Dimitris Skalkos

COPPER BENZOCHLORIN, A NOVEL PHOTOSENSITIZER FOR PHOTODYNAMIC THERAPY: EFFECTS ON A TRANSPLANTABLE UROTHELIAL TUMOR

Abstract— An iminum salt of octaethylbenzochlorin with copper in the aromatic ring, CDSI, was tested for its tumoricidal effects on theA–27 N‐[4‐(5‐notro‐2‐furyl)‐2‐thiazoly] formamide tumor line. CDSI was found to be an effective photosensitiz4r in vivo when used in combination with either a xenon arc lamp or a pulsed alexandrite laser. Hemodynamically, CDSI and light caused a rapid decrease in tumor blood flow. skin photosensitization was found to be minimal when drug‐injected mice werwe illuminated in a solar simulator.

Observations on the synthesis and in vivo photodynamic activity of some benzochlorins

Abstract— An improved synthesis of benzochlorins is reported. Demetallation of the meso‐hydroxymethylvinyl derivative of octaethylporphyrin, followed by treatment with sulfuric acid results in cyclization to generate the corresponding octaethylbenzochlorin in high yield. Prolonged treatment with acid generates the sulfonated derivative. These sensitizers were shown to be efficient photodynamic agents in vivo. Animals bearing a transplanted N‐{4‐(5‐nitro‐2‐furyl)‐2‐thiazolyl}formamide induced urothelial tumor were treated with either the benzochlorin or its sulfonated derivative. Irradiation of tumors 24 h later resulted in a significant tumoricidal effect in a short term assay. We conclude that benzochlorins warrant further examination as potential agents for use in photodynamic therapy.

Towards a consensus structure of hypericin in solution: direct evidence for a single tautomer and different ionization states in protic and nonprotic solvents by the use of variable temperature gradient 1H NMR

Abstract Hypericin, a meso -naphthodianthrone derivative, displays two types of electronic spectra in organic solvents, attributed to the existence of two tautomeric structures. Variable temperature gradient 1 H NMR studies demonstrate the occurrence of only one 7,14-dioxo tautomeric form, for the molecule of hypericin in protic and in nonprotic solvents, differing only in the degree of ionization of the 4-hydroxyl group in the bay region.

IMPAIRED ACCUMULATION OF A CATIONIC PHOTOSENSITIZING AGENT BY A CELL LINE EXHIBITING MULTIDRUG RESISTANCE

Abstract—Transport and accumulation of copper benzochlorin iminium salt (CDSl), a cationic photosensitizing agent, were examined using the P388/ADR murine leukemia, which exhibits the MDR (multidrug resistance) phe‐notype, and the wild‐type parent cell line, P388. The recent availability of radioactive CDSl permitted kinetic studies at drug levels in the submicromolar range. Exclusion of CDSl by P38WADR cells could be demonstrated, indicating that this agent is a substrate for the outward transport system associated with MDR. These results have implications with regard to the efficacy of cationic photosensitizers against this common neoplastic phenotype. The CDSl was readily accumulated by P388 cells and by P388/ADR cells: when the outward transport system was inhibited. Under these conditions, CDSl was tightly bound and could not be washed out even when the outward transport system was reactivated.

Iminium salt benzochlorins: structure-activity relationship studies.

An iminium salt of copper(II) octaethylbenzochlorin (CDS1) is an effective new photosensitizer despite the fact that it does not produce singlet oxygen, does not fluoresce and the triplet state lifetime can only be less than 20 ns. A number of octaethylbenzochlorin derivatives were synthesized in order to determine the structural component(s) that is(are) responsible for the photodynamic action of these new photosensitizers. Studies utilizing the N‐(4‐[5‐nitro‐2‐furyl]‐2‐thiazolyl)formamide‐induced urothelial tumor revealed that the coexistence of the copper inside the aromatic ring and the iminium group at the meso position are required for the photodynamic effect.

TIN ETIOPURPURIN DICHLORIDE-SENSITIZED LIPID PHOTOOXIDATION OF ERYTHROCYTE MEMBRANES

Abstract— Tin(IV) etiopurpurin dichloride (SnET2‐2Cl) is a photosensitizer which has been shown to be an effective photodynamic agent for the treatment of transplantable animal tumors in vivo. The purpose of this study was to understand the effect of SnET2‐2Cl on membrane lipid peroxidation. When erythrocyte membranes were exposed to visible light in the presence of SnET2‐2Cl, lipid peroxidation was observed. An accumulation of lipid hydroperoxides and an increase in lipid fluorescence were also observed. Thin layer chromatography of lipid extracts from photooxidized membrane revealed photoperoxide products derived from phospholipid. Investigations into the mechanism(s) of lipid peroxidation by SnET2 2Cl and light‐sensitized membranes were also performed. Results indicate that singlet oxygen (1O2) plays a major role in lipid peroxidation.

