Targeting phosphatidylinositol 3-kinase signaling in acute myelogenous leukemia.

INTRODUCTION: Despite continuous advances in our knowledge of the biology of acute myelogenous leukemia (AML), the prognosis of AML patients treated with standard chemotherapy is still poor, especially in the elderly. Therefore, there is a need for novel targeted and less toxic therapies, particularly for patients who develop resistance to traditional chemotherapeutic drugs. Constitutively active phosphatidylinositol 3-kinase (PI3K) signaling characterizes many types of tumors, including AML, where it negatively influences response to therapeutic treatments. AREAS COVERED: The literature data showed that small inhibitor molecules targeting PI3K signaling induced cell cycle arrest, apoptosis and decreased drug-resistance in AML cells. PI3K inhibitors were also capable of targeting leukemic initiating cells (LICs), the most relevant target for leukemia eradication, whereas they tended to spare healthy hematopoietic stem cells. EXPERT OPINION: Data emerging from pre-clinical settings suggest that the PI3K pathway is critically involved in regulating proliferation, survival and drug-resistance of AML cells. Therefore, we propose that novel drugs targeting this signaling pathway may offer a novel and less toxic treatment option for AML patients, most likely in combination with a lower dosage of traditional chemotherapeutic agents or other innovative therapeutic agents.

Effects of a new photoactivatable cationic porphyrin on ciliated protozoa and branchiopod crustaceans, potential components of freshwater ecosystems polluted by pathogenic agents and their vectors.

The increasing use of photosensitized processes for disinfection of microbiologically polluted waters requires a precise definition of the factors controlling the degree of photosensitivity in target and non-target organisms. In this regard, tests with protozoa and invertebrates which have a natural habitat in such waters may be used as first screening methods for the assessment of possible hazards for the ecosystem. A new cationic porphyrin, namely meso-tri(N-methyl-pyridyl)mono(N-dodecyl-pyridyl)porphine (C12), is tested in this work on the protozoan Ciliophora Colpoda inflata and Tetrahymena thermophila and the Crustacea Branchiopoda Artemia franciscana and Daphnia magna. The protocol involved 1 h incubation with porphyrin doses in the 0.1-10.0 µM range and subsequent irradiation with visible light at a fluence rate of 10 mW cm(-2). The results indicate that C12 porphyrin has a significant affinity for C. inflata and T. thermophila; this is also shown by fluorescence microscopic analyses. C. inflata cysts were resistant to the phototreatment up to a porphyrin dose of 0.6 µM. The effects of C12 on cysts have been evaluated at 3 and 24 h after the end of the phototreatment; a delay in the excystment process was observed. T. thermophila was fairly resistant to the phototreatment with C12 porphyrin. The data obtained with the two crustaceans indicated that the effects of dark- and photo-treatment with C12 need to be closely examined for every organism. A. franciscana is more resistant, probably owing to its ability to adapt to extreme conditions, while the high level of photosensitivity displayed by Daphnia magna represents a potential drawback, as this organism is often selected as a reference standard for assessing the environmental safety. Thus, while C12 photosensitisation can represent a useful tool for inducing a microbicidal or larvicidal action on polluted waters, the irradiation protocols must be carefully tailored to the nature of the specific water basin, and in particular to its biotic characteristics.

Photodynamic inactivation of microbial pathogens: disinfection of water and prevention of water-borne diseases.

Porphyrins have been shown to act as very efficient photosensitizing agents against a broad number of microbial pathogens, including bacteria, fungi, and protozoa. This property has promising applications at a clinical level for the treatment of infectious diseases by photodynamic therapy. Moreover, this technique is also being used to address environmental problems of high significance, such as the decontamination of wastewaters, the disinfection of fish-farming tanks, the protection of animal species (e.g., amphibians and reptiles) that are endangered by pathogens whose life cycle takes place largely in aqueous media, and the control of populations of noxious insects. Such diversified applications take advantage of the availability of a truly large number of porphyrin derivatives with chemical structures that can be tailored to comply with the physical and chemical properties as well as the biological features of several milieus. In addition, the property typical of porphyrins to absorb essentially all of the wavelengths in the sun emission spectrum allows the promotion of processes largely based on natural resources with significant energy savings and low impact on ecosystems.

