Evaluation of a (99m) Tc-labelled meso-bisphenylporphyrin as a tumour image agent.

Porphyrins are excellent agents for photodynamic treatment of various types of cancer and also good metal chelators that form highly stable metallo-complexes with different radionuclides. Therefore, radiolabelled porphyrins could also be potentially used as tumour imaging agents. In this context, the aim of this work was the radiolabelling of meso-bis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin, 2CPP, with Technetium-99 m ((99m) Tc) and the evaluation of its radiochemical and biological properties in vitro and in vivo. The labelling procedure was optimized resulting in an efficiency of 92.52 ± 0.48%. The complex (99m) TC-2CPP remained stable for more than 4 h. The biodistribution showed that (99m) Tc-2CPP is eliminated by gastrointestinal and urinary pathways. The tumour/muscle ratio increases over time, being 3.33 ± 1.22 and 3.55 ± 1.29 in WiDr-bearing tumours mice and in H1299-bearing tumours mice, respectively, 6 h post-injection, showing the tumour specificity of the (99m) Tc-2CPP complex. The favourable tumour/muscle ratio of (99m) Tc-2CPP shows that this complex could potentially be used as tumour imaging agent. Moreover, it could be used to follow the progression or regression of tumours before, during and after the radiotherapy, chemotherapy and photodynamic therapy.

Ultrasound-mediated synthesis of camphoric acid-based chiral salens for the enantioselective trimethylsilylcyanation of aldehydes.

New chiral salen ligands were prepared by the ultrasound-irradiated condensation of optically active (1R, 3S)-1,2,2-trimethyl-1,3-diaminocyclopentane with aromatic 1-hydroxyaldehydes. The ultrasound-mediated process is more convenient due to shorter reaction times, energy economy, and easier isolation of the products. The in situ formed Ti(IV)(salen) complexes, evaluated as catalysts in the enantioselective trimethylsilylcyanation of benzaldehyde, were found to be efficient for this process, originating the corresponding product in high yields (72-99%) and selectivities of up to 79%. The lowest energy transition states were determined by computational studies. These results were in qualitative agreement with the experimentally observed ones.

A new therapeutic proposal for inoperable osteosarcoma: Photodynamic therapy.

BACKGROUND: Osteosarcoma, a malignant tumor characterized by bone or osteoid formation, is the second most common primary bone neoplasm. Clinical symptoms include local and surrounding pain, unrelieved by rest or anesthesia. Osteosarcoma has a poor chemotherapeutic response with prognosis dependent on complete tumor excision. Therefore, for inoperable osteosarcoma new therapeutic strategies are needed. The present study aimed to develop murine models of cranial and vertebral osteosarcoma that facilitate simple clinical monitoring and real-time imaging to evaluate the outcome of photodynamic therapy based on a previously developed photosensitizer. METHODS: Balb/c nude mice were divided into two groups: the cranial and vertebral osteosarcoma groups. Each group was further subdivided into the photodynamic therapy-treated and untreated groups. Images were obtained by scintigraphy with 99mTc-MIBI and radiography. Tumor growth, necrotic area, osteoid matrix area, and inflammatory infiltration were analyzed. RESULTS: Cranial and vertebral tumors could be macroscopically observed and measured. Radiographic and scintigraphic images showed tumor cells present at the inoculation sites. After photodynamic therapy, scintigraphy showed lower tumoral radiopharmaceutical uptake, which correlated histologically with increased necrosis. Osteoid matrix volume increased, and tumor size decreased in all photodynamic therapy-treated animals. CONCLUSION: Cranial and vertebral osteosarcoma models in athymic mice are feasible and facilitate in vivo monitoring for the development of new therapies. Photodynamic therapy is a potential antitumoral treatment for surgically inoperable osteosarcoma.

Molecular analysis of apoptosis pathway after photodynamic therapy in breast cancer: Animal model study.

