from 1 to lOOmW and a beam diameter of from 1 to 10mm. The
light dose for laser irradiation is suitably from 5 to 333 J cm"2, preferably from 5 to
30 J cm"2 for laser light. For white light irradiation, a suitable dose is from 0.01 to
100 kJ/cm2, preferably from 0.1 to 20 kJc/m2, more preferably from 3 to 10 kJ/cm2.
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 (628nm, 10W output)
Tunable solid state laser (532-1060nm), including Sd:YAG
Light emitting diode (LED) (400-800nm)
Diode laser (630-850nm, 25W output), eg. gallium selenium arsenide
Tungsten filament lamp
Halogen cold light source
Fluorescent lamp.
In the method of the invention, the composition is suitably in the form of a
solution or a suspension in a pharmaceutically acceptable aqueous carrier, but may be
in the form of a solid such as a powder or a gel, an ointment or a cream. The
composition may be applied to the infected area by painting, spreading, spraying or
any other conventional technique.
The invention further provides the use of the composition for treatment of the
human or animal body. Suitably, the composition is provided for use in the treatment
of conditions resulting from bacterial infection, particularly by staphylococci, more
particularly by MRSA, EMRSA, VRSA, hetero-VRSA or CA-MRSA.
The invention may be used to treat bacterial infection, particularly by
staphylococcal bacteria, more particularly by MRSA, EMRSA, VRSA, hetero-VRSA
or CA-MRSA to treat or prevent skin infections such as boils, carbuncles, mastitis
and impetigo, to treat or prevent infections of acne, bums or wounds, or to treat or
prevent endocarditis, osteomyelitis, meningitis and pneumonia, arising as a result of
bacterial infection, to treat or prevent infections arising from the use of catheters,
implants or other medical devices, or to prevent infection following an operation,
such as a Caesarean section.
The invention may also be used in the prevention of carriage of the bacteria
by carriers who themselves show few, if any, symptoms.
Description of the Figures
Figure 1 shows the effect of aphage 75-SnCe6 conjugate on different EMRSA
strains.
Figure 2 shows the effects of conjugate, no conjugate, photosensitiser only or phage
only and presence or absence of irradiation on EMRSA-16 and S. epidermidis.
Figures 3 to 5 show the effect of the invention on EMRSA-16 and S. aureus 8325-4,
varying the light dose.
Figure 6 shows the effect of light dose using a fixed concentration of Φl1!-SnCe6
conjugate on EMRSA-16.
Figure 7 shows the effect of the invention on strains of VRSA (Mu3), hetero-VRSA
(Mu50)and CA-MRSA (MW2).
Figure 8 shows the effect of the invention on Streptococcus pyogenes.
Figure 9 shows the effect of the invention on Propionibacterium acnes.
EXAMPLES
Materials and Methods
The following 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/1 NaCl) to 1 litre of deionised,
distilled water. After mixing, the medium was autoclaved at 121 °C for 15 min.
Tryptone Soya Yeast Broth (TSY)
One litre of medium was made by adding 39g of Tryptone Soya Broth
(Oxoid) (17.0 g/1 pancreatic digest of casein, 3.0 g/1 papaic digest of soybean meal,
2.5 g/l glucose, 2.5 g/1 di-basic potassium phosphate, 5.0 g/1 NaCl) and 0.5% of yeast
extract (9.8 g/1 total nitrogen, 5.1 g/1 amino nitrogen, 0.3 g/1 NaCl) to 1 litre of
deionised, distilled water. After mixing, the medium was autoclaved at 121 °C for 15
min.
Nutrient Broth 2 Top Agar
0.35 % (w/v) of Agar Bacteriological (Agar No. 1, Oxoid) was added to NB2
medium. After mixing, the medium was autoclaved at 121 °C for 15 min.
Nutrient Broth 2 Bottom Agar
0.7% (w/v) of Agar Bacteriological was added to NB2 medium. After
autoclaving, 10 mM of CaCl2 was added (10ml 1M CaCl2 in 1 litre of NB2).
Columbia Blood Agar (CBA)
37.1g of Columbia Agar Base (Oxoid) (23.0 g/1 special peptone, 1.0 g/1
starch, 5.0 g/1 NaCl, 10.0 g/1 agar) was added to 1 litre of deionised, distilled water.
After autoclaving, the liquid agar was allowed to cool at room temperature until cool
enough to handle. 5% (v/v) defibrinated horse blood (E & O Laboratories, Scotland)
was then added.
Mannitol Salt Agar (MSA)
111g of Mannitol Salt Agar (Oxoid) (75.0 g/1 NaCl, 10.0 g/1 mannitol, 1.0 g/1
Lab-lemco powder, 10.0 g/1 peptone, 0.025 g/1 phenol red, 15.0 g/1 agar) was added
to 1 litre of deionised, distilled water.
All mixtures were autoclaved at 121 °C for 15 min. The liquid agar was then
poured into plates, covered and allowed to cool overnight.
Target organisms
The organisms used in the examples were as follows, given as names and
NCTC (National Collection of Type Cultures, UK) or ATCC (American Type
Culture Collection, USA) numbers:
Epidemic methicillin-resistant S. aureus (EMRSA)-l (NCTC 11939)
EMRSA-3(NCTC13130)
EMRSA-15(NCTC13142)
EMRSA-16(NCTC13143)
Mu3 (ATCC 700698), is a methicillin-resistant Staphylococcus aureus (MRSA)
strain with heterogeneous resistance to vancomycin, designated heterogeneously
vancomycin-resistant Staphylococcus aureus (hetero-VRSA) (Hanaki et al (1998). J.
Antimicrob. Chemother. 42:199-209)
Mu50 is the archetypal VRSA strain (Hiramatsu et al (1997). J. Antimicrob.
Chemother. 40:135-136)
MW2 is a Community-acquired MRSA 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 genome sequence of the prototypic CA-MRSA
strain, MW2, has revealed the presence of additional virulence factors not commonly
present in other S. aureus strains (Baba et al (2002), Lancet. 25;359(9320):1819-27).
