Sulphoselenide phthalocyanines as photosensitizers for photodynamic therapy


(57) Abstract:

The invention relates to the field of medicine and for the application of sulfadimidine besmearing phthalocyanine and its magnesium complex of General formula (I) as a photosensitizer for photodynamic therapy (FDP). These phthalocyanines are more effective photosensitizers in PDT process. 3 table.

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where M=HH [PcsH2]; Mg[PcsMg].

The present invention relates to pharmaceutical chemistry, and more particularly to drugs for photodynamic therapy (PDT) of malignant tumors and other abnormal growths.

Photodynamic therapy is based on the use of substances of photosensitizers, which when introduced into the body mainly localized in the tumor, and when light, in particular laser excitation, produce cytotoxic substances, especially singlet oxygen. Currently in clinical practice are applied photosensitizers based on complex mixtures hematoporphyrin derivative, for example Fotofrin-II (E. Sternberg, D. Dolphin, S. Bruckner. Tetrahedron, 54, 4151 (1998)).

The lack of photosensitizers based on hematoporphyrin derivative is then the to the absorption desensibilisation tissues in this area very much. This leads to a significant loss of radiation outside the tumor tissue, leads to the small penetration depth of the radiation in the tissue, which complicates the treatment of tumors of large size and leads to the necessity to use a large dose in the body of the drug and therapeutic radiation. All in all, this leads to low efficiency of PDT.

These disadvantages are partially eliminated when using a photosensitizer for PDT - sulfonated aluminum phthalocyanine ("Photosense") absorbing in the spectral range 660-680 nm. The extinction coefficient "Photosense" at the maximum of the absorption band is more than 105lol-1cm-1the absorption desensibilisation tissues in this range decreases, therefore increasing the depth of the photodynamic effects on tissues, which improves therapeutic efficacy of PDT (in. A. Sokolov, E. F. Stranadko, N. N. Zharkova, R. I. Yakubovskaya, E. C. Filonenko, T. A. Astrakhankina. Oncology issues, 41,

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134 (1995)). However, "photosense" has still some drawbacks, the main of which is very high value used doses (0.5-0.8 mg per kg of body weight), resulting in nalichie would have been more effective in the process of PDT, than "photosense". To solve this problem it is proposed to use as photosensitizers for FTD phthalocyanines following formula:

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where M=HH [PcsH2]; Mg [PcsMg]

Know the use of similar link structure - TETRANITRATE salt besmearing Tetra-4-sulfitation as acid dye (Cl, CC-Blue 249)

Use as photosensitizers some sulfonated metal phthalocyanine complexes, such as aluminum complex, it is not possible to assert that other metal complexes, as well as bezmetallny phthalocyanine can be used for the same purpose. It is known, for example, that the complexes of paramagnetic metals (copper, vanadium, etc.,) because of their photophysical properties (short-lived triplet state) do not generate singlet oxygen and can not be used as photosensitizers. (N. li, J. E. van Lier, Chem. Rev. 1999, v. 99, No. 9, pp. 2379-2450).

As for besmearing compounds in aqueous solutions it is in the fully aggregated state and accordingly absorbs in a different region of the spectrum (see page 3 of the description), has no fluorescence and does not generate singlethe about what sulfonated bezmetallny phthalocyanine does not have photosensitizing properties and cannot be used as a drug for PDT (N. Brasseur, N. Ali, R. Langlois, J. R. Wagner, J. Rousseau and J. E. van Lier, Photochem. Photobiology, 1987, v. 45, No. 5, pp. 581-586). However, as was shown by us, in biological environments due to the interaction with elements of cellular structures is a disaggregation of these two compounds (which is not obvious) and as a consequence manifestation of photosensitizing properties.

