Alfreda Graczyk

Diamino acid derivatives of PpIX as potential photosensitizers for photodynamic therapy of squamous cell carcinoma and prostate cancer: In vitro studies

Photodynamic therapy (PDT) is an alternative treatment modality involving light activated drugs, called photosensitizers (PSs), to treat cancer and non-cancerous conditions. The search for new compounds which might become effective PSs is the major direction for PDT development. In the present work we have studied the dark toxicity, intracellular localization and photodynamic properties of four potential, water soluble, second generation PSs--PP(Arg)(2), PP(Ser)(2)Arg(2), PP(Ala)(2)Arg(2), PP(Phe)(2)Arg(2), all diamino acid derivatives of protoporphyrin IX. Human prostate cancer (DU-145) and squamous carcinoma (A431) cells were used as experimental model. Among investigated compounds PP(Ser)(2)Arg(2) exhibited the lowest dark toxicity and the highest PDT effectiveness towards both cell lines. Fluorescence microscopy revealed the time-dependent changes in intracellular localization of the PS which were related to the phototoxicity. The results show that PP(Ser)(2)Arg(2) may be a potential PS for PDT.

Photodynamic therapy with 5‐aminolevulinic acid and diamino acid derivatives of protoporphyrin IX reduces papillomas in mice without eliminating transformation into squamous cell carcinoma of the skin

Photodynamic therapy (PDT) is used to treat malignant and nonmalignant diseases. It is also used for cosmetological skin treatment. PDT is generally considered to have a low risk of carcinogenicity. However, instances of nonmalignant human tumors turning malignant have been linked to PDT. In this study, we used 5‐aminolevulinic (ALA) acid and 3 water soluble photosensitizers‐PP(Arg)2, PP(Ser)2Arg2, PP(Ala)2Arg2, all diamino acid derivatives of protoporphyrin IX‐to treat benign papillomas in FVB/N mice induced by 7,12‐dimethylbenz(a)anthracene (DMBA)‐12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Of these drugs, ALA and PP(Arg)2 were found the most efficient. PDT reduced the number of papillomas, but with increasing effectiveness of the drugs, the risk of malignant transformation of the papillomas into squamous cell carcinomas increased. The underlying mechanisms are not clear and further investigations are needed.

Diaminoacid derivatives of protoporphyrine used as photosensitizers in photodynamic method of tumor diagnosis and treatment

Observation and selective destruction of biological tissues, due to use of photochemical reaction sensitised with photosensitive dyes, are applied in modern method of tumor diagnosis and treatment as well as destruction of atheromatous plaques and restenosis prevention. This method, called photodynamic method (PDT), has been developed for recent 30 years in USA, Canada, Japan, and China and in majority of European countries. It has many advantages that distinguish it from among other currently used diagnostic and therapeutic methods. Both, the applied photosensitizers and photodynamic effect influence on active biological compounds present in an organism, mainly on enzymatic proteins, hormones, and particular elements of immunological system. The PDT method can be used not only for in situ tumor treatment but also for the treatment in metastasis state. It was thought previously that direct activity of cytotoxic radicals and singlet oxygen causes tissues necrosis. At present, additional investigations are performed on destruction mechanisms, i.e., occlusion of blood vessels and lymphatic vessels and PDT influence on immunological system revealing in cytokines release. In Poland, the technology of new class of photosensitizer - diaminoacid derivatives of protoporphyrin PP(AA)2Arg2 as well as the basic half-product - hemis of very high purity has been developed and activities for their industrial production have been performed. In vitro investigations on quantum luminescence yield and singlet oxygen quantum yield of the obtained preparations were made. These compounds do not show toxic properties and they are neither mutagenic nor teratogenic. Three aminoacid protoporphyrine derivatives were chosen for industrial scale production, i.e., PP(Ala)2Arg2, PP(Ser)2Arg2, PP(Plu)2Arg2 that were efficient for the largest number of tumors investigated on cell lines. A preparation being the mixture of these derivatives is called Sensyphyrine. In the first stage of Sensyphyrine examinations in surgery, otolaryngology, and teracosurgery clinics over 100 patients were investigated. During the years 1999-2001, the technologies of Polish photosensitizer and dermatological preparation have been developed. This photosensitizer - IX(PPIX) protoporphyrine is 5-aminoavulenic acid (ALA) of pharmaceutic purity (99.5%) and the final form of dermatological preparation is in form of a cream (FOTOACID). The obtained preparation and designed diagnostic systems and therapeutic sources enabled us to carry out initial investigations on animals and next clinical in 400 patients.

Magnetic properties of oxidation products of phthalocyanine complexes with metal chlorides with electron acceptor properties

Abstract The effect of charge transfer complex formation on the oxidisability of various phthalocyanines complexes with Lewis acids has been investigated. Multistep electron transfer between phthalocyanine as electron donor and the molecules of electron acceptor was found to be possible, leading to the formation of mono- and polycation radicals with characteristic magnetic properties. The structure of the transition products has been established and an oxidation mechanism is suggested.