Staphylococcus epidermidis (NCTC 11047)
Streptococcus pyogenes (ATCC 12202)
Propionibacterium acnes (ATCC 29399)
Staphyloccus aureus 8324-5 (Novick (1967) Virology 33; 156-166).
All were maintained by weekly subculture on CB A.
Bacteriopbage
Phage 75 (Public Health Laboratory Service, UK) is a serogroup F
staphylococcal phage, capable of infecting EMRSA-16, EMRSA-3 and weakly
infectingEMRSA-15.
Bacteriophage 411 (landolo et al, (2002), Gene 289 (1-2); 109-118) is a
temperate bacteriophage of serological group B.Φl 1 is a transducing phage with a
low lysogenisation frequency. It infects S.aureus lytic group III strains which include
many human and animal pathogens.
Bacteriophage propagation
Mid-exponential EMRSA-16 (300ul) was added to 15ml Falcon tubes.
Approximately 10s pfu of phage 75 were added to the tubes and allowed to incubate
at room temperature for 30 min to allow the phage to infect the bacteria. 9ml of
cooled molten top NB2 agar (with lOmM CaC^), was added to the tubes, and the
mixture poured onto undried NB2 base agar plates. The plates were left to incubate
at 37 °C overnight.
The next morning 1 ml of NB2 with 10 mM CaCl2 was added to each plate,
and the top agar with the liquid medium was scraped into a small centrifuge tube.
The collected agar was then spun in a centrifuge at 15000 rpm for 15 min at 4°C.
The supernatant was collected and passed through a 0.45 μm (Nalgene) filter to
remove any bacterial cells. The resulting solution of phage 75 was stored at 4°C.
Bacteriophage precipitation
Phage precipitation was carried out to purify the phage 75 from the NB2
medium after propagation. To 5ml of phage 75 in NB2, 1.3 ml of 5M NaCl (1M
final concentration) and 0.2 ml Ix phosphate buffered saline (PBS) (8.0g/l NaCl,
0.2g/l KC1, 1.15 g/1 Na2HPO4t 0.2g/l KH2PO4) were added, and 20% PEG
(polyethylene glycol 8000, Sigma) was added to the solution and stirred slowly
overnight until completely dissolved. The solution was then placed on ice overnight
and the next morning the solution was centrifuged at 8OOOrpm for 20 min at 4°C.
The supernatant was removed and the remaining pellet was resuspended in 2.5ml 1x
PBS, and filtered through a 0.45 urn filter.
Photosensitiser
The photosensitiser used was tin (IV) chlorin e6 (SnCe6) (Frontier Scientific,
Lancashire, UK), which is photoactivatable at 633 nm.
Preparation of conjugate
2mg of SnCe6 was dissolved with stirring in 800μl of activation buffer (0.1
M MES (2-(N-morpholino(ethanesulphonic acid) (Sigma)), 0.5 M NaCl, pH 5.5). An
EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiirnidehydrochloride) (Sigma)
solution (4mg in 1 ml activation buffer) and a S-NHS (Nhydroxysulphosuccinimide)
(Fluka) solution (2.7 mg in 250 ul activation buffer)
were made.
To the dissolved SnCe6, 200ul of dissolved EDC and S-NHS were added,
and the mixture was left for 1 to 4 hours at room temperature with stirring to provide
a stable amine-reactive intermediate. The mixture was covered in aluminium foil as
SnCe6 is a light sensitive reagent. The reaction was quenched by adding 1.4μl (3-
mercaptoethanol (Sigma).
Experiments were carried out using the reagents at a molar ratio of
SnCe6: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 M NaOH. 1.5ml of phage 75 was then added to the amine-reactive solution to
allow the amino groups on the phage to react with the carboxyl groups of the SnCe6,
and then mixed for 4 to 16 hours. The reaction was quenched with 2.5ul
ethanolamine (Sigma).
The photosensitiser-phage conjugate (PS-phage) was separated from free PS
after conjugation by precipitating the PS-phage twice, as described above in
Bacteriophage Precipitation. The PS-phage was then dialysed against PBS.
In the examples below, the concentration of phage 75 is 7.3x106 pfu/ml and
the concentration of SnCe6/bacteriophage-SnCe6 is 1.5 μg/ml.
Laser
The laser used was a Model 127 Stabilite helium-neon (He/Ne) laser (Spectra
Physics, USA) with a power output of 35 mW. The laser emitted radiation in a
collimated beam, diameter 1.25 mm, with a wavelength of 633nm.
Example 1
A culture of EMRSA-16 in the mid-exponential growth phase was diluted to
Ixl07cfu/ml. 20μl samples of the diluted bacteria were then placed into wells of a
96-well plate (Nunc), together with a magnetic stirrer bar.
100 ul of the phage 75-SnCe6 conjugate prepared above and calcium chloride
(CaCl2) to a final concentration of 10 mM was added to the bacteria. The contents of
the wells were left to incubate at room temperature for 5 min, with stirring. Controls
were performed with 100 ul IxPBS added to the bacteria and used as a reference for
experimental samples. The experiment was carried out in duplicate.
After incubation, the contents of the well were directly exposed to the laser
light for 5 min, with stirring, corresponding to an energy density of 21 J/cm2.
Aluminium foil was placed in the surrounding wells to allow any escaping laser light
to be reflected back into the target well. Controls were performed with no laser
irradiation.
After exposure to the laser, 100 ul samples were immediately taken from each
well and serially diluted, from" 10' to 10"4, in 1 ml TSY in 1.5 ml Eppendorf tubes.
Aliquots of 50 ul of each dilution were then placed and spread out on half a CB A
plate. The plates were placed in a 37°C incubator overnight. The following morning
the number of survivors was counted, the average between the four sets was taken
