BACKGROUND TO THE INVENTION It is known that certain organic compounds ("photosensitisers") can induce cell death by absorption of light in the presence of oxygen. The cytotoxic effect involves Type I and/or Type II photooxidation. Such photosensitisers find use in the treatment of cancer and other diseases or infections with light (photodynamic therapy) and in the sterilisation (including disinfection) of surfaces and fluids by the light-induced destruction of microbes. In this context, the term sterilisation is taken to mean the reduction or elimination of microbes in a particular situation.

It is also known that certain coloured phenothiazinium compounds, (e. g. methylene blue) can take part in Type I and Type II photooxidation processes, but compounds of this type thus far have proved unsuitable or of low efficacy as sensitisers for photodynamic therapy, or have shown low photochemical antimicrobial activity.

For application in PDT, a good sensitiser must have at least some and preferably all of the following properties. Most importantly, it should cause the destruction of target cells (for example tumour cells or bacterial cells) efficiently on exposure to light (preferably wavelengths ca. 600-800 nm). The PDT treatment using the photosensitiser should show a high degree of selectivity between target and normal

tissues. The sensitiser should have relatively little dark toxicity and it should cause little or no skin photosensitivity in the patient.

For applications in photosterilisation, a good sensitiser must show a strong phototoxic effect in a wide range of microrganisms, ideally using ambient light, and should not photobleach readily.

In oncology, several different types of photosensitiser have been used to treat both solid tumours and thin tumours of hollow organs such as the oesophagus and bladder.

However, the use of these photosensitisers has been restricted partly because of lack of selectivity between tumour and healthy tissue and partly because of the prolonged skin photosensitivity which can be caused. There is a need for new photosensitisers which cause little or no skin photosensitivity and which selectively destroy tumour cells.

Although PDT has previously been used in the treatment of tumours, it has not yet been used against infections caused by bacteria and other microorganisms. The use of antibiotics to treat bacterial infections is becoming challenging due to the increasing resistance of many bacterial species to commonly used antibiotics, such as tetracyclines and beta-lactams. Hospital-acquired antibiotic resistant infections such as MRSA are especially problematic. Photodynamic antibacterial treatment is a promising alternative to antibiotics for local treatment.

When developing antibacterial agents a major problem which must be overcome is the uptake of the drug into the bacterial cell. Gram negative and Gram positive bacteria differ in the composition of their outer surface and respond differently to antimicrobial agents, especially in terms of uptake. Due to the high negatively charged surface of Gram negative bacteria they are relatively impermeable to neutral or anionic drugs, including most commonly used photosensitisers. Development of antimicrobial photosensitisers which are effective against Gram negative bacteria, as

well as Gram positive bacteria would be highly beneficial to replace commonly used antibiotics and drugs which are becoming increasingly ineffective due to resistance.

A number of different types of photosensitiser have been investigated in bacteria. These include phenothiazinium compounds, phthalocyanines, chlorins and naturally occurring photosensitisers. For uptake into Gram negative bacteria, it is accepted that the cationic derivatives are the most effective.

Phenothiazinium compounds are blue dyes with maximum absorption at wavelengths between 600-700 nm. They have been studied for their non-photodynamic antibacterial properties but few apart from methylene blue and toluidine blue have been investigated photodynamically.

Wainwright et al (1998) investigated the effect of a series of phenothiazinium methylene blue derivatives in tumour cell lines and bacteria. New methylene blue (NMB) and di methyl methylene blue (DMMB) were effective at inactivating MRSA and were shown to be more effective photosensitisers than methylene blue when acting on pigmented melanoma cell lines. Wagner et al (1998) studied these dyes and in addition a hydrophobic derivative for the inactivation of enveloped viruses.

The precise mode of antibacterial action of methylene blue is unknown, but one hypothesis is that because of its stereochemistry it can intercalate into DNA, and that photodynamic action causes DNA damage. Methylene blue itself has been shown to be ineffective as an anti-tumour agent. In addition, methylene blue is known to be susceptible to photobleaching, which could be a serious disadvantage in some applications. Because of the recognised limitations of methylene blue, both anti- tumour PDT and antimicrobial PDT would benefit from development of new phenothiazine-based photosensitisers.

STATEMENTS OF THE INVENTION According the present invention there is provided a phenothiazinium compound of Formula (I) :

wherein: A and B are each independently in which Z is CH2, O, S, SO2, NH, NCH3, NC2H5, NCH2CH2OH, or NCOCH3 and R1 and R2 are each independently linear or branched CnH2nY, where n is 1-6, Y is H, F, Cl, Br, I, OH, OCH3, OC2H5, OC3H7, CN or OCOCH3, and where XP-is a counteranion and P is 1,2 or 3 but not including a compound where A and B are both either-N (CH3) 2 or -N (CH2CH3) 2.

Preferably the counteranion is selected from any of Cl, Br, I-, F-, N03', HSO4-, CH3CO2-, or is a dianion, namely, S042-or HP042-, or trianion, namely PO43-.

Preferably A and B are the same and both R1 and R2 are n-propyl, n-butyl or n-pentyl.

The compound may be used against microorganisms. Preferably the compound is used against against bacteria. More preferably the compound is used against antibiotic resistant bacteria.

In one embodiment of the invention the compound is for use as a PDT agent or a photodiagnostic agent.