1. Synthesis of photosensitizers

Sulfonic acids besmearing phthalocyanine obtained by the sulfonation unsubstituted RSN2equimolecular or close to equimolecular amounts of chlorosulfonic acid in a high boiling inert solvent (trichlorobenzene, o-dichlorobenzene, etc.,) at 190-210oC. the Yield of the target products is 92-65%, Sulfonic phthalocyanine magnesium obtained by metallation RSsH2.

The following examples illustrate the invention.

Example 1. The disodium salt of dissolvability besmearing phthalocyanine. To a suspension 6,15 g (0.012 mol) besmearing phthalocyanine in 45 ml of trichlorobenzene added under stirring and 2.79 g (1.6 ml, 0,024 moloC, the precipitate is filtered off hot, washed successively with toluene, acetone, 5% hydrochloric acid, dried in vacuum, to obtain 8.5 g of technical sulfonic besmearing phthalocyanine. Technical complex is dissolved in water with 0.5% NaOH solution, filtered to remove insoluble impurities, and then acidified with diluted hydrochloric acid. The precipitation is filtered off, washed with 5% aqueous NaCl solution until neutral, then washed with 80% aqueous ethanol, to lack of chlorine ions in the wash water, and then absolute hot alcohol, dried, get of 8.04 g (93%) disodium salt of dissolvability besmearing phthalocyanine. After further purification of the complex by presidenial from water-ethanol and 7.5 g (87%) disodium salt of dissolvability besmearing phthalocyanine.

Found, %: N 14,99; S AT 9.53

WITH32H16PA2H806S2< / BR>
Calculated, %: N 15,59; S 8,92.

According to chromatographic analysis of the products of hydrolysis, the resulting complex contains 2 sulfopropyl maxnm (lg) in dimethyl sulfoxide: 696 (5,06), 663 (5,04), 645, 602.maxnm (lg) in buffer pH 6,86: 610 width. (4,45).

Example 2. The disodium salt of dialettologia or dimethylformamide.

To a solution of 0.35 g (0.49 mmol) of sodium salt of dissolvability besmearing phthalocyanine in 15 ml of dimethylsulfoxide added a solution of 0.11 g (0.51 mmol) of magnesium acetate tetrahydrate in 5 ml of dimethylsulfoxide. The mixture was stirred at 50-55oWith 1.5 h, filtered. To the solution was added chloroform, precipitated precipitate is filtered off, washed successively with chloroform, acetone, isopropanol, ethanol, dried, obtain 0.35 g (96%) disodium salt of dissolvability phthalocyanine magnesium. After additional purification by presidenial from water-isopropanol obtain 0.25 g (69%) disodium salt of dissolvability phthalocyanine magnesium.

Found, %: S 8,50

C32H14N8ABOUT6MgNa2S2< / BR>
Calculated, %: S 8,65.maxnm (lg) in dimethylformamide: 674 (5,25), in 4% aqueous dimethylformamide: 677 (5,15).

Mass spectral analysis method (MALDI) samples, and mass spectral analysis of the fractions obtained from the original sample separation using column liquid chromatography shows that the samples represent a mixture of mono-, di - and tri - sulfated derivatives. In a mixture dominated by desulfuromonas derived (70-80%).

Using this method were that the SUB> and PcSMg in vitro

2.1. Analysis of pharmacodynamics accumulation and discharge cells PcSH2and PcSMg

The studies were performed on cells adenocarcinoma easy person A. Cells were cultured in the medium Needle-MEM with the addition of 100 μg/ml gentilicia, 2 mm L-glutamine and 10% V / V heat inactivated fetal calf serum at 37oC in humidified atmosphere containing 5% C02. Cells were grown on cover glasses in 24-hole flat-bottomed plates (seeding 5104CL/cm3. After 24 h, after the formation of the loose layer on Wednesday introduced the study drug.