and multiplied by the appropriate dilution factor, and graphically analysed.
Phageat7.3xl06pfu/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 EMRSA-1 in place of EMRSA-16. It was
found that 99.98% of the bacteria were killed.
Example 3
Example 1 was repeated, using EMRSA-3 in place of EMRSA-16. It was
found that over 99.99% of the bacteria were killed.
Example 4
Example 1 was repeated, using EMRSA-15 in place of EMRSA-16. It was
found 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
found that over 99.99% of the bacteria were killed.
Result for Examples 1 to 5 are presented in Figure 1.
Example 6
Example 1 was repeated, using lOul each EMRSA-16 and S. epidermidis in
place of the 20^1 samples of EMRSA-16. Samples were plated on MBA plates for
enumeration.
Phage at 7.3xl06pfu/ml
SnCe6/phage at 1.5 μg/ml
21 J/cm2 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 strains 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 Φll-SnCe6 Conjugates against
Staphvlococcus aureus and a laser light source
Bacteriophage 011 was propagated and precipitated as described above for
phage 75, except.that S aureus strain 8325-4 was used as the propagating strain. Tin
chlorin e6 (SnCe6) was conjugated onto Staphylococcus phage Φ1l using the method
described above, achieving bound concentrations of 2.3 and 3.5 ug ml"1 SnCe6 with
the phage 011 at 4.7 x 107 pfu.ml"1. These Φ11-SnCe6 conjugates were then
incubated with various strains of Staphylococcus aureus and exposed to laser light at
633nm from a 35mW HeNe laser (21 J/cm2) for 5 minutes. The final concentration
of conjugated SnCe6 was 1.15 μg ml"1.
The results show that Φll-SnCe6 conjugates achieved 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 concentration (1.15μg ml"1) did
not achieve any kill. The results are presented in Figure 3.
We have also shown that this Φ11-SnCe6 conjugate is effective against a
methicillin-resistant strain of the organism (EMRSA-16), achieving 88.11% kill,
even though Φ11 only infects this strain under stringent optimal conditions. A range
of control experiments such as; light without photosensitiser (L+S-), photosensitiser
without light (L-S+), and unconjugated phage at 1 x 107 pfu ml"1 (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 (lOmM)
we are now achieving 99.88% kills against S. aureus 8325-4 using Φ1 1-SnCe6
conjugates (1.75μg ml"1). The results are presented in Figure 5.
For Figures 3 to 5 the photosensitiser (either SnCe6 or Φ11-SnCe6) was
added to give a final concentration of 1.15 ug ml"1 (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 minutes in the case of Figure 5.
The effect of varying the light dose on the kills obtained with the SnCe6-
phage Φ1l conjugate was investigated. The experiments were carried out as
described above except that the bacterial suspensions were exposed to light from the
Helium/Neon laser for different periods of time - these were 1, 5, 10, 20 and 30
minutes. In each case, the concentration of the Φ11-SnCe6 conjugate (final
concentration equivalent to 3.5 ug ml"1 of SnCe6) was the same.
Incubation of the organism with the Φ11-SnCe6 conjugate for upto 60
minutes in the dark had no significant effect on the viable count. However,
significant reductions in the viable count were obtained when the suspensions were
exposed to laser light in the presence of theΦ11-SnCe6 conjugate - greater kills were
obtained with the longer exposure times. Using an exposure time of 30 minutes, a
reduction in the viable count of approximately 99.9999% was obtained.
Φ11-SnCe6 was used to give a final concentration of 3.5 μg ml"1 (with respect to
SnCe6). The light source was a 35 mW Helium/Neon laser and irradiation (when
used) was for 1, 5,10,20 or 30 minutes. The results are presented in Figure 6.
In Figures 3 to 6
SnCe6 = tin chlorin e6
Φ11-SnCe6 = tin chlorin e6 conjugated to bacteriophage Φ11
PBS = Phosphate buffered saline
L+S+ = bacteria irradiated in the presence of conjugate
L+S- = bacteria irradiated in the absence of conjugate
L-S+ = bacteria exposed to conjugate in the absence of light
L-S- = bacteria exposed neither to light nor conjugate
Example 8
Lethal Photosensitisation of Staphylcoccus aureus using a
phage 75-tin (IV) chlorin e6 conjugate and a white light source
Bacterial strains: S. aureus 8325-4
EMRSA-16
Light 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 directed onto a 96 well
plate at a distance of 5 cm. A square of 4-wells is placed at the centre of the light
source.
Approx light intensity = 44,000 lux or 470^W/nm
Phage 75 was conjugated to SnCe6 as described above. Phages were used at a
concentration of 1 x 107 pfu/ml.
Ovemight cultures of S. aureus grown in nutrient broth were centrifuged,
resuspended in PBS and adjusted to an OD of 0.05 at 600nm (approximately 4 x 107
cfu/ml)
50ul of bacterial culture was aliquoted into a 96-well plate and 50μl of the one of the
following solutions added to the wells:
1) 3.5μg/ml SnCe6-phage 75 (final concentration 1.75μg/ml, 1 xl06pfu/well) in PBS
2) 1.75μg/ml SnCe6-phage 75 (final concentration 0.875ug/ml, 5 xlO5 pfu/well) in
PBS
3) 3.5μg/ml SnCe6 in PBS (final concentration 1.75μg/ml)
4) 1.75μg/ml SnCe6 in PBS (final concentration 0.875μg/ml)
5) PBS
6) Phage 75 at a concentration of 5 x 10s or 1 xlO6 pfu/well in PBS
Wells were either exposed 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 each well, serially
diluted and spread onto Columbia blood agar. Agar plates were incubated overnight