Progress in photodynamic method of tumor diagnosis and treatment

Development of photodymanic method for tumors diagnosis and therapy (PDT) was observed in the early 70s. The third stage of clinical investigations in the USA, Canada, Japan, and in European countries has begun since 1988. Tumors treatment consists in selective oxidation of biological material of a tumor tissue by a singlet oxygen or radical forms. Such forms are generated due to molecular oxygen, soluted in cells, exogeneously introduced photosensitiser which is better accumulated in the diseased tissues than in healthy ones and delivered light of adequate power and wavelength. Such therapeutic method allows selective destruction of tumor tissues, simultaneously protecting the healthy ones. Tumors diagnostics relies on localisation of photosensitisers, absorbed in tumor tissues, by means of fluorometric methods. Investigations with PDT method are carried out in several direction, i.e., synthesis and application of new photosensitisers, design of new light sources, radiation dosimetry in tissues, mechanisms of photochemical reaction, and clinical applications of PDT method. PDT method for tumors treatment compared to traditional ones (surgery, irradiation, chemotherapy) is much more selective but is constrained by many factors limiting its intensive development and clinical applications. In Poland the PDT method is applied in several clinics for treatment of skin tumors, lungs, gynaecological sphere, and urinary bladder. The paper presents current status and perspectives of development of this method. Technology for production of our own photosensitisers (aminoacid complexes and protoporphyrines) is implemented and adequate diagnostic and therapeutic systems have been constructed. Investigations required for specimens admission to clinical application were carried out.

Solvent effects on the electronic structure of bis(4-dimethylaminodithiobenzyl) nickel [NiII(4-DMAB)] from uv-vis and epr spectra

Abstract The UV-VIS and EPR spectra of solutions of Ni II (4-DMAB) in 10 solvents with donor number (DN) from 0.1 to 38.8 1 have been recorded. The changes in the spectra are in terms of a change in symmetry of the Ni II (4-DMAB) complex on increase in donor number of the solvent. The symmetry change is from square planar to rhombic bipyramidal with correspoding change in electronic configuration of the nickel ion from d 2 z 2 to d 1 z 2 , d 1 x 2 -y 2 . Light was observed to accelerate this structural change.

Interactions of protoporphyrin IX and its derivatives with benzodiazepine receptor

The aim of this study was to determine binding affinities of the Peripheral benzodiazepine receptor (PBR) with protoporphyrin IX, haematoporphyrin (Hp), and two arginized derivatives: Hp(Arg)(2) and PP(Arg)(2). The quenching of protein fluorescence was used to measure association constants during titration of PBR solution with PP derivatives in a function of concentration. The experimental data was fitted to theoretical curves, assuming strong and weak binding model with one binding site. As a result of this early study affinity constants were determined. The highest affinity toward PBR showed PPIX and Hp, Hp(Arg)(2), PP(Arg)(2) lower, respectively. Changes in pH of protein solution resulted in decrease of association constants in nearly all examined derivatives.

Interactions of peripheral-type benzodiazepine receptor with diamino acid derivatives of protoporphyrin IX

The aim of this study was to examine and determine binding affinities of the peripheral benzodiazepine receptor (PBR) with diamino acid derivatives of protoporphyrin IX with general structure PP(AA)(2) in a function of environmental pH changes. Fluorescence intensity was used as a measure of protein association constant. Decrease in protein fluorescence, after titration with increasing concentration of porphyrins, gave evidence of interactions between PBR and examined ligands. Experimental data were fitted to theoretical curves, assuming different models of interactions. All examined ligands were best fitted to weak binding model. Increase in environmental acidity resulted in changes in association constants. For all examined derivatives association constants were at least twice higher in pH 5.5 as compared to pH 7.4 and 6.5.

Influence of photodynamic effect on biological activity of PBR–PP complexes

The aim of this study was to examine the influence of photodynamic effect on biological activity of PBR-PP complexes. These measurements were performed in pH dependent environment. Constant concentration of solubilized receptor was titrated with increasing concentration of porphyrins (PPIX, Hp, PP(Arg)(2), Hp(Arg)(2), PP(Gly)(2), PP(Ala)(2), PP(Ser)(2), PP(Phe)(2)) and binding constants were calculated. PBP-PP mixtures were illuminated with 3 J, 5 J or 10 J of blue light and changes in protein fluorescence was recorded. Experimental data were fitted to weak and strong binding models. As a result for all derivatives weak binding model was the best fitted. The strongest binding showed PPIX in pH 7.4 and with pH drop binding constants showed greater values for all examined derivatives. Out of amino acid derivatives the strongest binding was noticed for PP(Gly)(2) and PP(Phe)(2) and for the last one pH influence was not observed.

Localization of porphyrine amino acid derivatives in superficial tumors using laser-induced fluorescence

The results of examinations of accumulation of bis-1(I- alanylo-N)-ethylo-deuteroprotoporfiryne (Ala-PP) in healthy tissues and superficial tumors of 10 patients are presented. Fluorescence imaging was carried out by means of a CCD camera, coupled with an image amplifier. As an excitation source a titanium laser was used. Contrary to healthy tissues, the tumor ones are characterized by the slight decrease in Ala-PP concentration during several days after injection of preparation what enables initial determination of a tissue character and next application of a selective therapeutic irradiation.