Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material.

Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant micro-organisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms.

Antimicrobial photodynamic therapy with RLP068 kills methicillin-resistant Staphylococcus aureus and improves wound healing in a mouse model of infected skin abrasion PDT with RLP068/Cl in infected mouse skin abrasion.

Photodynamic therapy (PDT) is an alternative treatment for infections that can kill drug resistant bacteria without damaging host-tissue. In this study we used bioluminescent methicillin-resistant Staphylococcus aureus, in a mouse skin abrasion model, to investigate the effect of PDT on bacterial inactivation and wound healing. RLP068/Cl, a tetracationic Zn(II)phthalocyanine derivative and toluidine blue (TBO) were used. The light-dose response of PDT to kill bacteria in vivo and the possible recurrence in the days post-treatment were monitored by real-time bioluminescence imaging, and wound healing by digital photography. The results showed PDT with RLP068/Cl (but not TBO) was able to kill bacteria, to inhibit bacterial re-growth after the treatment and to significantly accelerate the wound healing process (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

In vitro resistance selection studies of RLP068/Cl, a new Zn(II) phthalocyanine suitable for antimicrobial photodynamic therapy.

Resistance to antimicrobial agents is emerging in a wide variety of nosocomial and community-acquired pathogens. The development of alternative therapies against nosocomial infections caused by clinically relevant pathogens represents a major public health concern. RLP068/Cl is a novel Zn(II) phthalocyanine proposed as a photosensitizer suitable for antimicrobial photodynamic therapy (APDT) for localized infections. Its ability, following activation by light, to induce resistance in three major human pathogens after 20 daily passages was studied. Simultaneously for the same strains, the ability of daily sequential subcultures in subinhibitory concentrations of RLP068/Cl to develop resistant mutants without illumination was evaluated. We demonstrate that 20 consecutive APDT treatments with RLP068/Cl did not result in any resistant mutants and that, in dark conditions, only Staphylococcus aureus strains had increased MICs of RLP068/Cl. However, even in this case, the susceptibility of the mutated bacteria to APDT was not affected by their MIC increase.

Photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) appears to be endowed with several favorable features for the treatment of infections originated by microbial pathogens, including a broad spectrum of action, the efficient inactivation of antibiotic-resistant strains, the low mutagenic potential, and the lack of selection of photoresistant microbial cells. Therefore, intensive studies are being pursued in order to define the scope and field of application of this approach. RESULTS: Optimal cytocidal activity against a large variety of bacterial, fungal, and protozoan pathogens has been found to be typical of photosensitizers that are positively charged at physiological pH values (e.g., for the presence of quaternarized amino groups or the association with polylysine moieties) and are characterized by a moderate hydrophobicity (n-octanol/water partition coefficient around 10). These photosensitizers in a micromolar concentration can induce a >4-5 log decrease in the microbial population after incubation times as short as 5-10 minutes and irradiation under mild experimental conditions, such as fluence-rates around 50 mW/cm2 and irradiation times shorter than 15 minutes. CONCLUSIONS: PDT appears to represent an efficacious alternative modality for the treatment of localized microbial infections through the in situ application of the photosensitizer followed by irradiation of the photosensitizer-loaded infected area. Proposed clinical fields of interest of antimicrobial PDT include the treatment of chronic ulcers, infected burns, acne vulgaris, and a variety of oral infections.

A novel 10B-enriched carboranyl-containing phthalocyanine as a radio- and photo-sensitising agent for boron neutron capture therapy and photodynamic therapy of tumours: in vitro and in vivo studies.

The synthesis of a Zn(ii)-phthalocyanine derivative bearing four 10B-enriched o-carboranyl units (10B-ZnB4Pc) and its natural isotopic abundance analogue (ZnB4Pc) in the peripheral positions of the tetraazaisoindole macrocycle is presented. The photophysical properties of ZnB4Pc, as tested against model biological systems, were found to be similar with those typical of other photodynamically active porphyrin-type photosensitisers, including a singlet oxygen quantum yield of 0.67. The carboranyl-carrying phthalocyanine was efficiently accumulated by B16F1 melanotic melanoma cells in vitro, appeared to be partitioned in at least some subcellular organelles and, upon red light irradiation, induced extensive cell mortality. Moreover, ZnB4Pc, once i.v.-injected to C57BL/6 mice bearing a subcutaneously transplanted pigmented melanoma, photosensitised an important tumour response, provided that the irradiation at 600-700 nm was performed 3 h after the phthalocyanine administration, when appreciable concentrations of ZnB4Pc were still present in the serum. Analogously, irradiation of the 10B-ZnB4Pc-loaded pigmented melanoma with thermal neutrons 24 h after injection led to a 4 day delay in tumour growth as compared with control untreated mice. These results open the possibility to use one chemical compound as both a photosensitising and a radiosensitising agent for the treatment of tumours by the combined application of photodynamic therapy and boron neutron capture therapy.