Synthesis and in vivo activity of some porphyrindione derivatives with potential in photodynamic therapy

A number of synthetic bacteriochlorins were prepared from porphyrindiones. The potential of these compounds as sensitizers for photodynamic therapy was examined using the FANFT-induced urothelial cell carcinoma (AY-27) transplanted into rats. In a number of cases, tumor regression was significant following treatment with both drug and light.

Photodynamic Action Of Benzochlorins

A series of benzochlorins have been prepared and tested for tumoricidal activity using the FANFT-induced rat bladder tumor model. Results indicate that these chlorin derivatives, in combination with red light, can cause significant tumor regression at doses as low as 0.5 - 1.0 mg/kg body weight. This compares favorably with other sensitizers tested in the same model and suggests that further studies on the effectiveness of benzochlorins in photodynamic therapy be performed.

Innovation management technique (IMT) for very small-enterprises: concept, development and application

Open innovation suggests that the ability to absorb external knowledge has become a major driver for competition among enterprises. However, little attention has been paid to how very small enterprises (VSEs) deal, and handle innovation. In fact, most of the VSEs are not capable to implement innovation themselves. Innovation management techniques (IMTs) are methodological approaches aiming at the improvement of enterprises’ competitiveness by means of knowledge management. In this paper, we develop, test and evaluate a new methodology of IMT indented for VSEs of less developed regions such as the region of North Aegean of Greece. The method developed includes five phases of implementation and a plan for each VSE actions. The results of the pilot IMT application showed that there was a positive react by the entrepreneurs. This study proves the theory that the application of innovations depends on the human innovative resource rather the size of the company.

The dual effects of polar methanolic extract of Hypericum perforatum L. in bladder cancer cells

Introduction and background: We have reported on the polar methanolic fraction (PMF) of Hypericum Perforatum L as a novel photosensitizing agent for photodynamic therapy (PDT) and photodynamic diagnosis (PDD). PMF has been tested in human leukemic cells, HL-60 cells, cord blood hemopoietic progenitor cells, bladder cancers derived from metastatic lymph node (T-24) and primary papillary bladder lesion (RT-4). However, the mechanisms of the effects of PMF on these human cell lines have not been elucidated. We have investigated mechanisms of PMF + light versus PMF-alone (dark experiment) in T-24 human bladder cancer cells. Methods: PMF was prepared from an aerial herb of HPL which was brewed in methanol and extracted with ether and methanol. Stock solutions of PMF were made in DSMO and stored in dark conditions. PMF contains 0.57% hypericin and 2.52% hyperforin. The T24 cell line was obtained from American Type Culture Collection (ATCC). In PDT treatment, PMF (60&mgr;g/ml) was incubated with cells, which were excited with laser light (630nm) 24 hours later. Apoptosis was determined by DNA fragmentation/laddering assay. DNA isolation was performed according to the manufactures instructions with the Kit (Oncogene Kit#AM41). Isolated DNA samples were separated by electrophoresis in 1.5% in agarose gels and bands were visualized by ethidium bromide labeling. The initial cell cycle analysis and phase distribution was by flow cytometry. DNA synthesis was measured by [3H] thymidine incorporation, and cell cycle regulatory proteins were assayed by Western immunoblot. Results: The results of the flow cytometry showed PMF +light induced significant (40%) apoptosis in T24 cells, whereas Light or PMF alone produced little apoptosis. The percentage of cells in G0/G1 phase was decreased by 25% and in G2/M phase by 38%. The main impact was observed on the S phase which was blocked by 78% from the specific photocytotoxic process. DNA laddering analysis showed that PMF (60&mgr;g/ml) + light at 630nm induced DNA fragmentation in a light dose-dependent manner; in contrast, PMF or light alone did not induce DNA fragmentation. In separate experiments, PMF alone treatment produced a dose-dependent DNA synthesis with a 90% inhibition at a concentration of 25&mgr;g/ml (IC90 = 25&mgr;g/ml). Expression of p53 and p27 cell cycle regulatory proteins was not altered by PMF alone, however, a dose-dependent increase in p21 expression was observed that correlates with PMF concentrations. Cyclin A and cyclin B protein levels showed a clear decrease inverse to the concentration of PMF. In the absence of light treatment, flow cytometry analysis showed that PMF alone results in G0/G1 cell cycle arrest, with a 2-fold increase in G0/G1 cells concomitant with 50% decrease in cells in both S and G2/M phases. However, flow cytometry on PMF alone-treated cells did not show sub G0/G1 peak, further evidence of the lack of apoptosis as a mechanism of effect of PMF in the dark. Conclusions: With respect to light treatment, apoptosis appears to play a vital role in PDT-induced cytotoxicity. The flow cytometry and DNA laddering results revealed that T24 cells demonstrated apoptotic responses in PMF-mediated PDT. Experiments conducted with PMF alone showed a dose-dependent inhibition of DNA synthesis associated with G0/G1 cell cycle arrest and the extract is able to coordinate changes in key cell cycle regulatory proteins in human bladder cancer cells. Both experimental conditions suggest PMF as a potent and effect anti-proliferative agent in cancer chemoprevention and therapy of human urothelial carcinoma cells.