Furthermore, the present invention provides a conjugate or composite formed between a compound of Formula I and a polymer. The term composite as used herein refers to the situation wherein a compound of the invention is embedded in a polymer or physically occluded within or adsorbed onto a matrix or substrate. The polymer may be a biological polymer such as a peptide or a protein. Preferred polymers include those having anhydride and/or ester groups.

In addition, the present invention provides a compound formed by the reaction between a compound of Formula I and a chlorotriazine derivative. The chlorotriazine derivative may be a polymer having chlorotriazine groups attached thereto.

In one embodiment compounds of the present invention are used as a medicament.

The compounds of the present invention are useful as photosensitising drugs for PDT of conditions where treatment requires removal of unwanted tissue or cells such as cancer, precancerous disease, ophthalmic disease, vascular disease, autoimmune disease, and proliferative conditions of the skin and other organs. Specific and unpredicted advantages of these materials relate to their ability to be photoactive against target tissues at different times after systemic administration (depending upon the particular sensitiser used) and therefore their ability to be targeted to the vasculature or tumour cells (for example) directly. They also have a low tendency to sensitise skin to ambient light when administered systemically and a low tendency to colour skin.

The compounds may also be used in PDT as photoactivatable antimicrobial treatments for skin and other local infections, for sterilisation of burn wounds and other lesions, for sterilisation of both recipient tissue and donated tissue during organ including skin transplantation and for the treatment of dental microbial disease.

The said compounds are also useful as photoactivatable antimicrobial agents for general sterilisation of surfaces and fluids. Specific advantages of these compounds over existing known antimicrobial photosensitisers are their high photocytotoxicity at low light levels, combined with a low tendency to undergo photobleaching.

Furthermore, compounds of the present invention have the advantage, compared with other phenothiazinium photosensitisers, that they do not, in carrying out their cell- destroying activity, target the nucleus of the cell so that there is a much lower risk of the cells undergoing mutagenic transformations.

The present invention also provides compositions comprising a compound of the present invention together with a diluent or excipient.

Examples of uses of the compounds of the present invention are as photosensitising drugs for PDT to treat Barrett's oesophagus and cervical intraepithelial neoplasia (pre-cancerous diseases), bladder cancer and colon cancer, macular degeneration (an ophthalmological disease), vascular problems such as cardiovascular disease, arteriosclerosis and restenosis and autoimmune diseases such as rheumatoid arthritis, skin diseases such as psoriasis, acne and excema and other benign conditions such as endometriosis and menorrhagia. The compounds may also be used as anti-microbial treatments for skin and wound infections, other local infections as well as in the treatment of dental bacterial disease. The compounds may also be used in photochemical internalisation (the use of photosensitisers to assist the uptake and subcellular localisation of drugs) through their photosensitising properties and in non-therapeutic uses such as in photodiagnosis through their fluorescence properties.

The latter approach takes advantage of the fact that the photosensitiser concentrates

more in tumours than in surrounding healthy tissue and when induced to fluoresce (by application of blue light), the tumour fluoresces more strongly than the surrounding tissue.

The compounds may be used as a photoactivated antimicrobial agents for sterilisation of surfaces and fluids.

Accordingly, the present invention provides a method of treatment for cancer and other human or animal diseases through systemic or local administration of the photosensitiser, followed by application of light of an appropriate dose and wavelength or wavelength range.

Preferably the compound administered to a subject in need of treatment is that according to formula (I), where Rl and R2 are n-propyl and said light exposure is given up to 10 minutes from the point of drug administration.

In a preferred embodiment of the invention, light exposure is given within 1 minute of drug administration.

More preferably light exposure is give at the point of drug administration.

Alternatively the compound administered is that according to formula (I), where R and R2 are n-pentyl and said light exposure is given at longer times from the point of drug administration.

Accordingly, the present invention provides a method of treatment of microbial infections, burn wounds and other lesions and of dental bacterial disease, the method comprising systemic administration or applying to the area to be treated (for example by a spray) a therapeutically effective amount of a compound of the present invention and exposing said area to light to render active said compound.

Preferably the method comprises the step of administering a compound according to formula (I) where R'and R2 are n-butyl.

Furthermore, the present invention also provides a method of sterilising a surface or a fluid comprising applying the compound of the present invention to said surface or fluid and activating said compound by means of light.

Appropriate compounds of the present invention may be attached to polymeric surfaces, permanently by covalent bonds or reversibly by intermolecular interactions, thus affording a surface that can be sterilised whenever required by the application of light. This would be useful, for example, with intravenous lines in patients and in sutures and catheters and intravenous lines, where maintaining long-term sterility of the relevant surfaces is problematical. The resistance of the compounds to photobleaching is an advantage in such applications, where prolonged stability of the chromophore is required.

Accordingly the present invention also provides an article having at least one surface to which is attached a compound of the present invention.

Preferably the article is a medical device such as a venous, urinary or balloon catheter, suture, orthopaedic or artifical implant, heart valve, surgical screw or pin, pacemaker lead, feeding or breathing tube, vascular stent, intraocular lens, or small joint replacement. The article may also be of use in wound care and for packaging materials for medical use, for example, materials for medical equipment A compound of the present invention may be applied to walls, floors and ceilings of hospitals, clinical surfaces such as operating tables, abattoirs and clean rooms in scientific laboratories. Fibres may be converted into woven, knitted or non-woven textile articles such as cleaning cloths, wipes, surgical gowns, bed linen, wound dressings and bandages.