BACKGROUND: Molecular investigation of breast tumors has permitted better understanding about interaction of genes and pathways involved in tumor progression. OBJECTIVE: The aim of this study was to evaluate the association between genes belonging to the pathway of apoptosis with tumor response to photodynamic therapy. STUDY DESIGN/MATERIALS AND METHODS: The mammary tumors were induced in twenty-four Spraguey-Dawley female rats by oral gavage of 7,12-dimethylbenz(a)anthracene (8mg/Kg body weight). Animals were divided into three groups: G1 (normal tissue), G2 (tumors without treatment), G3 (animals euthanized 48h after treatment). The photosensitizer used was a chlorin, 5,15-bis-(2-bromo-5-hydroxyphenyl) chlorin in the dose of 8mg/kg for each animal. Light source of diode laser at a wavelength of 660nm, fluence rate of 100mW/cm, and light dose of 100J/cm was delivery to lesions for treatment. A sample from each animal was investigated by quantitative real time PCR using Rat Apoptosis RT(2) Profiler™ PCR Array platform. RESULTS: Pro-apoptotic BAK1, CARD6, CASP8, CIDEA, CIDEB, DAPK1, TNF, TNFRSF10B, FASLG, LOC687813, and TP73 genes showed increased expression, and CD40 anti-apoptotic gene showed decreased expression in the group who underwent PDT (G3) in relation to G2. CONCLUSION: The results indicated that these genes are involved more directly with cellular apoptosis induced by PDT using the Chlorin photosensitizer.

2-Bromo-5-hydroxyphenylporphyrins for photodynamic therapy: photosensitization efficiency, subcellular localization and in vivo studies.

BACKGROUND: Photodynamic therapy (PDT) is a therapeutic modality capable of inducing cell death by oxidative stress through activation of a sensitizer by light. Aryl-porphyrin with hydroxyl groups are good photosensitizers and presence of bromine atoms can enhance the photodynamic activity through heavy atom effect. These facts and our previous work made pertinent to compare the photodynamic capacity of tetraaryl brominated porphyrin (TBr4) with the corresponding diaryl (BBr2) derivative. METHODS: Cell cultures were incubated with the sensitizers, ranging from 50nM to 10µM and irradiated until 10J. Cell proliferation was analysed by MTT assay. Flow cytometry studies evaluated cell death pathways, mitochondrial membrane potential and ROS. For in vivo studies Balb/c nu/nu mice were injected with 4×10(6)cells. After PDT, monitoring was carried out for 12 days to establish Kaplan-Meier survival curves. Tumours were excised and histological analysis was performed. RESULTS: Both sensitizers seem to accumulate in the mitochondria. The molecules have no intrinsic cytotoxicity or in non-tumour cells at therapeutic concentrations. Both sensitizers induced a significant decrease of cell proliferation and growth of xenografts of melanoma and colorectal adenocarcinoma. Diaryl BBr2 is more efficient than tetraaryl TBr4, concerning intracellular ROS production, mitochondrial disruption and induction of cell death. The main cell death pathway is necrosis. CONCLUSIONS: TBr2 and BBr4 are promising sensitizers with good photodynamic properties and have the ability to induce cell death in human melanoma and colorectal adenocarcinoma in vitro and in vivo. We consider that BBr2 is a molecule that should be the subject of extensive studies towards clinical use.

Microwave synthesis and in vitro stability of diclofenac-ß-cyclodextrin conjugate for colon delivery.

The aim of this work was to synthesize an ester prodrug of diclofenac and ß-cyclodextrin suitable for colonic delivery. The synthesis of an ester linkage between diclofenac and ß-cyclodextrin was conducted by the nucleophilic substitution of mono-6-tosyl-ß-cyclodextrin under microwaves irradiation. After purification, the conjugate was characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry; infrared (IR) spectroscopy; proton nuclear magnetic resonance ((1)H NMR) spectroscopy; and two-dimensional rotating frame nuclear overhauser effect (ROESY) spectroscopy. The purity was qualified by high pressure liquid chromatography (HPLC). To assess its potential for colonic delivery, the conjugate was evaluated for stability in simulated gastric and small intestinal fluids, and in fecal material from humans processed within a slurry under anaerobic conditions. The conjugate was successfully synthesized with a yield of 20% following purification. The mass spectra showed the parent peak m/z 1434 corresponding to [conjugate+Na] adduct. IR and NMR results confirmed that the carboxyl group of diclofenac is covalently bound to one of the hydroxyl groups of cyclodextrin by an ester linkage. Moreover, ROESY data indicated that the formation of the conjugate is not accompanied by the inclusion of diclofenac within the cyclodextrin. The conjugate was otherwise stable in simulated gastric and small intestinal conditions, but was also readily hydrolyzed liberating diclofenac in less than 2h within the human fecal slurry. This confirmed the potential for this new prodrug as a carrier for colonic delivery.