The in vivo efficacy of phthalocyanine-nanoparticle conjugates for the photodynamic therapy of amelanotic melanoma.

The efficiency of a Zn(II)-phthalocyanine disulphide (C11Pc), a compound with both phthalocyanine units bearing seven hexyl chains and a sulphur terminated C11 chain, as a photodynamic therapy (PDT) agent was investigated in C57 mice bearing a sub-cutaneously transplanted amelanotic melanoma. The phthalocyanine was intravenously injected at a dose of 1.5 micromol/kg body weight either free or bound to gold nanoparticles, using a Cremophor emulsion as a delivery vehicle. Biodistribution studies at selected post-injection times showed that the nanoparticle-associated C11Pc was recovered in significantly larger amounts from all the examined tissues and the serum and yielded a greater selectivity of tumour targeting: thus, the ratio between the amount of phthalocyanine recovered from the amelanotic melanoma and the skin (peritumoural tissue) increased from 2.3 to 5.5 from the free to the gold nanoparticle-bound C11Pc at 24 h after injection. PDT studies with the C11Pc-loaded amelanotic melanoma showed a markedly more significant response of the tumour in the mice that had received the nanoparticle-bound photosensitiser; the PDT effect was especially extensive if the irradiation was performed at 3h after C11Pc injection when large phthalocyanine amounts were still present in the serum. This suggests that the PDT promoted by C11Pc predominantly acts via vascular damage at least in this specific animal model. This hypothesis was fully confirmed by electron microscopy observations of tumour specimens obtained at different times after the end of PDT, showing an extensive damage of the blood capillaries and the endothelial cells.

Synthesis and biological investigations of tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC).

We describe the total synthesis and biological properties of a new carboranyl-containing chlorin (TPFC) that might find application as a dual sensitizer in the PDT and BNCT treatment of cancer. TPFC was found to be non-toxic in the dark but showed extensive photosensitizing ability both in vitro and in vivo despite its relatively low singlet oxygen quantum yield. In particular, TPFC exhibited significant photosensitizing activity against highly pigmented melanotic melanoma tumors in mice.

Tumour-localizing and -photosensitising properties of meso-tetra(4-nido-carboranylphenyl)porphyrin (H2TCP).

A water-soluble meso-substituted porphyrin (H(2)TCP) bearing 36 boron atoms, which appeared to be an efficient photodynamic sensitiser (singlet oxygen quantum yield=0.44), was studied for its accumulation by murine melanotic melanoma cells (B16F1). The amount of H(2)TCP in the cells increased with the porphyrin dose in the incubation medium up to, and at least, 100 microM concentrations with no significant cytotoxic effect in the dark. Moreover, the H(2)TCP uptake increased with the incubation time reaching a plateau value corresponding with the recovery of 0.4 nmol of H(2)TCP per mg of cell proteins after 24h incubation. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly, exhibiting a discrete distribution in the cytoplasm with a pattern which was closely similar to that observed for the endosomal probe Lucifer yellow. The photosensitising efficiency of the H(2)TCP toward B16F1 cells was studied for different irradiation (1-15 min) and incubation (1-24 h) times. Nearly complete (>95%) cell mortality was obtained upon incubation with 20 microM H(2)TCP and 10 min irradiation with red light (600-700 nm, 20 mW/cm(2)). The porphyrin was also accumulated in appreciable amounts by the tumour tissue after intravenous injection to C57BL/6 mice bearing a subcutaneously transplanted melanotic melanoma. Maximum accumulation in the tumour was achieved by administration of H(2)TCP dissolved in the ternary mixture 20% dimethylsulfoxide (DMSO)-30% polyethyleneglycol (PEG 400)-50% water. Thus, this porphyrin could act as both a photodynamic therapy agent and a radiosensitising agent for boron neutron capture therapy.

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.

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 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.

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.