Stock solution of MSSH2and PcSMg (1 mm) were prepared in dimethyl sulfoxide. In culture medium, the concentration of DMSO in all experiments did not exceed 1%. When studying the kinetics of accumulation of cells incubated with RSSH2and PcSMg at a final concentration of 10 μg/ml for 10, 30 min, 1, 3 or 6 h, and then selected the medium, washed twice, the cells with warm saline solution and then spent Microspectral measurement. When studying the kinetics of excretion of the drug from cells cells were incubated with RSSH2and PcSMg in to the m and then placed in fresh culture medium for 30 min, 1, 6 or 24 h, again washed with warm saline solution and then spent Microspectral measurement. In the study of the concentration dependence of the accumulation of cells incubated with 0.4, and 0.8, 2, 10 or 50 μg/ml MSSH2and PcSMg for 30 min, then selected the medium, the cells were twice washed with warm saline solution and then spent Microspectral measurement.

For an analysis of accumulation, distribution and interaction RSSH2and PcSMg in cells used confocal Microspectroscopy and reconstruction of two - and three-dimensional spectral images (COMERCI).

Spectral analysis showed that RSSH2and PcSMg in cells are mainly associated with the membrane structures and, to a lesser extent, protein. The data obtained shows that the kinetics of penetration in cells RSSH2and PcSMg is relatively fast: beginning to plateau after 1 h and reaches a plateau after 3 h of incubation. The average intracellular concentration of photoactive form exceeds the concentration of the drug in the external environment for PCSH28-9 times, and for PcSMg in 1.5 times. At equal concentrations "Photosense" and MSSH2and Pcrevisit intracellular concentration "Photosense", that, obviously, is one of the main reasons for very high photodynamic activity RSSH2and PcSMg.

When removing the drug from the external environment of the intracellular concentration for 1 h falls: if RSSH21.7-2 times, for PcSMg - 1.3-1.5 times, and in the case of "Photosense" - 2-2,5 times. However, the intracellular concentration "Photosense" after that remains unchanged within 24 h, the intracellular concentration RSSH2continue to slowly decline after 24 h is only 5-10% from baseline.

2.2. The study of the cytotoxicity and photoactivity RSSH2and PcSMg

The study of the cytotoxicity and photoactivity RSSH2and PcSMg in vitro were performed on cell cultures epidermoid carcinoma of the hypopharynx man (ner-2). Test compounds were dissolved in distilled water with addition of 10% fetal calf serum, followed by titration to the working concentration was performed with step 2 in the environment of the Needle. To assess fototoestel cells before irradiation were incubated with serial dilutions of test compounds for 2 h under standard conditions (at 37oIn moist atmospheres the MCU power 500 W, filter KS-13 (640 nm). A light dose of 10 j/cm2Cell survival was assessed visually using light microscopy and a colorimetric method using the MTT-test. Determined the levels of inhibition of cell growth and IR50(the concentration of the cytotoxic agent, in which the observed inhibition of cell growth by 50%).

The experimental results showed that RSSH2and PcSMg is characterized by the absence of biologically significant levels of dark cytotoxic activity and exhibit high photodynamic activity, the level of which considerably exceeds the level of the photodynamic activity of the drug "photosense". So, IR50for RSSH2is 0,910-7M, PcSMg - 1,810-7M, whereas IR50"Photosense" equal 2,510-6M

3. Study MSSH2and PcSMg in vivo

3.1. Photodynamic therapy

RSSH2was injected intravenously at a dose of 0.5 mg/kg, a PcSMg - dose of 0.5 mg/kg and 2 mg/kg to mice BDF1with the growth of P-388, inoculated subcutaneously in the region of the gastrocnemius muscle of the right paw. After 24 h after administration of photosensitizers tumors were irradiated with red light (filters KC-10 and SES-26, 630 nm is th spot was 1.5 cm, the power density is 360 mW/cm2a light dose of 324 j/cm2.

The antitumor effect was evaluated by inhibition of tumor growth (SRW) relative to the corresponding values in animals not subjected to any stress. Biologically significant thought TO% (Experimental evaluation of anticancer drugs in the USSR and the USA. 1980., M, Medicine. - S. 71-106).