Synthesis of new 2-galactosylthiazolidine-4-carboxylic acid amides. Antitumor evaluation against melanoma and breast cancer cells.

A set of 2-galactosylthiazolidine-4-carboxylic acid amides was synthesized with different length for the carbon chain amide moiety. The cytotoxicity of the molecules was evaluated against A375 melanoma and MCF7 breast cancer cell lines. For the derivatives tested, the one that contains a C(16) amide carbon chain is the most active with an IC(50) of 17.0 µM for A375 and 5.8 µM for MCF7. This compound also shows cytotoxicity in the triple negative cancer cell line HCC1806. The selectivity of the compounds was assessed by comparing the cytotoxicity in cancer cell line versus in a fibroblast cell line. Flow cytometry studies show the activation of apoptotic pathways and also DNA damages with blockage of the cell cycle in the S-phase and appearance of peaks in G0/G1-phase.

Hypoxia-induced redox alterations and their correlation with 99mTc-MIBI and 99mTc-HL-91 uptake in colon cancer cells.

Colorectal cancer is one of the most common malignancies in the Western world and is an example of a solid tumour in which hypoxia is a common feature and develops because of the inability of the vascular system to supply adequate amounts of oxygen to growing tumours. Hypoxia effects on tumour cell biology can be detected and characterized using different methods. The use of imaging with gamma-emitting radionuclides to detect hypoxic tissue was first suggested by Chapman in 1979 [N Engl J Med 301 (1979) 1429-1432]. (99m)Tc-4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime, also known as (99m)Tc-HL-91, has been among the most studied hypoxia markers. The objective of this study was to correlate the uptake of (99m)Tc-HL-91 and (99m)Tc-MIBI in colon cancer cells under normoxic and hypoxic conditions and to compare this information with some parameters such as oxidative stress and mitochondrial dysfunction of the cells analyzed by flow cytometry. Our results show that the in vitro (99m)Tc-HL-91 uptake is higher in hypoxic conditions, which is confirmed by the decreased uptake of (99m)Tc-MIBI. Flow cytometry results demonstrate that hypoxic conditions used are not enough to induce cellular death, but are responsible for the alterations in the intracellular redox environment, namely, increase of ROS production, proteic pimonidazol-derived adduct formation and alteration in the mitochondrial membrane permeability. Therefore, these results confirm that (99m)Tc-HL-91 is a radiopharmaceutical with favourable characteristics for detecting hypoxia.

Synthesis of chiral pyrrolo[1,2-c]thiazoles via intramolecular dipolar cycloaddition of münchnones: an interesting rearrangement to pyrrolo[1,2-c]thiazines.

Intramolecular dipolar cycloaddition of bicyclic münchnones, 5H,7H-thiazolo[3,4-c]oxazol-4-ium-1-olates, derived from cyclodehydration of 2-substituted-N-acylthiazolidine-4-carboxylic acids are reported. A range of new pyrrolo[1,2-c]thiazole derivatives (7, 14, 15, 20, 23, and 26) were obtained as single enantiomers from 2-phenylthiazolidines, 2-benzoylthiazolidines, and 2-methylthiazolidine-4-carboxylates. Pyrrolo[1,2-c][1,4]thiazine derivative 27 was also obtained from pyrrolo[1,2-c]thiazole derivative 26. The structures of methyl (2R,4R)-2-(p-methoxybenzoyl)thiazolidine-4-carboxylate (17a), methyl (2R,4R)-2-(p-methoxybenzoyl)-N-(prop-2-ynyloxyacetyl)thiazolidine- 4-carboxylate (18), and 3-oxo-4-phenyl-3,4,6,8-tetrahydro-1H-furo[3',4':2,3]pyrrolo[1,2-c][1,4]thiazine (27) were determined by X-ray crystallography. Chirooptical studies of the pyrrolo[1,2-c]thiazoles were done by confirming the absolute configuration at the chiral center C-3.