at 37°C and counted the next day.
Results
(Table Removed)
this is calculated compared to bacteria incubated with PBS and kept in the
dark
All results are the average of replicate experiments.
Controls included bacteria incubated with SnCe6, phage 75-SnCe6 and phage 75
without exposure to white light. Phage 75 was also exposed to white light.
All controls had bacterial counts which were not significantly different to the control
suspension which had no photosensitiser added and was not irradiated.
Example 9
Further tests were carried out on S. aureus strains Mu3, Mu50 and MW2. To
suspensions of vancomycin-resistant strains of Staphylococcus aureus (Mu3 and
Mu50) or a community-acquired strain of MRSA (MW2), saline, phage 75, SnCe6 or
phage 75-SnCe6 was added and samples exposed to light from a 35 mW
Helium/Neon laser.
The concentration of SnCe6 used was 1.5 ug/ml, the phage concentration was
5.1 x 107 plaque-forming units/ml and the light energy dose was 21 J/cm2. The
numbers above the bars represent the % kill of the organism relative to the sample to
which saline only was added. The results are presented in Figure 7.
Example 10
Lethal photosensitization of Streptococcus pvogenes using tin cblorin e6 (SnCe6).
Streptococcus pyogenes ATCC 12202 was grown in Brain Heart Infusion
broth at 37°C in an atmosphere consisting of 5%C02 in air. The cells were harvested
by centrifugation and re-suspended in phosphate buffered saline (PBS) and diluted to
Ix 107cfu/ml in PBS. 20μl samples of the diluted bacterial suspension were then
placed into wells of a 96-well plate, together with a magnetic stirrer bar. 100μl of
different concentrations (1-50μg/ml) of the SnCe6 in PBS was added to the
bacterial suspensions. Controls were performed with 100 μ1 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 Helium/Neon laser emitting light with a wavelength of 633nm for 10
min, with stirring, corresponding to an energy density of 42 J/cm2. Aluminium foil
was placed in the surrounding wells to allow any escaping laser light to be reflected
back into the target well. Control wells were not irradiated with laser light.
After exposure to the laser light, 100 nl samples were immediately taken from
each well and serially diluted, from 10'1 to 10"5, in 1 ml TSY in 1.5 ml Eppendorf
tubes. Duplicate 50 μl 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 and 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 irradiation of the
organism with laser light in the absence of the photosensitiser. However, irradiation
of the organism in the presence of SnCe6 resulted in a concentration-dependent
decrease in the viable count. A 99.9997% kill of the organism was obtained using a
photosensitiser concentration of 50 μ/ml. The results are presented in Figure 8. In
Figure 8
L+ (open bars) = cultures irradiated with laser light in the absence of SnCe6
as well as in the presence of various concentrations of the photosensitiser;
L - (shaded bars) = cultures incubated in the dark in the absence of SnCe6 as
well as in the presence of various concentrations of the photosensitiser.
Example 11
Lethal photosensitization of Propionibacterium acnes using tin chlorin e6
(SnCefl.
Propionibacterium acnes ATCC 29399 was grown in pre-reduced Brain
Heart Infusion broth at 37°C in an anaerobic atmosphere. The cells were harvested by
centrifugation and re-suspended in phosphate buffered saline (PBS) and diluted to
1xl08cfu/ml in PBS. 20 \i\ samples of the diluted bacterial suspension were then
placed into wells of a 96-well plate, together with a magnetic stirrer bar. 100 ul of
different concentrations (1 - 50μg/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 Helium/Neon laser emitting light with a wavelength of 633nm for 10
min, with stirring, corresponding to an energy density of 42 J/cm2. Aluminium foil
was placed in the surrounding wells to allow any escaping laser light to be reflected
back into the target well. Control wells were not irradiated with laser light.
After exposure to the laser light, 100 μl samples were immediately taken from
each well and serially diluted, from 10'1 to 10'5, in 1 ml of pre-reduced TSY in 1.5 ml
Eppendorf tubes. Duplicate 50 ul aliquots of each dilution were then spread out on
half a CBA plate. The plates were incubated anaerobically at 37°C and 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 irradiation of the
organism with laser light in the absence of the photosensitiser. However, irradiation
of the organism in the presence of SnCe6 resulted in a concentration-dependent
decrease in the viable count. A 100% kill of the organism was obtained using a
photosensitiser concentration of 50 μg/ml. The results 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 the photosensitiser;
L - (shaded bars) = cultures incubated in the dark in the absence of SnCe6 as
well as in the presence of various concentrations of the photosensitiser.
Example 12
Preparation of conjugate of TBO and bacteriophage
1mg of toluidine blue 0 (TBO) was dissolved in 800 ul of activation buffer
(0.1M MES, 0.5M NaCl pH5.5) together with 0.4mg EDC and 0.6mg of S-NHS and 200
ul of phage (5 x 107pfu/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 proceed for a further 2 to 4 hours after
which time the reaction was quenched by adding hydroxylamine to a final concentration
of lOmM.
The TBO-phage conjugate was separated from free TBO by two rounds of phage
precipitation followed by dialysis against PBS.