The results of the conducted experiments have shown that PDT with PCSH2and PcSMg inhibits the growth of P-388 (table 1), and RSSH2when using a lower dose of photosensitizer than "photosense".

3.2. The study of the accumulation in the tissues of animals

The selectivity of accumulation RSSH2and PcSMg was studied in mice with Ehrlich tumor. The content of the photosensitizer in the tissue was estimated by fluorescence spectroscopy on laser diagnostic unit on the basis of He-Ne laser (wavelength generation - 633 nm) contact method ex vivo (N. N. Zharkova, D. N. Kozlov, Yu. N. Polivanov et al. "Laser-excited fluorescence spectrometric system for tissue diagnostics," Proc. SPIE, vol. 2328, pp. 196-201 (1994).

The dye was injected intravenously at a dose of 1 mg/kg as a control comparison was introduced "photosense" in a dose of 2 mg/kg (the dose at which the constant of Seleia" in Ehrlich tumor maximum) (Kazachkina N. I., Zharkova, N. N., Fomina G. I., et al. Pharmacokinetical study of Al - and Zn-sulphonated phthalocyanines. Proc. SPIE, vol.2924. pp. 233-249, 1996).

Exogenous fluorescence was recorded after 24 and 48 h after injection of the dye. It was shown that RSSH2and PcSMg in animals possess fluorescent properties in tissues of mice have a maximum fluorescence at 683-693 nm and selectively accumulate in Ehrlich tumor with the parameters of the selectivity of not less than the COP, "Photosense" (table 2).

3.1. Studies of the distribution and interactions of MSSH2in the various structures of tumor tissue

Cryogenic tissue sections were prepared as follows. The Ehrlich tumor transplanted mice subcutaneously. On the 14th day of tumor growth, the animals were injected intravenously once MSSH2at a dose of 5 mg/kg of body weight. After 24 h, animals were killed and dissected the tumor with surrounding tissues.

Serial cryostate slices were cut with a thickness of 10 μm. The sections were dried in air in the dark and stored at -20oC. One of the serial sections were fixed in 10% formalin and stained with hematoxylin/eosin. This cutoff was used for preliminary identification of tissue structures and their mapping. The cards are colored slices Prime method COMERCI was performed on unstained sections. Then these slices were fixed in 10%formalin and stained with hematoxylin/eosin Region, were investigated by means of COMERCE, were subjected to detailed histochemical analysis to identify tissue structures present in the spectral images.

Average relative concentration RSSH2and "Photosense" in various tissue structures are presented in table 3. Analysis of these data shows that the relative distribution of MSSH2in tissue structures significantly different from the distribution of "Photosense" and is characterized by a relatively homogeneous distribution in the tumor and its surrounding tissues, tumor cells, connective tissue and skin accumulate it in similar concentrations. The main difference relative distribution "Photosense" in tissue structures from distribution MSSH2is preferred (5-7 times) accumulation in the structures surrounding the tumor cells, but not in the cells.

If a divided total antitumor effect of PDT in vivo in two components: - the destruction of tumor cells by direct effects on tumor cells by fractions of a photosensitizer, loaysa and feeding the tumor tissue structures due to the fraction of a photosensitizer, localized in the surrounding tissue structures, it can be argued that photodynamic action "Photosense" will largely be determined by the effect of the Century. And in the case of MSSH2the relative importance of the effect And must be significantly higher than the "Photosense".

Thus, the synthesized sulphoselenide bezmetallny (RSSH2) and magnesium (RSSMD) phthalocyanines are effective photosensitizers in PDT process, and the effectiveness of sulfadimidine besmearing (RSSH2much greater than the efficiency of the famous "Photosense".

Application sulfisomidine phthalocyanines of General formula

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where M=HH [PcsH2]; Mg[PcsMg],

as photosensitizers for photodynamic therapy.


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