Alternatively the article is one for use in the food and beverage industry and may be an item of packaging, a wrapper or storage carton or a piece of processing equipment.

The article may be a refrigerator, vending machine, ice making machine, a piece of restaurant equipment or other kitchen appliance.

The present invention relates to phenothiazium sensitizers which show an unexpected and pronounced dependence of their photobiological properties in vitro and in vivo on the size and hydrophobic character of substituents on the terminal amino groups.

By careful selection of such structural features, photosensitisers with distinct advantages over existing materials are provided. Accordingly compounds of the present invention overcome the problems of the prior art by providing the following advantages in the field of oncology and in their antimicrobial effects: Advantages in oncology . Extremely strong photoactivity when compared with methylene and ethylene blue.

Low absorption of light in the UV/blue region. This results in a lower propensity of the compounds to skin photosensitivity.

Rapid skin clearance.

High selectivity for tumours.

Low dark toxicity.

'Low potential for DNA damage when compared with methylene blue.

Very short drug-to-light time interval compared with existing PDT drugs.

Antimicrobial advantages highly effective in deactivating a wide range of microorganisms, including Gram positive and Gram negative bacteria, MRSA and fungal infection.

Active against quiescent bacteria.

High selectivity for microorganisms with minimum damage to host tissue.

'Unexpectedly low level of photobleaching.

DETAILED DESCRIPTION OF THE INVENTION Compounds examined The phenothiazinium compounds used in this study were synthesised in Leeds University Department of Colour Chemistry by J. Griffiths. They included the following: R'-R4 = n-C3H7 : tetra-n-propyl Rl-R4 = n-C4H9: tetra-n-butyl R1 - R4 = n- C5H11 : tetra-n-pentyl R1 - R4 = n- C6H13 : tetra-n-hexyl Methylene blue (Rl-R4 = n-CH3) and ethylene blue (R1 - R4 = n=C2H5) were also examined for comparative purposes.

Stock solutions of photosensitisers were made up in water and/or DMSO and stored in the dark until required. Test solutions were made up in buffer or solvent or biological medium as required.

Spectral and physical properties of the phenothiazinium compounds Spectral data of the phenothiazinium compounds in water and in methanol (Table 1), show that all of the compounds have absorption peaks in the 650-700 nm region, but that there is considerable variability in the precise peak position. The phenothiazinium compounds with longer alkyl chains have absorption peaks at longer wavelengths and the peak positions in general are at longer wavelengths in water compared with methanol. These differences probably reflect the aggregation state of the photosensitisers.

The octanol-water partition coefficients (log p) for the various photosensitisers are also shown in Table 1, where log p = (mg/ml in octanol)/ (mg/ml in buffer) Table 1 Spectral and physical properties of the phenothiazinium compounds R Wavelength Wavelength Ex. Max Em. Max (nm) logP max in water max in (nm) (nm) methanol (nm) 1 667 656 640 690-1. 0 2 673 661 670 690 +0. 2 3 679 665 680 690 +1. 1 4 682 668 680 695 +1. 1 5 685 669 680 700 +1. 7 6 699 669 675 700 +1. 3 Ex. Max is the fluorescence excitation wavelength maximum and Em. Max is the fluorescence emission wavelength maximum The octanol buffer partition coefficient (logP) determines the lipophilicity of the drug. As might be expected, the lipophilicity increases with increasing value of R,

but it should be noted that even for higher values of R, the compounds remain soluble in biological media.

Phenothiazinium derivatives as PDT agents for mammalian cells and tumours The phenothiazinium derivatives were assessed for PDT efficacy in a series of mammalian cells in culture. RIF-1 murine fibrosarcoma cells were studied, using the MTT assay to assess remaining cell viability following PDT. The data from a series of experiments are summarised in Table 2, which shows the LDso values (concentration of photosensitiser needed to kill half of the cells under the conditions used) for four of the phenothiazine derivatives. Also shown for comparison are the data for methylene blue and ethylene blue. Some of these compounds are also able to kill cells in the dark and Table 2 also shows the ratio to LDSO for dark only controls.

It may be seen from Table 2 that the tetra-n-pentyl derivative, the tetra-n-butyl derivative and the tetra-n-propyl derivative are all efficient PDT agents under these conditions, being much more active than methylene blue or ethylene blue. The most efficient is the tetra-n-propyl derivative. Also, it is clear that, while for methylene blue there is only a small ratio between the LDso for dark and light toxicity, this ratio is much greater for the phenothiazinium derivatives.

The results illustrate the much increased photoactivity of these compounds, but also their relative lack of dark toxicity compared with methylene blue. This is a considerable advantage in therapeutic terms. Table 2 also shows the relative activity of the various derivatives in comparison with methylene blue and ethylene blue using a measure of their intrinsic ability to produce singlet oxygen. It may be seen that there is very little difference between the different compounds, showing that the marked differences observed in cells is almost entirely due to biological factors, the mechanisms of which are not yet known. Table 2 also shows the initial subcellular localisation of the different phenothiazinium compounds derivatives compared with that of methylene blue and ethylene blue, as well as any re-localisation which occurs following light administration. It is noteworthy that, whilst all of the derivatives initially localise in lysosomes, whilst methyene blue then relocalises to the nucleus

(with possible deleterious or mutagenic effects on DNA), the tetra-n-propyl, tetra-n- butyl, tetra-n-pentyl and tetra-n-hexyl derivatives relocalise to the mitochondria, which is a much better PDT target.