Zn(II)-phthalocyanines as phototherapeutic agents for cutaneous diseases. Photosensitization of fibroblasts and keratinocytes.

Two tetrasubstituted (RLP024 and RLP040) and one monosubstituted (MRLP101) Zn-phthalocyanines were readily accumulated by three skin-derived cell lines (HT-1080 transformed human fibroblasts, 3T3 mouse embryo fibroblasts and HaCaT human keratinocytes) upon 1 h-incubation with 0.5-5 microM phthalocyanine concentrations. The affinity was markedly larger for the tetra- as compared with the mono-substituted phthalocyanine, even though smaller phthalocyanine amounts were generally recovered from keratinocytes. As a consequence, the two tetra-substituted phthalocyanines exhibited a higher phototoxicity against all the three cell lines. Typically, the cell survival decreased by at least 80% after 1 min irradiation with 600-700 nm light at a fluence-rate of 50 mW/cm2 in the presence of 5 microM phthalocyanine. Fluorescence microscopy and caspase-3 activation studies indicate that cell death of fibroblasts largely occurred by a random-necrotic process while the keratinocytes underwent cell death predominantly via apoptosis in spite of a very similar pattern of subcellular distribution of the phthalocyanines.

A novel tetracationic phthalocyanine as a potential skin phototherapeutic agent.

An amphiphilic tetracationic derivative of Zn(II)-phthalocyanine (RLP068) was prepared by means of chemical synthesis and was showed to possess efficient photophysical and photosensitizing properties against model biological substrates. RLP068 was incorporated into a gel formulation, which allowed its ready penetration into the epidermal layers, but not into the dermis, of both Balb/c and hairless SKH1 mice after 1-2 h of topical deposition. Pharmacokinetic studies showed that the phthalocyanine thus formulated does not enter the general blood circulation. The epidermis-associated amount of phthalocyanine was sufficient to cause an important cutaneous damage upon irradiation with red light (600-700 nm; 100-180 mW/cm(2), 160 J/cm(2)); the latter was confined to the epidermal area with no apparent diffusion to the underlying dermal layers or appearance of photosensitivity in distal skin areas. A systematic investigation of the interplay among the different parameters (deposition time of the formulated phthalocyanine on mouse skin, irradiation fluence rate and total light fluence) allowed us to identify the minimal phototoxic dose, as well as to define irradiation protocols allowing the repeatability of the phototherapeutic treatment. The potential of RLP068 to act as a PDT agent for cutaneous diseases is briefly discussed.

LC-MS/MS determination in rabbit plasma of the main photoproduct of RLP068/Cl, a cationic sensitizer proposed for photodynamic therapy (PDT) of microbial infections.

The clinical development of a sensitizer for photodynamic therapy (PDT) requires the structural identification of the photoproducts and their quantification in biological fluids and tissues. We describe the LC-MS identification of the most important photoproducts of a cationic phthalocyanine sensitizer (RLP068/Cl) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the main photoproduct (the cationic phthalimide derivative 3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl)oxy]-N,N,N-trimethylbenzenaminium chloride) in rabbit plasma. The tri-deuterated product was used as co-eluting internal standard. The cationic photoproduct was isolated from plasma samples by protein precipitation with perchloric acid in methanol (7%, v/v). HPLC step was performed on a Phenomenex Synergi Hydro-RP column (20 mm x 2.0 mm, 2 microm particles) with a mobile phase of 0.5% (v/v) aqueous TFA/methanol (85:15, v/v). Flow rate was 0.2 mL/min and 40 microL injection were performed. Run time was 10 min. Detection was achieved by means of a Bruker Esquire 3000+ ion trap mass spectrometer equipped with an ESI source working in positive mode. A multiple reaction monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1+285.1 for the internal standard was used. The analytical method was validated over the concentration range 0.46-91.2 ng/mL and lower limits of detection (LLOD) and quantification (LLOQ) respectively of 0.2 and 0.5 ng/mL were found.