I/We claim:
1. A composition comprising a conjugate of a photosensitiser selected from a group consisting of porphyrins, phthalocyanines, chlorins, bacteriochlorins, phenothiaziniums, phenazines, acridines, texaphyrins, anthracyclins, pheophorbides, sapphyrins, fullerene, halogenated xanthenes, perylenequinonoid pigments, gilvocarcins, terthiophenes, benzophenanthridines, psoralens and riboflavin and a staphylococcal bacteriophage, wherein the photosensitiser is covalently linked to the bacteriophage, the concentration of the photosensitiser is from 0.01 to 200 µg/ml, and the concentration of the bacteriophage is from 1x105 to 1xl010 pfu/ml.
2. The composition as claimed in claim 1, wherein the photosensitiser is tin (IV) chlorin e6 (SnCe6).
3. The composition as claimed in claim 1 or claim 2, wherein the bacteriophage is chosen from phage 53,75, 79,80, 83, Φ11, Φ 12, Φ13, Φ147, ΦMR11, 48,71, Φ 812, SK311,Φ 131, SB-land U16.
4. The composition as claimed in claim 3, wherein the bacteriophage is phage 75 or phage Φ 11.
5. The composition as claimed in any of the preceding claims, which optionally comprises a source of Ca2+ ions, preferably calcium chloride.
6. The composition as claimed in any of claims 1 to 5, in the form of a solution in a pharmaceutically acceptable carrier.
7. The composition as claimed in any of claims 1 to 6, wherein the composition optionally comprises one or more of a buffer, salt, antioxidant, preservative, gelling agent or remineralisation agent.