Table 3 shows LDso values for some of the derivatives in a series of different cells in culture, representing different human tissues and cancers. It is clear that the tetra-n- propyl, the tetra-n-butyl and the tetra-n-pentyl derivatives are again highly active compared with methylene blue and that they are also active in all cell lines tested.

Several asymmetric phenothiazinium derivatives (where Rl= R2 t R3 = R4) have been prepared and tested in cells in culture. Several of these have proved superior photosensitisers to methylene blue, both in terms of absolute activity and in terms of the light to dark toxicity ratio. Sample data for these compounds are shown in Table 4.

Table 4. Chemical properties of the asymmetric phenothiazines in comparison to methylene blue and phototoxicity and dark toxicity in SiHa human cervical squamous cell carcinoma cells.

#max in Singlet Log P PDT LDso Ratio dark : methanol oxygen (µM) 2 PDT LDso2 (nm) generation' Methylene blue 656 47-1.0 18.7 1. 0 1 Di-n-butyl 661 29 +1.0 4.6 2. 0 7 morpholino Di-n-butyl 660 13 +1.3 1. 7 0. 3 87 diethanolamine Di-n-pentyl 663 32 +1. 1 0.43 0. 03 39 diethanolamine

l % photo-oxidation of 1,3-diphenylisobenzofuran after 10 min red light illumination with lOOmg/ml of the phenothiazinium compounds in 90% DMF: 10% water.

2 Cells were incubated with the phenothiazinium compounds for 2h. For measurement of phototoxicity, cells were illuminated with 3J/cm2 665nm light.

Dark toxicity was measured in parallel. Cell survival was assessed after 48h using the sulforhodamine B (SRB) assay.

Figure 1 shows the anti-tumour photodynamic efficacy (% tumour necrosis) of symmetrically substituted thiazines of type (I). Female CBA/Gy mice with CaNT subcutaneous tumours were injected with a solution of the drug at a dose of 16.6 pmol per kilogram. They were treated with the optimum wavelength of light (determined in separated experiments) 1 hour after injection. The light source was a Patterson lamp with appropriate filters giving a bandwidth of 30 nm, and treatment was 60 J cm~2 at a rate of 50 mW cm-2. It can be seen from Figure 1 that tumour response is very dependent on the nature of the alkyl groups, and the tetra-n-pentyl

and tetra-n-butyl derivatives were particularly effective compared with methylene blue.

Figure 2 shows the anti-tumour photodynamic efficacy (% tumour necrosis) for the tetra-n-propyl and tetra-n-pentyl derivatives as a function of the time interval between drug and light administration. These data show a quite unexpected difference between the two compounds. The tetra-n-propyl derivative is very active at very short drug-light time intervals (ie by giving light almost immediately after giving drug) whereas the tetra-n-pentyl derivative has very low activity at very short times, but much higher activity after 1 hour. The explanation for this finding is as yet unknown, but clearly these differential properties could be exploited for different applications. For example, the fast acting drug could be used for vascular treatments and the slower acting drug could be used for tumour cell treatments.

Figure 3 shows the relative skin photosensitivity caused by the tetra-n-butyl and tetra- n-pentyl derivatives, compared with polyhaematoporphyrin, PHP (equivalent to Photofrin). Photofrin is the current leading PDT agent for oncology, but has the major disadvantage of causing prolonged skin photosensitivity in patients. In this model, the skin photosensitivity is measured in terms of the increase in ear thickness 24 hours after exposure to drug and light. Figure 3 shows that, as expected, PHP shows a high level of skin photosensitivity, but the two phenothiazinium derivatives show little or no skin photosensitivity. These two derivatives also caused very little skin colouration after administration.

Photo-antimicrobial activity General Methods The methods given below are in respect of E coli, but were essentially the same for other bacteria studied ie P. aeruginosa, S. aureus and MRSA.

Standard preparation of bacteria The standard protocol outlined below was modified as appropriate to study variation of various experimental parameters.

100ml of nutrient broth (0.5% yeast extract 1.0% tryptone w/v) was aseptically inoculated with E. coli strain DH5, and incubated in a shaking incubator overnight at 37°C. The incubator was set to 250 strokes per minute and a rotary motion of a 2.5cm circle. Following incubation 10ml of the culture was aseptically transferred to 200ml of nutrient broth, and grown in the shaking incubator detailed above until in mid log phase. Cells were collected by centrifugation (3000rpm, 10 minutes), and resuspended in 0. 1M potassium phosphate buffer (pH7.0) and centrifuged again (3000rpm, 10 minutes). The supernatant was discarded and the pellet resuspended in 0. 1M potassium phosphate buffer (pH7.0) to an absorbance of 0.85-0.90 at 650nm.

In the experiments involving illumination in nutrient media, bacterial cells were resuspended in nutrient media at this stage.

Bacterial cell inactivation experiments 25ml of the prepared cell suspension was incubated with 0.25mls of a O. ImM stock solution of photosensitiser in a 250ml sterile foil covered conical flask. The suspension was incubated for 30 minutes in the dark in a 37°C shaking incubator at 250rpm. The suspension was irradiated by a 500W halogen lamp, from a distance of 75cm, for 60 minutes, the power of the lamp was 1.3mW/cm2 giving 4.68J/cm2 over the hour illumination. 50ml of the illuminated and nonilluminated samples of the suspension were removed and diluted in 0.1 M pH 7.0 potassium phosphate buffer.