Determination of tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum by liquid chromatography-tandem mass spectrometry.

The development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum is described. The dodecadeuterated product (RLP068-D12) was used as co-eluting internal standard. RLP068 was isolated from serum samples by solid-phase extraction using weak cationic exchange cartridges (WCX). An oxidative derivatisation was used in order to simplify the peculiar HPLC and MS behaviour of the analyte and thus increasing sensitivity. Liquid Chromatography was carried out on a Polaris C18 Ether column (50 mm x 2.0 mm) with an isocratic run of 0.5% aqueous TFA/methanol. Detection was achieved by means of a Bruker Esquire 3000+ Ion Trap Mass Spectrometer equipped with an ESI source working in positive mode. A Multiple Reaction Monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1 + 285.1 for the internal standard was used. The analytical method was validated over the concentration range 2-65 ng/mL. lower limits of detection (LLOD) and quantification (LLOQ) were respectively 1 and 2 ng/mL. The method is innovative and applicable to pharmacokinetic studies.

Phthalocyanine-photosensitized inactivation of a pathogenic protozoan, Acanthamoeba palestinensis.

Incubation of Acanthamoeba palestinensis cells with a tetracationic phthalocyanine (RLP068) at concentrations ranging between 0.2 and 1.0 microM, caused a ready uptake of the photosensitizer with recoveries of the order of 0.5-2.5 nmol per mg of cell protein. The amount of cell-bound phthalocyanine did not appreciably change with incubation times ranging between 0.5 and 3 h. Fluorescence microscopic investigations showed an obvious accumulation of the phthalocyanine at the level of the vacuolar membranes. A nearly complete photoinduced cell death occurred upon irradiating A. palestinensis cells with 600-700 nm light with a total energy of 15-30 J cm(-2) using 1.0 microM RLP068 in the incubation medium. DAPI staining of the photosensitized cells indicates significant damage of the nucleus. On the other hand, photosensitization of the protozoan cells does not directly involve the mitochondria as shown by the lack of photoinduced decrease in the activity of typical mitochondrial enzymes, such as NADH dehydrogenase and citrate synthase.

Photophysical, photochemical and antibacterial photosensitizing properties of a novel octacationic Zn(II)-phthalocyanine.

A novel Zn(II)-phthalocyanine (1). peripherally substituted with four bis(N,N,N-trimethyl)amino-2-propyloxy groups prepared by chemical synthesis is shown to be an efficient photodynamic sensitizer with a quantum yield of 0.6 for singlet oxygen generation in neat water, which is reduced to about 0.3 in phosphate-buffered saline. The physicochemical properties of 1 in both the ground and the electronically excited states strongly depend on the nature of the medium; in particular, aggregation of 1 was favoured by polar media of high ionic strength. Compound 1 exhibited an appreciable affinity for a typical Gram-positive bacterium (Staphylococcus aureus) and a typical Gram-negative bacterium (Escherichia coli). Both bacterial strains were extensively inactivated upon 5 min-irradiation with 675 nm light in the presence of 1 microM photosensitizer, even though the binding of 1 to the two bacterial cells appears to occur according to different pathways. In particular, E. coli cells underwent initial photodamage at the level of specific proteins in the outer wall, thus promoting the penetration of the photosensitizer to the cytoplasmic membrane where some enzymes critical for cell survival were inactivated.

Approaches to selectivity in the Zn(II)-phthalocyanine-photosensitized inactivation of wild-type and antibiotic-resistant Staphylococcus aureus.

A number of Zn(II)- phthalocyanines bearing peripheral substituents of cationic nature due to the presence of quaternarized anilinium or ammonium groups were shown to be efficient photoantimicrobial agents: a 4-5 log decrease in the survival of both wild-type or methicillin-resistant Staphylococcus aureus was obtained upon short irradiation times in the presence of phthalocyanine concentrations as low as 0.1 microM. A careful selection of the experimental protocol, and in particular the use of short (5 min) incubation times and mild irradiation parameters, allowed one to achieve a high selectivity of S. aureus photoinactivation as compared with important constituents of potential host tissues, such as human fibroblasts and keratinocytes. The efficiency and selectivity of the photoprocess were not affected by the presence of 5% human serum.