Documents:

1991-delnp-2006-Abstract-(12-11-2010).pdf

1991-delnp-2006-abstract.pdf

1991-DELNP-2006-Assignment-(24-09-2010).pdf

1991-delnp-2006-Claims-(05-05-2011).pdf

1991-delnp-2006-Claims-(11-07-2011).pdf

1991-delnp-2006-Claims-(12-11-2010).pdf

1991-delnp-2006-claims.pdf

1991-delnp-2006-Correspondence Others-(01-07-2011).pdf

1991-DELNP-2006-Correspondence Others-(04-11-2011).pdf

1991-delnp-2006-Correspondence Others-(05-05-2011).pdf

1991-delnp-2006-Correspondence Others-(11-07-2011).pdf

1991-delnp-2006-Correspondence-Others-(12-11-2010).pdf

1991-DELNP-2006-Correspondence-Others-(24-09-2010).pdf

1991-delnp-2006-correspondence-others-1.pdf

1991-delnp-2006-correspondence-others.pdf

1991-delnp-2006-description(complete).pdf

1991-delnp-2006-drawings.pdf

1991-delnp-2006-form-1.pdf

1991-delnp-2006-Form-13-(05-05-2011).pdf

1991-delnp-2006-form-18.pdf

1991-delnp-2006-form-2.pdf

1991-DELNP-2006-Form-3-(24-09-2010).pdf

1991-delnp-2006-form-3.pdf

1991-delnp-2006-form-5.pdf

1991-delnp-2006-GPA-(01-07-2011).pdf

1991-delnp-2006-pct-210.pdf

1991-delnp-2006-pct-304.pdf

1991-delnp-2006-pct-306.pdf

1991-DELNP-2006-Petition 137-(24-09-2010).pdf

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Patent Number

250289

Indian Patent Application Number

1991/DELNP/2006

PG Journal Number

51/2011

Publication Date

23-Dec-2011

Grant Date

21-Dec-2011

Date of Filing

12-Apr-2006

Name of Patentee

UCL BIOMEDICA PLC

Applicant Address

C/O FINANCE DIVISION GOWER STREET LONDON WC1E 6BT UK.

Inventors:

#	Inventor's Name	Inventor's Address
1	WILSON, MICHAEL	University College London, Gower Street, London WC1E 9BT (UK).
2	NAIR, SEAN	University College London, Gower Street, London WC1E 9BT (UK).

PCT International Classification Number

A61K 41/00

PCT International Application Number

PCT/GB2004/004305

PCT International Filing date

2004-10-08

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	0323699.9	2003-10-09	U.K.