50ml of the diluted suspension was then plated on nutrient agar (0.5% yeast extract, 1.0% tryptone, 2.0% agar w/v) and incubated overnight at 37°C to give a number of colony forming units between 30-300. Cell inactivation was then measured.

Control studies involving plating out of bacteria before and after the 30 minute incubation step with no phenothiazinium compounds but 0.25mls DMSO showed

no change in CFU/ml. Illumination of the bacterial culture alone with no phenothiazinium compounds but 0.25mls DMSO also demonstrated no change in CFU. For illumination in nutrient media, control tests showed a loglo increase in CFU/ml of 0.2 during the hour illumination.

Some irradiations were carried out with a laser, when the bacterial suspension was illuminated with a 665nm Ceramoptec diode array laser at 0.1 W.

Determination of the effect of phenothiazinium compounds on bacterial cell growth 200ml of nutrient media (0.5% yeast extract 1.0% tryptone w/v) in foil covered 250ml conical flasks was aseptically innoculated with 10ml of a fully grown bacterial culture. In addition the media contained 1.0 ml of a 1 mM stock solution of phenothiazinium compounds with a final concentration of 10wM, apart from the control which contained no phenothiazinium compounds but 1.0 ml DMSO.

The suspension was incubated at 37°C and 250rpm in a shaking incubator in the dark. Iml samples were taken every hour for 6 hours and turbidity based on apparent optical density at 550nm caused by light scattering was measured. Control studies show this wavelength is out of the region of photosensitiser absorption. Following optical density readings the 1. 0ml sample was spun in a MSE Micro-Centaur centrifuge (10 000g x 5 minutes) and the absorbance spectra of the supernatant read spectrophotometrically.

For the tetra-n-butyl derivative only, similar experiments were carried out where the bacteria were allowed to grow without photosensitiser for 3 hours, after which time the phenothiazinium compounds was added. Subsequent growth was monitored as a function of time, both for exposure to light and in the dark.

Preparation and light-induced inactivation of Candida albicans For the experiments involving the yeast Candida albicans the organism was grown in Sabouraud (oxoid) liquid media until in the log stage of growth (cells were harvested

after 4 hours growth). They were then resuspended to an optical density of 0.87 at 650 nm, this is equivalent to 7.0 log 10 CFU/ml compared to 8.5 log so CFU/ml used for the other organisms. The illumination procedure was the same as used in previous experiments. Following illumination the yeast were plated out on Sabouraud dextrose agar (oxoid) and incubated for 24 hours at 37 °C, to assay for colony forming units.

Photobleaching 0. 25mls of a 1mM solution of photosensitiser, 0.25mls of 10mM tryptophan was added to 25mls of 60% methanol, 40% potassium phosphate buffer (pH7.0). Experiments were also carried out in the absence of tryptophan where this was replaced by 0.25mls of the 60% methanol, 40% potassium phosphate buffer (pH7.0).

The mixture was illuminated as in the cell inactivation experiments above (1. 3mW/cm2) for 60 minutes, samples were taken every 15 minutes and spectra recorded on a UV-Visible spectrophotometer between 500nm and 700nm. For high light dose, illumination was at 9mW/cm for 60 minutes.

Results Antibacterial properties ofphenothiazinium derivatives Figure 4 shows log change in Colony Forming Units (CFU)/ml of E. coli incubated for 30 minutes with IOVM phenothiazinium compound and illuminated for 60 minutes at 1. 3mW/cm2 Data were recorded of cell survival following a 60 minute illumination by a halogen lamp. It may be seen that there is substantial bacterial inactivation with the trend in the group being a decrease from methylene blue to ethylene blue, followed by an increase of almost 1000 fold up to the tetra-n-butyl phenothiazinium derivative. The longer chain phenothiazinium compounds then show reduced bacterial cell kill ability such that the tetra-n-hexyl derivative is almost inactive. The tetra-n-butyl phenothiazine led to the largest change in colony forming units per ml of 5.1 log, 0 equivalent to a percentage cell survival of 0.001%. The

lowest change of 0.19 logic CFU was using the tetra-n-hexyl derivative which is a cell survival of 65.3%. There was no cell inactivation with a light only control.

Figure 5 shows the log change in CFU/ml of E. coli incubated for 30 minutes with different concentrations of tetra-nbutyl phenothiazinium derivative and illuminated for 15 minutes at 1. 3mW/cm~2 101lM was the most effective concentration tested for bacterial inactivation using tetra-n-butyl phenothiazinium derivative. The log change in CFU/ml with this concentration was 4.59 logic units. Cell kill effects were achieved with all of the concentrations tested but were reduced at the lower drug doses. At 5011M there is a log change of 2.15 units which is reduced compared to 101lu. This could be due to photosensitiser aggregation and therefore lower drug doses to the cell.

Many antibiotics are only poorly effective against non-growing or stationary bacteria and it is important to assess the ability of the phenothiazinium compounds to inactivate stationary phase bacteria. During the stationary period the cell has a thicker peptidoglyan cell wall and differences in protein metabolism and therefore might be less susceptable to the photodynamic effect. Figure 6 shows the log change in CFU/ml of Ecoli in the stationary phase of growth following incubation for 30 minutes with 101lM phenothiazinium compounds and illuminated for 60 minutes at 1. 3mW/cm2. It may be seen that the effectiveness of the tetra-n-propyl and tetra-n- butyl derivatives is only slightly reduced against stationary phase bacteria, with again the tetra-n-butyl derivative being the most effective.

Inactivation of bacteria may be more challenging in a therapeutic environment, because the sensitiser may bind preferentially to extracellular proteins rather than the bacterial lipopolysaccharide membrane. This was tested by resuspending the bacteria in nutrient medium containing many factors which might compete with bacterial cells for photosensitiser binding. Figure 7 shows the log change in CFU/ml of E. coli resuspended in nutrient medium, from which it may be seen that there is little

reduction in the level of cell kill. Again, the tetra-n-butyl phenothiazinium derivative has the highest antibacterial activity.

Figure 8 shows the log change in CFU/ml of E. coli following incubation with 101lM phenothiazinium compound for 30 minutes and illumination with laser light (665nm) for only 4 minutes at 0.1 W. Again, the same pattern of activity among the phenothiazine derivatives is seen, showing that the effects are present with coherent laser light. The potential advantages of a laser source are increased accuracy of light doses and shorter illumination times.

Further studies with laser light showed that a log change of 5.69 CFU/ml can be achieved with a 14 minute illumination at 0.1 W with the tetra-n-butyl phenothiazinium derivative and that after a 20 minute illumination there is a log change of almost 8.5 units, though the number of CFU are too small to make this figure statistically reliable Uptake of the photosensitisers into bacterial cells is clearly important in determining photo-activity. Figure 9 shows uptake of 101lM phenothiazinium compounds into E. coli cells following a 30 minute incubation. Cells were washed twice in 0. 1M pH7.0 potassium phosphate buffer to remove extra-cellular or loosely bound sensitiser. It may be seen that uptake of the phenothiazinium compounds is somewhat correlated with phototoxicity in that the tetra-n-butyl derivative is taken up the most by the bacterial cells. However, the correlation between uptake and photoactivity is far from exact. For example, the ratio between the photoactivity and the uptake for the tetra-n-butyl derivative is far greater than that for the tetra-n-hexyl derivative. These ratios would be expected to be the same for all of the derivatives if the photoactivity could be explained only on the basis of uptake. It is therefore clear that the extremely high activities of the tetra-n-butyl and tetra-n-propyl derivatives must be due to some additional factors, as yet unknown.

Data not shown have proved that the tetra-n-butyl derivative is taken up very quickly into the E coli ; there are no differences in uptake between incubation times of 5 minutes and 30 minutes. However, in the presence of nutrient medium, the uptake was found to be somewhat slower and reduced in extent.

Figure 10 shows the log change in CFU/ml of E. coli cells incubated with 10pM tetra - n-butyl phenothiazinium derivative and then washed twice with 0.1 M pH7 potassium phosphate buffer to remove any extra-cellular or loosely bound photosensitiser, which may have an effect on the phototoxicity. Illumination used laser light (665 nm) at 0.1 W for 4 minutes. The results show there is little difference between the log change in CFU/ml of washed and unwashed cells, indicating that it is the tightly bound photosensitiser which is causing cell death. At present, the precise location of the photosensitiser within the bacterial cell is not known but the photodynamic action is effective and non-recovering.

Data for the effect of the different phenothiazinium compounds on the growth of an E. coli culture in the dark as compared to a control are shown in Figure 11. Incubation was carried out in the dark at 37°C for 6 hours and measurements were based on apparent turbidity at 550nm as described earlier. All the cultures containing phenothiazinium compounds show reduced growth as compared to the control with the tetra-n-butyl phenothiazinium derivative showing the greatest inhibition in the number of cells in the bacterial suspension. It should be emphasised that this dark inhibition is many orders of magnitude less than that observed for cell inactivation in the light.

Further work was carried out with the tetra-n-butyl derivative alone to determine the effect of photosensitiser plus light on growth of bacteria. These data, shown in Figure 12 show clearly that for the growing bacteria with addition of photosensitiser after 3 hours, there is continuing growth in the dark, but complete elimination of growth in the light. The data again illustrate the very powerful photobacteriocidal effect of this photosensitiser.

Figure 13 shows percentage cell survival of Pseudomonas aeruginosa following incubation with 10tM tetra-n-butyl phenothiazinium derivative. Illumination was with laser light (665nm) at 0.1 W. P aeruginosa is a Gram negative organism which is commonly associated with a number of skin conditions including infections of ulcers and burn wounds. The figure shows that the tetra-n-butyl phenothiazinium derivative can photodynamically inactive this organism extremely efficiently. An illumination time of only 2 minutes with laser light (665nm) at 0.1 W leads to a greater than 99% cell kill, while increase of the illumination time to 10 minutes gives almost 4 logs of cell kill. Control studies showed that there is no reduction in cell number caused by the illumination alone in the absence of photosensitiser with lOnM tetra-n-butyl phenothiazinium derivative.

Figure 14 shows percentage cell survival of Staphylococcus aureus following incubation with 10tM tetra-nbutyl phenothiazinium. Illumination was with laser light (665 nm) at 0.1 W. S. aureus is a Gram positive organism which differs from Gram negative organisms in that it has a thick outer peptidoglycan layer and no external lipopolysaccharide. The bacterial structure is the same as in MRSA (Methicillin resistant Saureus) which is resistant to almost all commonly used antibiotics. The data show that after only a 1 minute illumination almost 99% of the bacteria are inactivated and that after 10 minutes there is almost 5 logs of cell kill, illustrating the very high photoactivity of the tetra-n-butyl derivative against this Gram positive organism.

It is important to determine if the photosensitiser would also be active against the antibiotic resistant form, MRSA, as this would have major health and industrial applications. Figure 15 shows percentage cell survival of MRSA following illumination with 665 nm laser light at 0.1 W and incubation with lOM tetra-n-butyl phenothiazinium derivative. The data clearly show that this photosensitiser is indeed highly photoactive in killing MRSA.

Anti-fungalproperties of phenothiazinium derivatives In order to test the ability of the tetrabutyl derivative to kill fungal cells in the light, the photosensitiser was incubated with cells of Candida albicans and the culture was subjected to laser light as described above. The results are shown in Figure 16, in which it is clear that this eukaryotic organism is also readily destroyed. This photosensitiser is therefore also highly photoactive against this fungal organism which is responsible for many common infections e. g. thrush.

Selectivity for bacterial cells versus mammalian tissues It is clearly important for therapeutic purposes that there is minimal damage to host tissues while microorganisms are being destroyed. This was tested by applying a solution of the tetra-n-butyl phenothiazinium derivative to the ears of experimental mice and illuminating, under conditions in which the total dose was almost 20 times that needed for bacterial or fungal elimination. The possible effects on the host tissue were assessed by measuring any increase in ear thickness. This is a standard model for detecting photodynamic reactions in the skin. Figure 17 shows the data obtained, compared with results from intravenous administration of PHP, a drug equivalent to Photofrin which is known to cause prolonged skin reaction. It is clear from Figure 17 that, while the reaction from PHP is very strong, as expected, there is little or no reaction from the tetra-n-butyl phenothiazinium derivative, suggesting that mammalian tissues would not be damaged during antimicrobial treatment.

Photobleaching Photobleaching removes detectable colour from the photosensitiser, rendering it inactive and is the result of the its instability to light and reduction or oxidation. Such photobleaching may have advantages or disadvantages depending on the potential application. For example, photobleaching is undesirable in the coating of lines and catheters. Two sets of experiments were carried out; one at a high light dose (9.0mW/cm2) and one at a low light dose (1. 3mW/cm2) with and without tryptophan as described above. Absorption spectra at low light dose, with and without tryptophan, showed no changes for any of the phenothiazinium compounds demonstrating they are stable at this dose. At the high light dose, spectral changes

were observed for the methylene blue, indicating photobleaching. The maximum absorbance decreased and the wavelength peak shifted over the one hour illumination. These changes occurred to the same extent with and without tryptophan. However, none of the other phenothiazinium compounds showed this degradation and remained stable to photobleaching at the high light dose.

Phenothioazinium compounds of structure I suitable for inclusion in polymers or attachment to, or adsorption on, polymer surfaces (a) Inclusion within polymers Example To a clear solution of cellulose triacetate (0.5 g) in dichloromethane (10 cm3) was added sensitiser (I, X = Y = n-Bu) (O. Olg) and the mixture was stirred until the sensitiser had dissolved completely. The solution was then cast on a glass plate and allowed to dry slowly, giving a clear blue film. The film showed typical singlet oxygen generating properties on exposure to light. Thus an aerated red solution of tetraphenylcyclopentadienone (a characteristic singlet oxygen detector) in toluene containing the film was rapidly bleached on exposure to light from a 40 w tungsten filament lamp. An identical solution showed no bleaching when irradiated for the same period of time in the absence of the film.

(b) Adsorption on polymers Phenothiazinium compound la and Ib were made according to the following reaction scheme and were isolated as dark blue solids. They were characterised by mass spectrometry.

la ; R = n-Pr Ib ; R =n-Bu Compounds (la and Ib) were extremely basic and readily protonated in dilute acids to give (IIa) and (lib) respectively, which could be adsorbed strongly on polymeric surfaces, e. g. polyamides, polyacrylates, polyesters, polycarbonates, polyurethanes, and strongly resisted removal by water or solvents. Alternatively la or Ib could be adsorbed directly onto acidic surfaces to give their corresponding cationic salts directly.

IIa ; R = n-Pr IIb ; R = n-Bu (c) Covalent attachment of the phenothiazinium sensitisers to polymer substrates Derivatives Ia and Ib Compounds la and Ib proved very reactive as nucleophiles in various substitution reactions that could provide a means of attaching the sensitiser unit covalent to polymers.

Thus reaction with anhydrides occurred, as exemplified by the following reaction: [polymer] CH2 [polymer] NH N O RZN S N J + O RzN S \ N J O NH 0 p r3N N \ N O ,-a') a 0 cc') a [polym, ] [polymer]

Example : Polyethylene-graft-maleic anhydride (1.0 g) was dissolved in toluene (25 cm3) with warming. The sensitiser la (0.20 g) was added and reaction mixture heated under reflux for 1 hour. The mixture was poured into methanol and the precipitate was filtered off, washed with methanol and dried, giving the sensitiser-bound copolymer as a dark blue powder (1.1 g). Covalent attachment of the sensitiser to the polymer was confirmed by dissolving the powder in dichloromethane and precipitating it by addition of methanol. No blue colour remained in the supernatant liquid.

A similar nucleophilic substitution reaction will occur with polymers containing ester (polymer) 0 r"NH a M/ J SNJ 0! j \ s % N + (polymer)-COZAIkyI-- RZN I s \ N o N N groups, i. e. :

Phenothiazinium derivatives Ia and Ib are also very reactive towards chlorotriazine derivatives, and their linkage to polymers can be carried out by the following procedure:

Where X =-NH- (Polymer) in the case of polyamide polymers X=-O- (Polymer) in the case of cellulosic polymers Alternatively, the residual chlorine in the previous example can be replaced by other reactive groups, as in the following reaction: y Y nuYeHphile o NN'X ç _ RzN S \ N J R2N s NJ I i zon N) a N

Where X and Y =-NH- (polymer) Or X and Y =-O-(polymer) Or X may be an amine-NHR or-NRR', and Y =-NH- (polymer), or-O- (polymer) These are not the only means of attaching the phenothiazinium derivatives to polymers, and other methods may by employed based on existing polymer-grafting chemistry known to those skilled in the art.

Example : Sodium carbonate (0.20 g) and cyanuric chloride (0.30 g) were added to a solution of the sensitiser la (0.26 g) in dry acetone (170 cm3) at room temperature, and the mixture was stirred for 15 minutes. A sheet of transparent cellulose film (2.2 g) was immersed in an aqueous solution of sodium hydroxide (1M ; 500 cm3) for 10 minutes and then washed free of sodium hydroxide. This was then introduced into the sensitiser solution and the stirred mixture heated at 50 °C for 15 minutes. Water (200

ml) was added and the mixture heated at 60°c for 30 minutes. The blue cellulose film was then removed and washed with water, and then heated in sodium carbonate solution (6%) to remove any unfixed dye. Covalent attachment to the cellulose was confirmed by heating the film in boiling sodium carbonate solution or boiling methanol, when no blue colour was removed. The film showed typical singlet oxygen generating properties on exposure to light, and when immersed in an air-saturated solution of tetraphenylcyclopentadienone in toluene and exposed to light from a 40 w tungsten filament lamp, the red dienone was bleached more rapidly than an identical solution containing no film.

REFERENCES Wainwright M, Phoenix DA, Laycock SL, Wareing DRA, Wright PA. (1998).

Photobactericidal activity of phenothiazinium dyes against methicillin-resistant strains of Staphylococcus aureus. FEMS Microbiology Letters 160,177-181.

Wagner SJ, Skripchenko A, Robinette D, Foley JW, Cincotta L (1998). Factors affecting virus photoinactivation by a series of phenothiazine dyes. Photochemistry and Photobiology 67,343-349.

Table 2. Chemical properties of the phenothiazines and phototoxicity, dark toxicity, cellular uptake and subcellular localisation in RIF-1 murine<BR> fibrosarcoma cells. R Singlet oxygen PDT LD50 Ratio dark : Uptake at PDT Initial Relocalisation LogP generation1 (µM)2 PDT LD502 LD50 (nmol / localisation4 + light4 mg protein)3 Methyl 47 54 3 > 4.6 Lysosomes Nucleus -1.0 Ethyl 42 3.9 27 2.9 Lysosomes Lysosomes +0.2 Propyl 40 0.42 19 0.9 Lysosomes Mitochondira +1.1 Butyl 41 1.1 7 3.1 Lysosomes Mitochondria +1.1 Pentyl 39 0.74 10 1.6 Lysosomes Mitochondria +1.7 Hexyl 35 2.1 4 1.6 Lysosomes Mitochondira +1.3 1% photo-oxidation of 1,3-diphenylisobenzofuran after 10 min red light illumination with 100mg/ml of the phenothiazine in 90% DMF: 10%<BR> water.<BR> <P>2Cells were incubated with the phenothiazine for 2h. For measurement of phototoxicity, cells were illuminated with 3J / cm2 (10mW / cm2)<BR> white light. Dark toxicity was measured in parallel. Cell survival was assessed after 24h using the MTT assay.<BR> <P>3Cells were incubated with the phenothiazine for 2h. Cells were solubilised in 2% SDS and the phenothiazine levels measured by fluorescencen.<BR> <P>4Cells were incubated with the PDT LD50 concentration of the phenothiazine for 2h and fluorescence images captrued before and during 10min<BR> illumination with 630nm light.

Table 3. Phototoxicity and dark toxicity of the phenothiazines in human tumour cell lines. R OE331 SiHa2 HT13763 HT29 Methyl -PDT LD50 (M) 43.5 ~ 1. 8 18.7 ~ 1. 0 37.9 ~ 10. 1 88.5 ~ 6. 1 - Ratio dark: PDT 2.0 1.0 1.6 1.5 LDso Propyl - PDT LD50 (µM) 0.30 ~ 0. 09 0.075 ~ 0. 015 0.20 ~ 0. 13 0.22 ~ 0. 04 - Ratio dark: PDT 11 80 13 18 LDso Butyl - PDT LD50 (µM) 0.28 0.06 - Ratio dark: PDT 18 LDso Pentyl - PDT LD50 (µM) 0.29 ~ 0. 06 0.75 ~ 0. 22 1.49 ~ 0. 41 - Ratio dark: PDT 11 6 4 LD5o l oesophageal adenocarcinoma 2 cervical squamous cell carcinoma 3 bladder transitional cell carcinoma 4 colon adenocarcinoma <BR> <BR> <BR> <BR> <BR> Cells were incubated with thephenothiazine for 2h. For measurement of phototoxicity, cells<BR> <BR> were illuminated with 3J/cm (lOmW/cm2) 665nm light. Dark toxicity was measured in parallel. Cell survival was assessed after 48h using the sulforhodamine B (SRB) assay.